i UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO DOCTORADO EN CIENCIAS BIOMÉDICAS INSTITUTO DE FISIOLOGÍA CELULAR REGULACIÓN DEL CANAL TRPV4 POR SALBUTAMOL T E S I S QUE PARA OPTAR POR EL TÍTULO DE: DOCTOR EN CIENCIAS P R E S E N T A : MIGUEL BENÍTEZ ANGELES DIRECTORA DE TESIS: DRA. TAMARA LUTI ROSENBAUM EMIR INSTITUTO DE FISIOLOGÍA CELULAR, UNAM COMITÉ TUTOR DRA. MARCIA HIRIART URDANIVIA INSTITUTO DE FISIOLOGÍA CELULAR, UNAM DRA. STÉPHANIE C. THÉBAULT INSTITUTO DE NEUROBIOLOGÍA, UNAM Cd. Universitaria, Cd. Mx., México, 2024 UNAM – Dirección General de Bibliotecas Tesis Digitales Restricciones de uso DERECHOS RESERVADOS © PROHIBIDA SU REPRODUCCIÓN TOTAL O PARCIAL Todo el material contenido en esta tesis esta protegido por la Ley Federal del Derecho de Autor (LFDA) de los Estados Unidos Mexicanos (México). El uso de imágenes, fragmentos de videos, y demás material que sea objeto de protección de los derechos de autor, será exclusivamente para fines educativos e informativos y deberá citar la fuente donde la obtuvo mencionando el autor o autores. Cualquier uso distinto como el lucro, reproducción, edición o modificación, será perseguido y sancionado por el respectivo titular de los Derechos de Autor. ii El presente trabajo de investigación fue realizado bajo la tutoría de la Dra. Tamara Luti Rosenbaum Emir en el Instituto de Fisiología Celular, UNAM. El proyecto fue financiado a través del Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) de la UNAM con clave IN200720 e IN200423 y por el Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT) con número A1-S-8760. Asimismo, contó con el apoyo de la Secretaría de Educación, Ciencia, Tecnología e Innovación del Gobierno de la Ciudad de México (SECTEI/208/2019). El sustentante contó con una beca de CONAHCyT para estudios de posgrado, número 1002182. iii Jurado de examen PRESIDENTE Dr. Alberto Darszon Israel SECRETARIO (A) Dra. Tamara Luti Rosenbaum Emir VOCAL Dra. Adela Rodríguez Romero VOCAL Dr. Gerardo Alfonso Corzo Burguete VOCAL Dr. Enoch Luis Baltazar iv Agradecimientos A la Dra. Tamara Rosenbaum, por la confianza depositada en mí desde el primer día, por su tiempo, paciencia y apoyo durante mi formación en el laboratorio, por mostrar con su ejemplo que el trabajo científico requiere de esfuerzo, así como los consejos que me brindó para diferentes aspectos y circunstancias. A la Dra. Marcia Hiriart y a la Dra. Stéphanie Thébault, por formar parte de mi comité tutor, por sus observaciones y comentarios cada semestre que ayudaron a mejorar mi trabajo, así como sus palabras de motivación para continuar en la ciencia. A los miembros del jurado de mi examen de candidatura: Dra. Nuria, Dra. Marcia, Dr. Takuya, Dr. Arturo y Dr. Ranier por sus preguntas y comentarios durante la evaluación que ayudaron a fortalecer mi trabajo; y a los miembros del jurado de mi examen profesional: Dra. Tamara, Dr. Alberto, Dra. Adela, Dr. Gerardo y Dr. Enoch por sus correcciones y sugerencias a mi escrito de tesis. A la Biól. Alejandra Llorente Gil por su apoyo técnico en la construcción de mutantes, cultivo y transfección celular durante mi estancia en el laboratorio. A Félix Sierra por compartir conmigo su conocimiento en el uso del equipo, por sus consejos y apoyo. A la Unidad de Biología Molecular, a la Unidad de Cómputo y a Sandra G. Moncada Hernández, por los servicios y apoyo técnico brindados durante mi estancia en el instituto. A mis padres, a mi abuela y a mi hermana por sus consejos, cariño y apoyo durante estos años que sin duda me han hecho crecer como persona. A Angélica por tu apoyo, paciencia, confianza, ánimos, consejos, compañía y más en este viaje. A Raúl por tu sincera amistad. A mis amigos de la preparatoria, la facultad y el instituto, por su amistad y por darme la oportunidad de conocerlos. v Abreviaturas b1-AR.- b1-adrenergic receptor / Receptor b1-adrenérgico b2-AR.- b2-adrenergic receptor / Receptor b2-adrenérgico 4a-PDD.- 4 alpha-phorbol 12,13-didecanoate / 4 alfa-forbol 12,13-didecanoato AA.- Arachidonic acid / Ácido araquidónico AIP4.- Atrophin-1-interacting protein-4 / Proteína-4 que interactúa con la Atrofina-1 ALI.- Acute lung injury / Lesión pulmonar aguda ARD.- Ankyrin repeat domain / Dominio de repetidos de anquirina ARDS.- Acute respiratory distress syndrome / Síndrome de dificultad respiratoria aguda ATP.- Adenosine triphosphate / Adenosín trifosfato BKCa.- Large-conductance Ca2+-activated K+ channel / Canales de K+ activados por Ca2+ de alta conductancia BR.- Bradykinin receptor / Receptor de bradicinina CaCl2.- Calcium chloride / Cloruro de calcio CaM.- Calmodulin-binding domain / Sitio de unión a calmodulina cAMP.- Cyclic adenosine monophosphate / Adenosín monofosfato cíclico CD.- Coupling domain / Dominio de contacto cDNA.- Complementary deoxyribonucleic acid / Ácido desoxirribonucleico complementario CNG.- Cyclic nucleotide–gated ion channel / Canales iónicos activados por nucleótidos cíclicos COPD.- Chronic obstructive pulmonary disease / Enfermedad pulmonar obstructiva crónica DAPL.- Ácido aspártico (D), Alanina (A), Prolina (P) y Leucina (L) DMEM.- Dulbecco’s modified Eagle’s medium / Medio Eagle modificado de Dulbecco DMSO.- Dimethyl sulfoxide / Dimetilsulfóxido DRG.- Dorsal root ganglion neurons / Neuronas del ganglio de la raíz dorsal EAG.- Ether‐à‐go‐go channels / Canales de potasio etér-à-go-go vi EDTA.- Ethylenediaminetetraacetic acid / Ácido etilendiaminotetraacético EET.- Epoxyeicosatrienoic acids / Ácidos epoxieicosatrienoicos GPCR.- G protein-coupled receptors / Receptores acoplados a proteínas G GSK.- GSK1016790A o N-[(1S)-1-[[4-[(2S)-2-[[(2,4-Dichlorophenyl)sulfonyl]amino]-3-hydroxy- 1-oxopropyl]-1-piperazinyl]carbonyl]-3-methylbutyl]benzo[b]thiophene-2-carboxamide GTP.- Guanosine-5'-triphosphate / 5’-trifosfato de guanosina HEK293.- Human embryonic kidney 293 cells / Células embrionarias de riñón humano 293 HEPES.- N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid / Ácido N-2-hidroxietilpiperazina- N'-2-etanesulfónico (amortiguador) hSkM1.- Human skeletal muscle sodium channel-1 / Canales de sodio del músculo esquelético humano-1 IK.- Intermediate-conductance calcium-activated potassium channel / Canales de K+ activados por Ca2+ de conductancia intermedia IP3.- Inositol 1,4,5-trisphosphate / Inositol 1,4,5-trisfosfato LABAs.- Long acting β2 agonists/ Agonistas del receptor β2-adrenérgico de larga duración. LB.- Luria-Bertani broth / caldo Luria-Bertani LPA.- Lysophosphatidic acid 18:1 / Ácido lisofosfatídico 18:1 Lyn.- Lck/Yes-related novel protein (tyrosine kinase) / Cinasa Lyn relacionada con Lck/Ye (tirosina cinasa) NaCl.- Sodium chloride / Cloruro de sodio NaOH.- Sodium hydroxide / Hidróxido de sodio PACSIN.- Protein kinase C and casein kinase substrate in neurons protein 3 / Proteína cinasa C y del sustrato de la caseína cinasa en las Neuronas PCR.- Polymerase chain reaction / Reacción en cadena de la polimerasa PDZ.- Postsynaptic density protein of 95 kDa (PSD95), Drosophila disc large tumor suppressor (DlgA), and Zonula occludens-1 protein (Zo-1) / Proteína de densidad postsináptica (PSD95), vii Dominio supresor de tumores grandes del disco de Drosophila (Dlg1) y proteína Zonula occludens 1 (Zo-1) pEGFP-N3.- Vector for fusing Green Fluorescent Protein to the C-terminus of a partner protein / Vector para fusionar Proteína Verde Fluorescente con el extremo C de una proteína asociada. PIBS.- Phosphoinositide binding site / Sitio de unión a fosfoinosítidos PIP2.- Phosphatidylinositol 4,5-bisphosphate / Fosfatidilinositol-4,5-bifosfato PKA.- Protein kinase A / Proteína cinasa A PKC.- Protein kinase C / Proteína cinasa C PRD.- Proline rich domain / Dominio rico en prolinas SABAs.- Short acting β2 agonists/ Agonistas del receptor β2-adrenérgico de corta duración. SGK1.- Serum and glucocorticoid-regulated kinase-1 / Cinasa-1 regulada por suero/glucocorticoides SK.- Small-conductance calcium-activated potassium channel / Canales de K+ activados por Ca2+ de conductancia pequeña STIM1.- Stromal interaction molecule-1 / Molécula de interacción estromal-1 TRP.- Transient receptor potential / Receptores del potencial transitorio TRPA.- Ankyrin TRP / Canal TRP miembros de la subfamilia de anquirinas TRPM.- Melastatin-related TRP / Canales TRP miembros de la subfamilia de melastatina TRPV.- Vanilloid TRP / Canales TRP miembros de la subfamilia vaniloide tsA201.- Transformed human kidney cell stably expressing an SV40 temperature-sensitive T antigen / Células de riñón humano transformadas para la expresión estable del antígeno T sensible a la temperatura SV40 Ultra-LABAs.- Ultra long acting β2 agonists / Agonistas del receptor β2-adrenérgico de ultra larga duración. VSLD.- Voltage sensor like-domain / Dominio similar al sensor de voltaje viii Índice Resumen 1 Abstract 2 Introducción A) Canales iónicos 3 B) Canales TRP 4 C) Generalidades del canal TRPV4 4 D) El canal TRPV4 y el sistema respiratorio 8 E) El salbutamol y otros agonistas del receptor b2-AR 10 F) El receptor b2-adrenérgico 12 G) Vía canónica de señalización del b2-AR 13 H) Interacción entre el b2-AR y el canal TRPV4 14 I) Efecto de los agonistas del b2-AR en canales iónicos 16 J) Mecanismos de inhibición de un canal iónico 17 Hipótesis 19 Objetivo general 19 Objetivos particulares 19 Métodos a) Cultivo de células HEK293 20 b) Mutagénesis dirigida en los posibles sitios de unión del salbutamol en el canal TRPV4 20 c) Modelaje computacional 22 d) Experimentos de electrofisiología 22 e) Reactivos y disoluciones 28 f) Análisis y estadísticos 29 Resultados 30 Discusión 50 Conclusiones 54 Consideraciones finales 55 Referencias 56 Resumen El Receptor del Potencial Transitorio Vaniloide 4 (TRPV4) es un canal catiónico no selectivo presente en diversos tejidos incluyendo al sistema respiratorio, donde participa en el mantenimiento de la barrera alvéolo-capilar y en la remodelación de las vías respiratorias. Así mismo, participa en la regulación de la presión osmótica celular y en la contracción muscular, entre otras funciones; al regular la entrada de Ca2+ y modificar el potencial de membrana. La función desregulada del canal TRPV4 se asocia a diferentes síntomas como la tos, la inflamación y la contracción de las vías aéreas que derivan en diferentes enfermedades. El asma es una patología que se caracteriza por la constricción de los bronquios y que, tradicionalmente, se ha tratado usando broncodilatadores, como los ligandos que interaccionan con los receptores b2- adrenérgicos (b2-AR). En este trabajo se demostró que el salbutamol, un broncodilatador y agonista del receptor b2-AR, inhibe parcialmente la actividad del canal TRPV4 al unirse de forma directa en una región cercana al poro. Estos resultados sugieren que el TRPV4 puede ser un posible blanco terapéutico para el tratamiento de algunas enfermedades respiratorias. 1 2 Abstract The Transient Receptor Potential Vanilloid V4 (TRPV4) is a non-selective cation channel, which is expressed in several tissues including the respiratory system where it participates in the maintenance of the alveolar-capillary barrier and in the remodeling of the airways. Moreover, this channel also participates in the regulation of cellular osmotic pressure, and muscle contraction, among other functions, by regulating the entry of Ca2+ and modifying the membrane potential. Dysregulation of the function of TRPV4 has been associated to several symptoms such as coughing, inflammation, and airway constriction. Asthma is a disease characterized by bronchial constriction and it is commonly treated with bronchodilators, such as ligands which interact with b2-adrenergic receptors (b2-AR). In this work, it was shown that salbutamol, a bronchodilator and b2-AR agonist, partially inhibits the activity of the TRPV4 channel by interacting directly in a region near its pore. These data highlight TRPV4 as a possible pharmacological target in the treatment of some respiratory diseases. 3 Introducción y Antecedentes A) Canales iónicos Las membranas celulares son estructuras hidrofóbicas conformadas principalmente por foslípidos, cuyo ordenamiento forma una barrera física semipermeable entre el medio extracelular y el intracelular de las células. Sin embargo, en dichas membranas también se encuentran proteínas que, a diferencia de los fosfolípidos, permiten el paso regulado de moléculas de gran tamaño, hidrofílicas o cargadas (Cooper, 2000; Kulbacka et al., 2017). Los canales iónicos son proteínas altamente especializadas que se encuentran en una gran diversidad de organismos desde bacterias hasta animales superiores, incluyendo los humanos, donde se expresan en diferentes tipos celulares y sistemas permitiendo la interacción del medio interno y externo de las células. Lo anterior es fundamental para varias funciones biológicas como el mantenimiento de la presión osmótica, la regulación del pH celular y el transporte de diferentes moléculas o iones como el agua, la glucosa, el Na+, el K+, el Ca2+ o el Cl- (Kulbacka et al., 2017). Las proteínas presentan una relación estrecha entre su estructura y su función, y los canales iónicos no son la excepción. Estas proteínas en respuesta a estímulos específicos, exhiben transiciones entre varios estados no conductores y conductores siendo el resultado de cambios conformacionales en su estructura, los llamados mecanismos de compuerta (o de apertura/cierre) (Hille, 2001; Petkov, 2009). Dichos mecanismos regulan el paso de ciertos iones a favor de un gradiente electroquímico, sin gasto de energía, (Hille, 2001); esta propiedad le permite a las células generar respuestas como la coordinación de la contracción muscular o la generación de los potenciales de acción en el sistema nervioso (Eisenberg, 1998; Petkov, 2009). 4 B) Canales TRP Los receptores de potencial transitorio (TRP) son canales catiónicos no selectivos que participan en la transducción de señales de las células, regulando el potencial de membrana y las concentraciones intracelulares de varios iones, principalmente Ca2+ (Samanta et al., 2018). La familia TRP se ha dividido por la homología de sus secuencias, en siete subfamilias: canónica (TRPC), vaniloide (TRPV), melastatina (TRPM), policistina (TRPP), mucolipina (TRPML), anquirina (TRPA) y nompC (TRPN) (Yin & Kuebler, 2010). Reportes recientes describen nuevas subfamilias: soromelastatina (TRPS), tipo-TRP (TRPL) y levadura (TRPY/TRPF); presentes únicamente en organismos invertebrados (Himmel et al., 2020; Himmel & Cox; 2020). Estas proteínas, que se consideran polimodales por su activación ante múltiples estímulos, se expresan en levaduras, animales invertebrados y vertebrados (Nilius & Owsianik, 2011), participando en procesos como la fototransducción, en la homeostasis del Ca2+, en la generación de dolor, en la respuesta a cambios moderados de temperatura y en la modulación del ciclo celular, entre otras funciones (Montell et al., 2002). C) Generalidades funcionales y estructurales del canal TRPV4 Dentro de la subfamilia de los canales TRPV, el TRPV4 se describió por primera vez en el año 2000 (Liedtke, et al., 2000 y Strotmann et al., 2000) como un canal catiónico no selectivo, con mayor permeabilidad a iones divalentes como el Mg2+ y el Ca2+. En presencia del Ca2+ extracelular, el canal se comporta como un rectificador saliente (Voets et al., 2002) y, ante un aumento de la concentración intracelular de Ca2+, el canal presenta una potenciación seguida de una inhibición (Strotmann et al., 2003; Strotmann et al., 2010). 5 El canal TRPV4 se activa por condiciones hipoosmóticas, por cambios en la temperatura ambiental (temperaturas cercanas a los 27 ºC), por estrés mecánico (Liedtke et al., 2000; Liedtke & Friedman, 2003) y por compuestos químicos endógenos como el ácido lisofosfatídico o LPA (Benítez et al., 2023), los derivados del ácido araquidónico (AA), endocanabinoides y el modulador fosfatidilinositol-4,5-bifosfato o PIP2 (Garcia-Elias et al., 2013). También responde a compuestos de origen natural como el bis-andrografólido A de la planta Andrographis paniculate, el flavonoide apigenina (Smith et al., 2006; Ma et al., 2012) y a moléculas sintéticas como el 4 alfa-forbol 12,13- didecanoato (4a-PDD) y el GSK1016790A, al cual me referiré en este escrito únicamente como GSK (Watanabe et al., 2002; Goldenberg et al., 2015). Comúnmente, el canal TRPV4 se expresa como un homotetrámero (Schaefer, 2005) aunque puede formar heterotetrámeros con otros canales TRP como TRPC1 y TRPP2, en una estequiometría 2:2 alternada (Stewart et al., 2010; Ma et al., 2011). Recientemente también se ha descrito su ensamble con el canal TRPV3 (Hu et al., 2022). Las subunidades del canal TRPV4 (871 residuos de aminoácidos en el canal de humano) cuentan con seis cruces transmembranales (S1-S6), donde las primeras cuatro hélices (S1-S4) forman un dominio similar al sensor de voltaje (VSLD) de los canales de K+, que interacciona con el S5 y S6 (dominio del poro) de la subunidad adyacente en un intercambio de dominios (Deng et al., 2018; Kwon et al., 2023); además cada subunidad cuenta con un extremo amino terminal y uno carboxilo terminal que se ubican en el citoplasma (Fig. 1) (Garcia- Elias et al., 2013; Goldenberg et al., 2015). En el extremo amino terminal de cada subunidad del canal TRPV4 se encuentra un dominio rico en prolinas, en donde los residuos de prolina 142 y 143, interactuan con la proteína cinasa C y del sustrato de la caseína cinasa en las neuronas 3 (PACSIN 3), que actúa como un modulador negativo de TRPV4 ante estímulos térmicos e hipoosmóticos (Cuajungco et al., 2006; D'hoedt et al., 2008), reorientando y estabilizando la región N-terminal del canal lejos del PIP2 (Goretzki et al., 2018). 6 De igual manera se han descrito un sitio de unión a fosfoinosítidos (++W++), al que se une el PIP2, sensibilizando al canal ante estímulos térmicos u osmóticos (Garcia-Elias et al., 2013); un dominio con seis repeticiones de anquirina (ARD) involucradas en el transporte, anclaje y localización del canal y que también permiten la interacción del canal con otras proteínas (Denker & Barber, 2002, Sedgwick & Smerdon, 1999). Recientemente, se ha descrito la interacción del canal TRPV4 con la GTPasa RhoA que se encuentra anclada a la membrana, esta relación implica una modulación mutua, en la cual el TRPV4, además de regular la entrada de Ca2+, interactúa con la región catalítica de RhoA y ésta, a su vez, limita el movimiento del canal previniendo su apertura espontánea hasta su activación por los estímulos correctos (McCray et al., 2021; Nadezhdin et al., 2023). Fig. 1. Representación esquemática de una subunidad del canal TRPV4 y su ubicación en el tetrámero. Cada subunidad del tetrámero está conformada por seis cruces transmembranales (cilindros rojos, VLSD y amarillos, poro), un extremo amino (NH2) y carboxilo (COOH) terminal en los que se encuentran dominios que participan en la interacción y regulación de la proteína. La estructura se resolvió con la técnica de Cryo-EM a una resolución de 3 Å (8T1B). 7 Entre el ARD y el S1, se ubican dos hebras b que, junto con una tercera hebra b del extremo carboxilo terminal, forman una hoja b antiparalela del dominio de contacto (CD), este acoplamiento permite la interacción de regiones transmembranales e intracelulares claves en la regulación del canal como los enlazadores S2-S3 y S4-S5 así como la caja TRP (Nadezhdin et al., 2023; Kwon et al., 2023; Deng et al., 2019). Por otra parte, en el extremo carboxilo terminal se localiza la caja TRP, una región conservada en varios miembros de esta familia de canales iónicos, que regula los procesos de compuerta del canal al interactuar con el enlazador S4-S5 (Teng et al., 2015; Teng et al., 2016); un dominio de unión a calmodulina, que participa en la potenciación, y posterior inactivación del canal TRPV4 en función de la concentración de Ca2+ intracelular o [Ca2+]i (Strotmann et al., 2003; Strotmann et al., 2010; Watanabe et al., 2003). Además, este dominio funciona como sitio de unión para el inositol trisfosfato (IP3), que sensibiliza la respuesta del canal a los estímulos mecánicos e hipotónicos y modula su respuesta a los ácidos epoxieicosatrienoicos o EET (García-Elias et al., 2008). Finalmente, se ha descrito un motivo similar al dominio de la Proteína de Densidad Postsináptica (PSD95) o PDZ, que consiste en cuatro aminoácidos (DAPL) y participa en la interacción del canal con distintas proteínas, influyendo en su tráfico hacia la membrana (Garcia-Elias et al., 2008; Nilius & Voets, 2013). Después de su síntesis, el canal TRPV4 sufre varias modificaciones postraduccionales que incluyen, la fosforilación de los residuos S162, T175, S184 y S189 por las proteínas cinasa A (PKA) y cinasa C (PKC), y del residuo Y253, del extremo amino terminal, por la cinasa Lyn relacionada con Lck/Ye, lo que sensibiliza al canal ante variaciones en el volumen celular favoreciendo su respuesta a cambios en las condiciones hipotónicas (Fan et al., 2009; Xu et al., 2003). También se ha descrito la ubiquitinación de los residuos 411–437 por la proteína-4 que interactúa con la Atrofina-1 (ubiquitina ligasa) o AIP4 que promueve la endocitosis del canal, reduciendo su expresión en la membrana celular (Wegierski et al., 2006). 8 En el asa extracelular adyacente al poro, el residuo N651 es glicosilado y esto influye en el tráfico del canal hacia la membrana (Xu et al., 2006). Por su parte, en el extremo carboxilo terminal la fosforilación del residuo S824, por la cinasa PKA (Peng et al., 2010) o por la cinasa-1 regulada por suero/glucocorticoides o SGK1 (Shin et al., 2015), es importante para la sensibilización del canal a condiciones hipotónicas (Peng et al., 2010). Además, este residuo participa en la respuesta ante el ácido araquidónico y sus derivados (Cao et al., 2018); en su interacción con otras proteínas, como la calmodulina, la molécula de interacción estromal-1 (STIM1) y la actina que modulan la actividad de TRPV4 (Shin et al., 2015); así como en su transporte a la zona del núcleo y posterior exportación/localización hacia la membrana (Méndez-Gómez et al., 2022). Las características del canal TRPV4, así como su respuesta a estímulos de diferente naturaleza lo convierten en una proteína importante que se expresa en los sistemas nervioso, cardiovascular, respiratorio, urinario, esquelético, inmune y digestivo, así como en el hígado, el páncreas y la piel (Plant & Strotmann, 2007), en donde contribuye a la osmorregulación y el control en el flujo de Ca2+, favoreciendo el mantenimiento de la homeostasis (White et al., 2016). Además, el canal TRPV4 participa en la hiperactivación del espermatozoide humano, permitiendo su movilidad (Mundt et al., 2018). En este trabajo, nos hemos enfocado en estudiar los efectos de algunos broncodilatadores sobre la función del canal TRPV4, por ende, a continuación se describe el papel de este canal sobre la función respiratoria. D) El canal TRPV4 en el sistema respiratorio El canal TRPV4 se expresa en el sistema respiratorio, principalmente en el músculo liso, los fibroblastos, las glándulas submucosas, las células endoteliales vasculares y los epitelios traqueal, bronquial y alveolar, así como en los macrófagos (Jia & Lee, 2007; Palaniyandi et al., 2020). 9 Se sabe que distintos estímulos activan al canal TRPV4 en el sistema respiratorio y esta activación se traduce en la generación de señales de Ca2+ que promueven diferentes respuestas dependiendo del tipo celular y la región. Algunas de las principales funciones en las que participa el canal TRPV4 son: el mantenimiento de la barrera alvéolo-capilar, la remodelación de las vías respiratorias, la regulación de la presión osmótica celular, la contracción muscular. Este canal además genera fuerzas mecánicas dinámicas importantes durante el desarrollo pulmonar en el estado embrionario (Jesudason, 2007; Morgan et al., 2018; Yu et al., 2019). Se ha demostrado que la sobreactivación del canal promueve síntomas como la tos, la secreción de moco, la inflamación y la contracción de las vías aéreas (Fig. 2) (Andrade et al., 2007; Balakrishna et al., 2014; Bonvini et al., 2016; Bonvini et al; 2020). Lo anterior contribuye, en grados variables, a distintas patologías pulmonares como la lesión pulmonar aguda (ALI), el síndrome de dificultad respiratoria aguda (ARDS), la enfermedad pulmonar obstructiva crónica (COPD), el edema y el asma (Li et al., 2011; Balakrishna et al., 2014; Scheraga et al., 2017; Bihari et al., 2017; Palaniyandi et al., 2020). Fig. 2. La sobreactivación del canal TRPV4 produce tos, secreción de moco y contracción de las vías aéreas, síntomas en patologías como la ALI, el ARDS, la COPD, el edema y el asma. 10 Consecuentemente, se ha descrito que el uso de antagonistas generales o específicos del canal TRPV4 como el GSK2193874, el GSK222069 y el GSK2337429A, desarrollados por GlaxoSmithKline, ayudan en la mejora de algunos síntomas respiratorios agudos (Huh et al., 2012; Balakrishna et al., 2014; Bihari et al., 2017). E) El salbutamol y otros agonistas del receptor b2-AR Los broncodilatadores son medicamentos que permiten la entrada de aire al sistema respiratorio al relajar el músculo liso, liberando la obstrucción en las vías aéreas. Los agonistas de los receptores β- adrenérgicos (β-AR), cuyas características y acciones se describen a detalle más adelante, los agentes anticolinérgicos y las metilxantinas son los tres tipos principales de broncodilatadores empleados en el tratamiento de ciertas enfermedades respiratorias como el asma o la COPD (Creer & Levstek, 1998; Couëtil et al., 2016). El salbutamol es un agonista del b2-AR con actividad broncodilatadora de acción corta, empleado para tratar episodios agudos de broncoespasmo. Debido a la presencia de un carbono quiral en su estructura, se presenta en forma de dos isómeros R- y S- (Fig. 3) (Brittain et al., 1973). Fig. 3. Isómeros de salbutamol que componen la mezcla racémica empleada en el tratamiento de los síntomas en patologías como la COPD, el edema y el asma. R-Salbutamol o levalbuterol S-Salbutamol 11 El R-salbutamol, también conocido como levalbuterol, es el isoméro efectivo con actividad farmacológica y exhibe una afinidad por el b2-AR 150 veces mayor (EC50 = 0.3 - 0.6 µM; Skeberdis et al.; 1997; Scott et al., 1999; Matera et al., 2011) que el isómero S (Cullum et al., 1969; Ameredes & Calhoun, 2009). Por su parte, el S-salbutamol se ha descrito como un compuesto inerte (Penn et al., 1996), y en algunos reportes con efectos adversos en el tratamiento de los síntomas respiratorios (Perrin-Fayolle et al., 1996). Además del salbutamol, existen otros agonistas del b2-AR, que se agrupan comúnmente en tres clases, de acuerdo con el tiempo de acción dentro del organismo (Billington et al., 2017) y que son los siguientes: • SABAs (Short Acting β2 Agonists): agonistas de acción corta (duración de 3 a 6 h). Se utilizan como tratamiento inicial contra síntomas agudos de algunas enfermedades respiratorias como el asma; dentro de este grupo se encuentran el salbutamol, la terbutalina, el metaproterenol y el isoproterenol. • LABAs (Long Acting β2 Agonists): agonistas de acción prolongada (con una duración de 12 h). Se utilizan como parte del tratamiento de primera línea contra la enfermedad pulmonar obstructiva crónica (COPD); ejemplos de estos compuestos son el salmeterol, el formoterol, el olodaterol y el clenbuterol. • Ultra-LABAs (Ultra Long Acting β2 Agonist): agonistas de acción muy prolongada (con una duración de 24 h). Son indicados en el tratamiento broncodilatador en pacientes con COPD, un ejemplo de estos compuestos, es el indacaterol. Los betabloqueantes son un grupo de antagonistas de los receptores β-AR, que compiten con las catecolaminas norepinefrina y epinefrina por el sitio de unión del receptor, impidiendo las respuestas dependientes de los b-adrenoceptores (Haeusler, 1990). Se clasifican como selectivos, cuando se unen 12 únicamente al receptor β1, o no selectivos cuando tienen afinidad por los receptores β1 y β2, como el carvedilol, el propranolol o el nadolol (Ying et al., 2013). Son comúnmente utilizados en el tratamiento de padecimientos del sistema vascular como la angina de pecho (Wahl, 2005; Mason & Malhotra, 2019). F) El receptor b2-adrenérgico Los b-adrenoceptores son una familia de receptores acoplados a proteínas G (GPCR), conformada por los subtipos: b1, b2 y b3, que se expresan en el sistema cardíaco, respiratorio y tejido adiposo, principalmente (Johnson, 1998). Son proteínas cuya actividad está asociada a desencadenar los mecanismos que llevan al aumento de la frecuencia cardíaca, la relajación del músculo liso de las vías aéreas y de los vasos sanguíneos, así como los procesos de glucogenólisis y lipólisis en respuesta a estímulos simpáticos del sistema nervioso periférico autónomo en situaciones de peligro (Bylund, 2007). Aunque en este trabajo no se estudiaron las relaciones entre la estructura y la función del b2-AR, mismas que se han descrito ampliamente, es importante mencionar que este receptor está conformado por siete dominios transmembranales. Su extremo amino terminal es extracelular y el extremo carboxilo terminal es intracelular. Además, cuenta con tres asas extracelulares y tres asas intracelulares (Fig. 4); la tercera asa intracelular forma el sitio de interacción con la proteína G (McGraw & Liggett, 2005). El sitio de unión para los agonistas se ubica dentro de la región transmembranal y los residuos S203, S204, S207, D113, N312 juegan un papel crítico en el reconocimiento de los ligandos (Ambrosio et al., 2000; Del Carmine et al., 2002). Así mismo, el segmento S5 (Fig. 4) es fundamental para la función del receptor ya que posee una gran flexibilidad que le permite adoptar cambios conformacionales favorables para la unión de los ligandos (Katritch et al., 2009). 13 Es particularmente importante mencionar que el b2-AR se expresa en diferentes tejidos y tipos celulares del sistema respiratorio como el músculo liso, las células alveolares del tipo II, las glándulas mucosas, las células epiteliales, el endotelio vascular y los mastocitos así como en el músculo cardíaco y el músculo liso uterino (Johnson, 1998; McGraw & Liggett, 2005). G) Vía de señalización del b2-AR en el músculo liso Cuando un agonista activa al b2-AR, la subunidad Gα se disocia de las subunidades Gβγ de la proteína G y, junto con el trifosfato de guanosina (GTP), forman un complejo que se recluta en balsas lipídicas de la membrana celular y activa a la adenilato ciclasa, enzima encargada de catalizar la conversión de adenosín trifosfato (ATP) a adenosín monofosfato cíclico (cAMP) (Lorton & Bellinger, 2015). Fig. 4. Representación esquemática de la estructura del b2-AR (PDB 3NY8). La proteína está conformada por siete cruces transmembranales, un extremo amino-terminal (NH2) extracelular y uno carboxilo (COOH) terminal intracelular. El sitio de unión a los ligandos se encuentra entre los cruces transmembranales 3, 5 (morado) y 7 donde participan los residuos D113, S203, S204, S207 y N312 (azul) para establecer interacciones con diferentes moléculas. 14 De manera canónica, se ha establecido que el cAMP se une a la subunidad reguladora de la PKA dependiente de cAMP, facilitando la disociación de la subunidad catalítica, lo cual favorece la transferencia de fosfatos desde el ATP a residuos de serina o treonina de proteínas diana. En el caso del músculo liso, promueve la apertura de los canales de K+ activados por Ca2+ (BKCa), lo que implica una hiperpolarización de la célula y, como consecuencia de esto, ocurre una reducción en la entrada de Ca2+ extracelular a través de los canales de Ca2+ dependientes de voltaje. Todo esto se suma a la recaptura de Ca2+ por el sarcolema donde el fosfolambán, un inhibidor proteico que, una vez fosforilado, se une y regula positivamente a la bomba de Ca2+, termina por relajar a las vías aéreas (MacLennan & Kranias, 2003; Feher, 2017). Cabe mencionar que el cAMP también funciona como segundo mensajero y puede unirse a otras proteínas entre las cuales destacan los canales iónicos; ejemplo de esto es su interacción con los canales sensibles a nucleótidos cíclicos (CNG) o con los canales ether-à-go-go (EAG) (Brüggemann et al., 1993; Liao et al., 2012). H) Interacción entre el b2-AR y el canal TRPV4 Desde la década pasada se tiene conocimiento sobre la modulación de los canales TRP por la acción de las vías de señales asociadas a las GPCR. Una de las interacciones más estudiada es la del receptor de bradicinina (BR) que, al activarse, estimula diferentes canales TRP (TRPA1, TRPV1, TRPV4 y TRPM8) en neuronas del ganglio de la raíz dorsal (DRG), donde se expresan estos canales iónicos, y se producen potenciales de acción que resultan en la generación de dolor e inflamación (Levine et al., 2007; Bessac et al., 2008; Liu et al., 2010). 15 Actualmente, existe escasa información sobre posibles interacciones entre las vías de señalización de los b2-AR y los canales TRP. Sin embargo, se ha descrito que en túbulos contorneados y conectores distales tardíos del riñón, la activación del β1-AR y la consecuente fosforilación de PKA, estimulan el transporte transepitelial de Ca2+ mediado por el canal TRPV5, un miembro de la misma subfamilia de canales a la que pertenece el TRPV4 (van der Hagen et al., 2014). En células epiteliales conjuntivas, la 3-yodotironamina que es un derivado endógeno de la hormona tiroidea, modula la actividad del β2-AR estimulada por isoproterenol que conlleva a una regulación de Ca2+ intracelular; mediada por TRPM8 y/o TRPV1 (Dinter, et al., 2015). Aunque actualmente no existen reportes de la interacción directa entre el canal TRPV4 y el b2-AR, se ha descrito un mecanismo que implica la participación del canal y de un receptor acoplado a la proteína Gq (GqPCR) en la relajación del músculo liso vascular. La activación de GqPCR promueve un aumento de Ca2+ e IP3, y este último activa y aumenta los tiempos de apertura de los canales TRPV4 en las células endoteliales. A su vez, la activación del canal TRPV4 promueve la vasodilatación a través de los canales de potasio conductancias baja (SK) e intermedia (IK) sensibles al Ca2+ (Hong et al., 2018). De igual forma, en macrófagos alveolares, se ha investigado el efecto del cAMP sobre el canal TRPV4, donde se ha observado que esta molécula suprime la entrada de Ca2+ a través del canal, previniendo una reacción inflamatoria exacerbada y la consecuente lesión pulmonar aguda o ALI (Rayees et al., 2019). 16 I) Efecto de los agonistas del b2-AR en canales iónicos. Es importante considerar que los agonistas de los receptores b2-AR también podrían interactuar directamente con otras proteínas. En este sentido, se ha demostrado que el clenbuterol y el propanolol (ligandos del b2-AR) son capaces de generar una inhibición rápida y reversible de las corrientes de los canales de sodio dependientes de voltaje de tipo hSkM1 que se encuentran presentes en fibras de músculo esquelético de rata y de humano, pero también en modelos de expresión heteróloga como las células tsA201 (células humanas modificadas de riñón embrionarias) transfectadas con este canal de sodio (Desaphy et al., 2003). El efecto de los ligandos, independientemente de su naturaleza agonista (clenbuterol) o antagonista (propanolol), ocurre de forma directa sobre el canal sin la modulación del b2-AR o subproductos de la vía de señalización como las PKA o PKC, ni por la participación de cAMP, que podrían tener un papel importante en la inhibición de los canales de sodio de las fibras musculares (Desaphy et al., 2003). En este sentido, en el mismo estudio, también se reportó que el salbutamol (agonista del b2- AR) y el nadolol (antagonista del b2-AR) no tienen un efecto inhibitorio sobre los canales de sodio. Lo anterior llevó a los autores a concluir que esto se debía a las características estructurales particulares de estos compuestos y se propuso que la presencia de los grupos hidroxilo en el anillo bencénico disminuye la hidrofobicidad del compuesto, interfiriendo con la correcta interacción entre las moléculas y el canal (Desaphy et al., 2003). 17 J) Mecanismos de inhibición de un canal iónico Los compuestos inhibidores de los canales iónicos utilizan diferentes mecanismos para impedir el flujo de los iones a través del poro. Estos se pueden clasificar en dos grupos principales: Los bloqueadores, que se unen en alguna región dentro del poro o compiten con los iones permeantes, obstruyendo físicamente su transporte a través de los canales iónicos (Hille, 2001). Por lo general, aunque no en todos los casos dependiendo de la afinidad de unión, el bloqueo del poro se considera un proceso reversible, ya que eliminando al compuesto bloqueador es posible restaurar la función normal del canal (Petkov, 2009). Por su parte, la inhibición alostérica implica la unión específica del compuesto en alguna región intracelular, extracelular o transmembranal, que estabiliza a la proteína en una conformación cerrada no conductora (Hille, 2001; Petkov, 2009). Es importante mencionar que el sitio de unión al ligando es un factor determinante en el tipo de modificaciones conformacionales que adoptará la proteína, el cambio de las barreras energéticas y como consecuencia, del efecto que se produzca (Hogg et al., 2005). A partir de todo lo anterior, se deriva la importancia de identificar y estudiar activadores, inhibidores y reguladores de la actividad del canal TRPV4. También es importante estudiar las relaciones que existen entre su estructura y su función, ya que es un canal fisiológicamente importante cuya activación alterada como consecuencia de mutaciones en sitios clave, resulta en enfermedades severas del sistema nervioso y del sistema musculoesquelético en humanos (Nilius & Owsianik, 2010; Nilius & Voets, 2013; Loukin et al., 2015; Taga et al., 2022). Así, este proyecto se centra en el estudio de la regulación del canal TRPV4 por ligandos (agonistas y antagonistas) del b2-AR que pueden modular la actividad a la baja del canal (Fig. 5). Los resultados que se obtuvieron en este estudio podrían contribuir a diseñar terapias para ciertas patologías asociadas a la función incorrecta o 18 hiperactivación del canal TRPV4 utilizando inhibidores como se ha propuesto recientemente para los pacientes afectados por Covid-19 (Kuebler et al., 2020). Fig. 5. Los ligandos del b2-AR regulan a la baja la actividad del canal TRPV4. Este resultado podría favorecer la mejoría de pacientes con ciertas patologías respiratorias e incluso con otras afecciones. 19 Hipótesis El salbutamol, así como otros broncodilatadores, modulan la actividad del canal TRPV4 interactuando de manera directa con la proteína. Objetivo general Determinar el posible efecto inhibitorio del salbutamol y de moléculas estructuralmente similares a éste, sobre la actividad del canal TRPV4 y las modificaciones que generan en las propiedades biofísicas del canal. Objetivos particulares • Caracterizar los efectos del salbutamol y moléculas similares a éste, sobre las propiedades biofísicas del canal TRPV4 a nivel de las corrientes macroscópicas. • Determinar el mecanismo por medio del cual el salbutamol y moléculas similares modulan al canal TRPV4. o Descartar o confirmar si la modulación del canal TRPV4 por salbutamol es por la activación de vías de señalización intracelulares asociadas al b2-AR. • Establecer modelos alostéricos (por medio del registro de canales únicos) para los efectos del salbutamol sobre el canal TRPV4. 20 Métodos a) Cultivo de células HEK293 Las células embrionarias de riñón humano, HEK-293 (ATCC CRL-1573), se cultivaron en medio DMEM (Dulbecco’s Modified Eagle’s Medium, Gibco) suplementado con 10% de suero fetal bovino (HyClone), 1% de penicilina-estreptomicina (Gibco) y se mantuvieron a una temperatura de 37 ºC y a 5 % de CO2, hasta alcanzar una densidad del 85%. Las células se resuspendieron con 1 mL de tripsina-EDTA (Gibco), se centrifugaron a 12,000 rpm durante 3:30 m y se sembraron en vidrios pretratados con poli-D-lisina (Sigma-Aldrich), para usarlos en los registros electrofisiológicos descritos más abajo. Después de 24 h de la siembra, las células se transfectaron transitoriamente con cDNA de TRPV4 humano (silvestre o alguno de los canales mutantes) y pEGFP-N3 usando el reactivo JetPei (Polyplus Transfection; siguiendo las instrucciones del fabricante), para identificar las células fluorescentes y poder realizar los registros electrofisiológicos. b) Mutagénesis dirigida en el canal TRPV4 Se propusieron varios sitios de unión directa del salbutamol sobre el canal TRPV4, seleccionándolos por la similitud que mantienen con el bolsillo de unión presente en el b2-AR, su ubicación extracelular en TRPV4 y los modelajes computacionales realizados por la Dra. Ariela Vergara (Universidad de Talca, Chile), cuya metodología se describe abajo. Los residuos que se mutaron fueron los siguientes: S557, S563, E572, D613, Y621, S630, S634, S667, D682, S687, S688 y T689; sustituyendo el aminoácido nativo por una alanina con la técnica de mutagénesis dirigida por la reacción en cadena de la polimerasa (PCR) en dos pasos, misma que se describe a continuación. 21 Se identificó la secuencia del canal TRPV4 de humano en el plásmido y se diseñaron cuatro oligonucleótidos con el programa VectorNTI (Thermo Fisher) para la sustitución del aminoácido seleccionado en cada canal mutante: dos oligonucleótidos (directo y complementario) que contenían la sustitución, así como un sitio nuevo de restricción y dos oligonucleótidos silvestres que delimitaron la secuencia en la que se encontraba el sitio para la mutación. En la primera PCR, se insertó la mutación deseada, así como un nuevo sitio de restricción en la secuencia silvestre del canal TRPV4. En la segunda PCR se tomó el producto de la primera PCR y se produjeron varias copias del DNA mutado. Con ayuda de enzimas digestivas, se cortaron los DNAs obtenidos en los dos sitios de restricción que delimitaban la secuencia con el sitio de la mutación, se separaron los fragmentos de DNA con la mutación al igual que el vector de expresión en un gel de agarosa al 0.8% y, por medio de una reacción de ligación, se reintrodujeron en el sitio adecuado del canal contenido en un vector (pcDNA3) con un casette de resistencia a ampicilina para expresión en células de mamífero. El vector se transformó en bacterias competentes (E. coli) usando choque térmico. Estas bacterias se sembraron en cajas de agar suplementadas con antibiótico a 37º C durante una noche. Después de una noche se seleccionaron las colonias que lograron crecer en presencia de amplicilina y se cultivaron en medio de cultivo Luria-Bertani o LB (10 g de triptona, 5 g de extracto de levadura, 5 g de NaCl, 1 mL de NaOH [1N] y 15 g de agar). Se extrajo el DNA, el cual se secuenció en la Unidad de Biología Molecular del Instituto de Fisiología Celular (IFC) para cada canal mutante generado. 22 c) Modelaje computacional Brevemente, se describe la metodología utilizada por la Dra. Ariela Vergara (Universidad de Talca, Chile) para predecir el acoplamiento molecular entre el salbutamol y el canal TRPV4 (Benítez- Angeles et al., 2022). A partir del canal TRPV4 de X. tropicalis (»85% de identidad), la única estructura resuelta para el canal TRPV4 hasta ese momento (Deng et al., 2018), se construyeron un total de 2000 modelos de TRPV4 humano con el software Modeller 9.25 (Eswar et al., 2007) y se seleccionó el que obtuvo el valor más bajo de energía molpdf (molecular PDF) y la puntuación más alta de Procheck (Laskowski et al., 1993). En el programa AutoDockTools 1.5.7 (Morris et al., 2009) se acoplaron las moléculas de TRPV4 y el isómero R-salbutamol asignando cargas parciales a Gasteiger. El espacio de búsqueda inicial se delimitó a través de una cuadrícula generada con AutoGrid4.2.6 (Morris et al., 1998) que cubría toda la superficie del canal y se modelaron las conformaciones de unión TRPV4-salbutamol utilizando el algoritmo genético lamarckiano de AutoDock4.2.6 (Morris et al., 1998, 2009). Finalmente, se realizó un refinamiento del acoplamiento reduciendo gradualmente el área de búsqueda dando un total de 1000 conformaciones que se agruparon y se analizaron con el programa VMD 1.9.3 (Humphrey et al., 1996) d) Experimentos de electrofisiología El equipo de registro electrofisiológico consistió en: un microscopio invertido con epifluorescencia (Nikon), un amplificador EPC10 (HEKA Elektronik), un micromanipulador motorizado (Sutter Instruments) y un sistema de recambio rápido de soluciones RSC-200 (Bio-Logic Science Instruments). Los datos se capturaron con el programa PatchMaster (HEKA Elektronik) y tanto se analizaron como se graficaron en el programa Igor Pro (Wavemetrics Inc.). 23 Se utilizó la técnica de patch-clamp o técnica de fijación de voltaje en micro-áreas de membrana que consiste en establecer un sello de alta resistencia (GW) entre la membrana celular y la punta de una pipeta de vidrio (3-5 MW). Para los experimentos aquí reportados, se utilizaron dos configuraciones de la técnica de patch-clamp: la configuración inside-out, donde la pipeta se separó para obtener una porción de membrana escindida con la parte intracelular de los canales TRPV4 expuesta hacia las soluciones del baño y la configuración outside-out, donde la membrana se rompió con una ligera succión pero, cuando se escindió el parche, se formó el sello nuevamente ahora con la parte extracelular de los canales TRPV4 expuesta a las soluciones del baño, donde es posible cambiar los compuestos de intéres de acuerdo con las necesidades del experimento. Las corrientes se generaron usando el amplificador controlado por una computadora, por medio de la aplicación de pulsos cuadrados de -120 mV a +120 mV, con una duración de 10 ms, desde un potencial de mantenimiento de 0 mV. d.1) Obtención de curvas de respuesta ante diferentes dosis de compuestos que interaccionan con el canal TRPV4 Para obtener la respuesta del canal ante cambios en la dosis de los diferentes compuestos que se probaron en este estudio, se realizaron los siguientes experimentos: para el caso de GSK se obtuvieron las curvas de respuesta al agonista para el canal silvestre y para tres canales mutantes (S667A, S687A y S667A-S687A). Así, se registraron las corrientes de fuga o iniciales en ausencia de cualquier compuesto, después las corrientes a las concentraciones de 25, 50, 100, 250, 500 y 800 nM de GSK durante 1.30 min y, finalmente, las corrientes a la concentración saturante de GSK (1 μM) durante 1.30 min. Los datos se normalizaron al valor de las corrientes obtenidas con 1 μM de GSK. Estos experimentos donde se comparan las respuestas ante el agonista del canal silvestre y los canales mutantes son indispensables para definir que cualquier cambio por la aplicación de los broncodilatadores sobre la modulación de la actividad del canal TRPV4 se deben a un efecto directo 24 por la interacción entre la mólecula y el canal y no por cambios globales en las propiedades de compuerta de las proteínas mutadas (por ejemplo, a su agonista GSK). Todos los ligandos del b2-AR aquí estudiados, tuvieron efectos inhibitorios sobre la actividad del canal TRPV4. Para obtener las curvas de inhibición del canal ante salbutamol, así como del antagonista del b2-AR ICI 118,551 (referido de ahora en adelante como ICI), se realizaron experimentos electrofisiológicos en estado cerrado (en ausencia de GSK) y en la configuración de outside-out. Para generar dichas curvas de inhibición, primero se obtuvieron las corrientes de fuga y las corrientes en presencia del agonista de la concentración saturante de GSK 300 nM después de 1.30 min. Posteriormente, se lavó el GSK para cerrar a los canales y se aplicó el salbutamol a las concentraciones de 10, 25, 50, 100, 250, 500 μM y 1 mM por 5 min, mientras que el efecto de ICI (antagonista) se evaluó a las concentraciones de 0.001, 0.025, 0.1, 5, 10, 50, 100, 500 𝜇M por 5 min. Posterior al tiempo de aplicación de ambos compuestos, se midió nuevamente la respuesta del canal al agonista GSK 300 nM por 1.30 min. Las corrientes remanentes de las diferentes concentraciones de salbutamol y de ICI respectivamente, se promediaron y se normalizaron al valor de las corrientes obtenidas con GSK y las curvas dosis a respuesta de los tres compuestos (GSK, salbutamol e ICI) se ajustaron a la ecuación de Hill: ! !max =! " "# ["] $% " $ Los parámetros que arroja el ajuste de las curvas a la ecuación de Hill son: n, el coeficiente o número de Hill (número de moléculas de GSK, salbutamol e ICI que interaccionan con el canal); [X], la concentración de GSK, salbutamol o ICI y Kd, la constante de disociación aparente o EC50 para GSK e IC50 para salbutamol e ICI. 25 d.2. Registro del efecto de los agonistas y antagonista del β2-AR sobre las corrientes macroscópicas del canal TRPV4 Primero, se obtuvieron las corrientes de fuga (o en ausencia del agonista) y las corrientes en presencia del agonista GSK 300 nM después de 1.30 min. Después, se lavó el GSK para cerrar los canales y se aplicó el vehículo (configuración inside-out o in-out y outside-out o out-out, estado cerrado y abierto), el salbutamol (configuración in-out y out-out, estado cerrado y abierto), la terbutalina, el clenbuterol, el metaproterenol, el isoproterenol, el levalbuterol o el ICI (configuración out-out, estado cerrado) a una concentración de 500 μM por 5 min. Posteriormente, los parches de membrana se expusieron de nuevo al agonista GSK 300 nM por 1.30 min y se midieron las corrientes remanentes. Como controles experimentales, evaluamos los efectos de los compuestos en un canal relacionado al TRPV4, el TRPV1. Para dichos experimentos, se registraron las corrientes de fuga y las corrientes inducidas por capsaicina (200 nM), uno de los agonistas de alta afinidad de este canal. A continuación, se lavó la capsaicina hasta cerrar los canales y se aplicó salbutamol, terbutalina o isoproterenol a 500 µM durante 5 min. Finalmente, se volvieron a medir las corrientes en presencia de capsaicina (200 nM). Los demás experimentos controles consistieron en comparar el efecto del salbutamol en células sin tratamiento y células pretratadas (24 h previas) con el antagonista del b2-AR, ICI; en estado cerrado y en configuración outside-out. Para cada agonista y antagonista del b2-AR probados, se normalizaron las corrientes remanentes al valor de las corrientes iniciales obtenidas con GSK (o capsaicina) en cada experimento, se promediaron y se presentan como 1-(I/Imax), a partir de lo cual, se puede calcular el porcentaje de inhibición, como se menciona a lo largo del texto. 26 d.3 Registro de las corrientes macroscópicas de las mutantes de TRPV4 en presencia de salbutamol Se registró la actividad del canal TRPV4 en su forma silvestre (WT) y de las diferentes mutantes obtenidas (S557A, S563A, E572A, S630A, S634A, S667A, D682A, S687A, S688A y T689A) ante una concentración de 500 µM de salbutamol (concentración saturante) durante 5 min, como se describe arriba. Para cada mutante se obtuvieron sellos pareados usando células trasnfectadas con canales WT, es decir, se alternaron los sellos para asegurar que el salbutamol funcionaba y que una posible falta de efecto sobre los canales mutantes no se debía a la descomposición del compuesto. Para cada canal mutante, las corrientes de GSK obtenidas después del tratamiento con salbutamol, se normalizaron al valor de las corrientes iniciales obtenidas con GSK y se promediaron. Los datos del efecto del salbutamol en estado cerrado y en configuración de outside-out se compararon entre los canales mutantes y el canal WT. d.4. Constante de tiempo del efecto de inhibición del salbutamol en el canal TRPV4 Para la obtención de la constante de tiempo (t) de inhibición por salbutamol en estado cerrado, primero se aplicó GSK, se lavó con solución de registro, luego se aplicó el salbutamol y, finalmente, se volvió a aplicar GSK, en la configuración de outside-out. Los pulsos de voltaje fueron de -60 mV a diferentes tiempos de aplicación de salbutamol a una concentración de 500 µM (30s, 1min, 1min 30s, 2min, 2min 30s, 3min, 3min 30s, 4min, 4min 30 s, 5min, 5min 30s y 6min). La gráfica se reconstruyó usando el promedio normalizado de los parches para cada punto en el tiempo y se ajustaron los datos a una exponencial simple. 27 d.5. Constante de tiempo de la recuperación de las corrientes activadas por GSK después del tratamiento con salbutamol Para la obtención de la constante de tiempo (t) de la recuperación de las corrientes activadas por GSK después de la exposición a salbutamol, se aplicaron pulsos de -120 mV a diferentes tiempos (1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min) en presencia de GSK (300nM) después de que los parches fueron expuestos a salbutamol 500 µM por 5 min. Se reconstruyó la gráfica con el promedio normalizado de los parches para cada punto en el tiempo y se ajustaron los datos a una doble exponencial. d.6. Registro de canales unitarios de TRPV4 en presencia de salbutamol Para los registros de canales unitarios se modificaron algunos parámetros con la finalidad de aumentar la probabilidad de aislar un solo canal por sello, por ejemplo se aumentó la resistencia de las pipetas (8 a 10 MW) y se utilizó una menor concentración de DNA del canal TRPV4 de humano (5 a 10 ng) en la transfección. Los registros de canal unitario se realizaron usando la configuración outside-out, donde a cada sello se le aplicó un pulso continuo a +60 mV durante 5.30 min en promedio. Los primeros 20 s correspondieron a la medición de las corrientes de fuga, los 90 s posteriores correspondieorn al efecto del agonista GSK y, finalmente, el tiempo restante a la aplicación conjunta de GSK + salbutamol (estado abierto) para determinar sus efectos en el comportamiento de las corrientes unitarias. Cabe señalar que las corrientes fueron filtradas a 2 kHz con una frecuencia de muestreo de 50 kHz. En el análisis de los registros obtenidos, se restó el valor de las corrientes de fuga y posteriormente se utilizaron de 5 a 10 trazos por sello para construir histogramas que fueron ajustados a una función Gaussiana, cuyo pico corresponde a la amplitud promedio de la corriente del canal unitario analizado, como se había hecho anteriormente en el laboratorio (Islas, 2015). 28 La probabilidad de apertura de cada sello para las dos condiciones (GSK y GSK + salbutamol) se obtuvo utilizando un programa personalizado en el software IgorPro (Wavemetrics Inc.), diseñado en colaboración con el laboratorio del Dr. León Islas de la Facultad de Medicina de la UNAM. Así, cada trazo de los registros unitarios se idealizó empleando un umbral equivalente a la mitad de la amplitud de la corriente (Colquhoun & Sigworth, 1985), para detectar los eventos de valor los cuales se promediaron y graficaron; como valor máximo 1 correspondiente a todo el tiempo abierto y valor mínimo 0 equivalente a ninguna apertura. Los tiempos de permanencia y los histogramas de amplitud en los estados cerrado o abierto se recopilaron en histogramas de tiempo logarítmicos de acuerdo con la transformación Sine-Sigworth (Sigworth y Sine, 1987). e) Reactivos y disoluciones Se emplearon soluciones isométricas (mismas concentraciones en la pipeta y el baño) compuestas por (en mM): NaCl 130, el amortiguador HEPES 3 y el agente quelante de iones divalentes EDTA 1, a un pH 7.2 (ajustado con NaOH) y libre de Ca2+; para evitar el bloqueo del canal TRPV4 por este ion (Nilius et al., 2004); con excepción de uno de los controles donde en la solución de baño omitimos al EDTA, y agregamos 2 mM de CaCl2. La solución stock de GSK1016790A (GSK, Sigma-Aldrich) se preparó a una concentración de 15.25 mM en dimetilsulfóxido (DMSO), almacenada y congelada a -20º C. Para los experimentos de electrofisiología, una alícuota del stock se descongeló y preparada a la concentración necesaria para cada experimento. Es importante mencionar, que se realizaron experimentos control exponiendo a los canales TRPV4 a la concentración más alta de DMSO utilizada, luego activándolos con GSK y finalmente normalizando la corriente al final de la exposición a DMSO con la corriente de GSK, para demostrar el mínimo efecto de este vehículo sobre el canal. 29 El hemisulfato de salbutamol, de terbutalina y de metaproterenol, así como el hidrocloruro de isoproterenol, clenbuterol, levalbuterol y de ICI-118,551 (Sigma-Aldrich) se prepararon para cada experimento a una concentración de 100 mM en agua desionizada y se disueltos posteriormente a la concentración necesaria para cada experimento. f) Análisis estadísticos Los datos se muestran como el promedio ± el error estándar del promedio y se analizaron con la prueba estadística T de Student no pareada y de dos colas o con un ANOVA de una vía y la prueba post-hoc de Tukey, según fuese el caso p<0.05 (*) se consideró como una diferencia estadísticamente significativa. 30 Resultados 1) Efecto del salbutamol sobre el canal TRPV4 en configuración outside-out Primero, se evaluó el efecto del vehículo en el que se disuelve el salbutamol sobre la actividad del canal TRPV4. Los experimentos mostraron la inhibición de las corrientes activadas por GSK en un 21.2 ± 2.7% para el estado cerrado y del 16.2 ± 2.1% para el estado abierto (Fig 6. cuadrados y círculos vacíos, respectivamente). Este efecto probablemente se debe a un proceso de decaimiento o “rundown” ya descrito para la actividad de este canal en parches escindidos por su dependencia a la presencia de PIP2 (Garcia-Elias et al., 2013). Sin embargo, al aplicar salbutamol (500 µM) por 5 min se obtuvo una inhibición de las corrientes activadas por GSK del 54.3 ± 3.9% cuando el broncodilatador se aplicó en estado cerrado (Fig. 6, cuadrados morados) y una inhibición del 32.5 ± 4.8% cuando fue aplicado en el estado abierto (Fig. 6, círculos rosas), lo que sugiere una dependencia al estado del canal para la inhibición por salbutamol. Fig. 6. Efecto del salbutamol en el canal TRPV4 en los estados abierto y cerrado en la configuración de outside-out del patch-clamp. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol en estado cerrado (morado) y abierto (rosa). Se representan los valores promedio para: vehículo en estado cerrado=21.2 ± 2.7% (n=13); salbutamol en estado cerrado=54.3 ± 3.9 % (n=14); salbutamol + GSK (estado abierto)=32.5 ± 4.8 % (n=15) y vehículo en estado abierto=16.2 ± 2.1% (n=15). ANOVA de una vía, vhc cerrado vs salb cerrado p<0.0001; salb cerrado vs salb abierto p=0.0011; vhc abierto vs salb abierto p=0.023 (*). 31 2) Efecto del salbutamol sobre el canal TRPV4 en configuración inside-out Para determinar si el salbutamol podía afectar diferencialmente la actividad del canal TRPV4 cuando era aplicado por el lado intracelular o extracelular del canal, se realizaron experimentos en el estado cerrado y en la configuración inside-out. Los resultados de estos experimentos indican que la inhibición de las corrientes del TRPV4 en presencia del vehículo fue de 12.8 ± 3.5%, lo cual indica diferencias estadísticamente relevantes en comparación con el 36.0 ± 6.2% de inhibición que se observó en presencia de 500 µM de salbutamol por 5 min (Fig. 7). Así mismo, la inhibición por salbutamol en la configuración de inside-out fue menor al porcentaje inhibido (54.3 ± 3.9%) en la configuración outside-out, indicando una preferencia del compuesto por la cara extracelular del canal, además de una mayor afinidad aparente por el estado cerrado del mismo. Fig. 7. Efecto del salbutamol sobre el canal TRPV4 en configuración de inside-out y estado cerrado. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol (morado). Se representan los valores promedio para las corrientes bajo los distintos tratamientos: vehículo = 12.8 ± 3.5% (n=11); salbutamol (inside-out) = 36.0 ± 6.2% (n=13), salbutamol (outside-out) = 54.3 ± 3.9 % (n=14). ANOVA de una vía, vhc vs salb in-out p=0.0057; salb in-out vs salb out-out p=0.0218; vhc vs salb out-out p<0.0001(*). 32 3) Efecto del salbutamol sobre el canal TRPV4 en presencia de Ca2+ extracelular Aunque el canal TRPV4 presenta una doble respuesta, de potenciación y luego de inhibición por Ca2+, como ya se mencionó anteriormente, es un canal cuya selectividad es mayor por Ca2+ que por Na+, por ende fue necesario determinar que en presencia del Ca2+ también existieran efectos del salbutamol para continuar con nuestro estudio. Al aplicar salbutamol [500 µM] por 5 min en presencia de Ca2+ [2mM] a los parches de membrana escindidos en la configuración outside-out y en el estado cerrado, los resultados mostraron una inhibición de las corrientes en respuesta a GSK del 64.5 ± 5.0% (Fig.8), un porcentaje mayor al inhibido en ausencia de este ion (54.3 ± 3.9%), lo que concuerda con los efectos inhibitorios reportados para el Ca2+ (Nilius et al., 2004). Por ende, para evitar una sobreestimación del efecto del salbutamol en TRPV4, todos los experimentos posteriores se realizaron con soluciones de registro isométricas libres de Ca2+. Fig. 8. Efecto del salbutamol sobre la actividad del canal TRPV4 en presencia de Ca2+ en el estado cerrado y en la configuración de outside-out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol en presencia de Ca2+ (azul) y libre de Ca2+ (morado). Se obtuvieron los siguientes valores promedio para la inhibición de las corrientes: salbutamol + Ca2+=64.5 ± 5.0% (n=6); salbutamol (outside-out)=54.3 ± 3.9% (n=14). Prueba T de Student no pareada de dos colas sin diferencias estadísticamente significativas (ns). 33 4) Efecto del salbutamol y otros SABAs sobre el canal TRPV1 Una vez que se estableció el efecto inhibitorio del salbutamol sobre las corrientes del canal TRPV4, se decidió determinar si estos efectos se limitaban a este canal o si podía afectar la activación de un canal relacionado como el TRPV1, miembro de la misma subfamilia. Para estos experimentos, se expresó al canal TRPV1 en células HEK293 y se probaron tres SABAs para determinar sus efectos. Los resultados mostraron que la respuesta del canal TRPV1 ante capsaicina, agonista de este canal, no se modificó después de los tratamientos con los SABAs que fueron aplicados en el estado cerrado del canal. En particular, en estos experimentos, primero se midieron las corrientes de fuga, luego las corrientes activadas por una concentración subsaturante de capsaicina de 200 nM, los canales se cerraron lavando con solución de registro y luego se aplicaron los diferentes compuestos por 5 min, finalmente se volvió a aplicar capsaicina para evaluar si existía inhibición de las corrientes. Los valores de inhibición de las corrientes de capsaicina fueron del 2.6 ± 5.3 % para salbutamol, 6.3 ± 3.5 % para terbutalina y 3.9 ± 5% para isoprenalina (Fig. 9), lo cual indica que, a diferencia de lo que observamos con el canal TRPV4, este canal no es modulado por estos compuestos. Fig. 9. Efecto del salbutamol, terbutalina e isoprenalina sobre las corrientes del canal TRPV1 en el estado cerrado y en la configuración de outside-out. Trazo representativo de la corriente inicial (gris), en presencia de capsaicina (negro) y al aplicar un SABA como el salbutamol (amarillo). Los valores promedio para las corrientes fueron: salbutamol = 2.6 ± 5.3 % (n=10), terbutalina = 6.3 ± 3.5 % (n=4), isoprenalina = 3.9 ± 5% (n=4). ANOVA de una vía sin diferencias estadísticamente significativas (ns). 34 5) Dependencia de la inhibición del canal TRPV4 sobre la dosis de salbutamol Primero se determinaron las características de activación del canal TRPV4 en presencia del agonista GSK en parches escindidos en la configuración de outside-out bajo nuestras condiciones experimentales. Se determinó el efecto del DMSO, vehículo en el que estaba disuelto el GSK, a una concentración de 915 nM o 0.006% (0.7 ± 0.8%, n=7). También se obtuvo la curva de respuesta ante la dosis para GSK (Fig. 10), los datos fueron ajustados a la ecuación de Hill la cual arrojó que el canal se activa con una EC50= 112.3 nM y un número de Hill de 3.2. Con base en estos valores, se utilizó la concentración saturante de GSK 300 nM para activar el canal, en todos los experimentos. Dentro de los experimentos que se incluyeron para la caracterización de los efectos del salbutamol sobre la actividad del TRPV4 a nivel de las corrientes macroscópicas, se realizó una curva de inhibición por salbutamol en la configuración de outside-out (Fig. 11). El ajuste de la ecuación de Hill a la curva de inhibición por salbutamol arrojó una IC50 de 81.3 µM y un número de Hill de 1.4. La concentración de 1 mM, que fue la más alta que se utilizó, redujo alrededor de un 50% a las Fig. 10. Trazo representativo de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar DMSO (rosa). El valor promedio de la corriente al aplicar DMSO (0.006%)=0.7 ± 0.8%, n=7. Curva de respuesta en función de la dosis de GSK en parches escindidos en la configuración de outside-out para el canal TRPV4. La línea azul representa el ajuste a la ecuación de Hill: EC50=112.3 nM y número de Hill = 3.2 (n=5). 35 corrientes activadas por GSK, por lo tanto, el salbutamol presenta efectos de antagonismo parcial sobre la actividad del TRPV4. 6) Curso temporal de la inhibición del canal TRPV4 por salbutamol Los compuestos tienden a producir sus efectos de inhibición (o activación) en los canales iónicos con cursos temporales característicos. Para determinar el curso temporal de la inhibición del canal TRPV4 por salbutamol, se realizaron experimentos donde se midieron las corrientes inicialmente activadas por GSK en parches con configuración outside-out, se lavó el agonista y luego se aplicó salbutamol a una dosis de 500 µM por una duración determinada y se volvieron a medir las corrientes en presencia de GSK. El cálculo de la constante de tiempo para la inhibición del canal TRPV4 por salbutamol en el estado cerrado, se obtuvo ajustando el promedio de los valores obtenidos para cada tiempo a una ecuación exponencial que arrojó un valor de tau (t, inhibición del 63% de la corriente) igual a 225.6 s (n=4-10; Fig. 12). Fig. 11. Curva de inhibición de las corrientes del canal TRPV4 ante diferentes concentraciones de salbutamol. La línea representa el ajuste a la ecuación de Hill: IC50=81.3 µM y número de Hill = 1.4 (n=3-12) 36 7) Reversibilidad de la inhibición por salbutamol en el canal TRPV4 Para determinar si la inhibición del canal TRPV4 por salbutamol era reversible, primero se midieron las corrientes iniciales en presencia de GSK, luego se lavó el agonista y se aplicó salbutamol por 5 min a la concentración de 500 µM en estado cerrado y en la configuración de outside-out. Posteriormente se volvió a aplicar GSK con un seguimiento de la reactivación de las corrientes por 10 minutos. Las corrientes remanentes fueron de aproximadamente el 50%, valor que se alcanzó en t=46.6 s (n=5; Fig. 13 línea morada) después de haber retirado el salbutamol. Esta constante de recuperación de la corriente fue 5 veces más rápida en comparación con el curso temporal de la inhibición (t=225.6 s, Fig. 12). Sin embargo, el canal no alcanzó en ningún caso el valor inicial de las corrientes, incluso después de 3 minutos se presentó una ligera reducción de las corrientes sin la presencia del salbutamol (rundown, que no fue significativamente distinto a los valores de las corrientes en los primeros minutos). Así mismo, se ajustó una exponencial al decaimiento de las corrientes en ausencia de salbutamol y en presencia de GSK que se muestra como una línea rosa y que arroja un valor de t para este efecto de 291 s (n=5; Fig. 13). Así, se concluye que el efecto de salbutamol es básicamente irreversible. Fig. 12. Curso temporal del efecto de inhibición de 500 µM salbutamol sobre las corrientes el canal TRPV4 en estado cerrado. t= 225.6 s (n=8-10 sellos por cada punto temporal). Los datos fueron obtenidos a -60 mV y ajustados a una exponencial simple. 37 8) Efectos del salbutamol sobre las corrientes unitarias de canales TRPV4. Con el fin de conocer más sobre los detalles biofísicos del efecto inhibitorio del salbutamol sobre el canal TRPV4, se realizaron registros de canales unitarios. La caracterización del efecto de salbutamol sobre TRPV4 a nivel de corrientes microscópicas incluyó la determinación de la amplitud de un solo canal en presencia de GSK y salbutamol, así como el estudio de cambios en la probabilidad de apertura del canal. En estos experimentos se registró la corriente unitaria de TRPV4 a un voltaje de +60 mV en la configuración de outside-out, en ausencia de compuestos o corriente de fuga (trazo negro), en presencia del agonista GSK (trazo azul) indicando que el canal se abre con una amplitud de corriente de 7.7 pA y en presencia de GSK + 500 µM de salbutamol (trazo morado). Los registros en presencia del salbutamol demuestran que el canal se abre con una amplitud de 8.9 pA, similar a la que se observa solo en presencia de GSK (Fig.14). Esto sugiere que este compuesto no actúa como un bloqueador del poro sino más bien como un modulador alostérico de la actividad del canal. Fig. 13. Curso temporal de la recuperación de las corrientes después de la inhibición por 500 µM de salbutamol. t= 46.6 s (n=5) después de la aplicación de salbutamol 500 µM en estado cerrado y t= 291 s (n=5) para el efecto de decaimiento. 38 En cuanto a los cambios en la probabilidad de apertura, en respuesta al GSK, el canal pasa la mayor parte del tiempo en el estado abierto, exhibiendo una probabilidad de apertura de 0.84, mientras que la presencia de salbutamol reduce la probabilidad de apertura a 0.45 (Fig.15), que es una dismiminución de alrededor de un 50%, lo cual concuerda con lo observado para las corrientes macroscópicas. Fig. 14. Trazos de canal unitario de TRPV4 silvestre de humano a +60 mV en ausencia de ligando (trazo negro), en presencia de GSK [300 nM] (trazo azul) con una amplitud de corriente de 7.7 pA y de GSK [300 nM] + salbutamol [500 µM] (trazo morado) con una amplitud de corriente de 8.9 pA. La letra O indica el estado abierto y la letra C el estado cerrado. 39 Tomando en cuenta la disminución en la probabilidad de apertura y la nula alteración en la amplitud de la corriente, es posible sugerir que el salbutamol es un antagonista alostérico y no un bloqueador del poro cuyos efectos, en general, se acompañarían de una reducción en la amplitud de la corriente unitaria más que una disminución gradual en la probabilidad de apertura, como lo que se observa. 9) Efecto de otros SABAs sobre la actividad del canal TRPV4. Para determinar si moléculas similares al salbutamol también podían promover cambios en la activación del canal TRPV4, se evaluaron los efectos de los agonistas del β2-AR: levalbuterol o R- salbutamol, terbutalina, isoprotenerol, metaproterenol, clenbuterol; así como del antagonista del β2- AR, ICI, sobre la actividad del canal TRPV4. Los resultados de los experimentos mostraron que la inhibición de las corrientes después de la aplicación de la terbutalina (54.4 ± 5.7 % n=7), el isoprotenerol (51.6 ± 11.6% n=5), el metaprotenerol (51.7 ± 11.4% n=6) y el levalbuterol o isómero R de salbutamol (42.6 ± 5.4% n=13), todos agonistas de acción corta o SABAs en una concentración de 500 µM (durante 5 min), es similar a la obtenida en presencia de salbutamol (52.1 ± 4.2% n=15), cuando los compuestos fueron aplicados en estado cerrado y en la configuración de outside-out (Fig.16). Fig. 15. Probabilidad de apertura del canal unitario de TRPV4 silvestre de humano mostrado en la figura anterior obtenida a +60 mV en presencia de GSK [300 nM] = 0.84 (puntos azules) y GSK [300 nM] + salbutamol [500 µM] = 0.45 (puntos morados). 40 Por su parte, el clenbuterol un agonista LABA del β2-AR, y el ICI antagonista de dicho receptor, inhibieron un mayor porcentaje de las corrientes de TRPV4 activadas por GSK (71.7 ± 4.2 %; n=7 y 78.3 ± 5.0%; n=13), respectivamente (Fig.17), en comparación con el salbutamol y el resto de los SABAs (Fig.16). Fig. 17. Efecto del clenbuterol e ICI 118,551 sobre el canal TRPV4 en el estado cerrado y en la configuración de outside-out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol (morado), clenbuterol (azul) e ICI (rosa). Los valores promedio para las corrientes fueron: salbutamol = 52.1 ± 4.2% (n=15); clenbuterol = 71.7 ± 4.2% (n=7) e ICI 78.3 ± 5.0% (n=13). ANOVA de una vía, salb vs clen p=0.0328 y salb vs ICI p=0.0005(*). Fig. 16. Efecto de los agonistas del b2-AR de corta duración (SABAs) sobre el canal TRPV4 en estado cerrado y en configuración de outside-out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar alguno de los SABAs probados (colores). Los valores promedio para las corrientes fueron: salbutamol = 52.1 ± 4.2% (n=15); levalbuterol = 42.6 ± 5.4% (n=13); terbutalina = 54.4 ± 5.7% (n=7); isoprotenerol = 51.6 ± 11.6% (n=5) y metaprotenerol = 51.7 ± 11.4% (n=6). ANOVA de una vía, sin diferencias estadísticamente significativas (ns). 41 Es importante destacar que todos los compuestos SABAs son estructuralmente semejantes entre ellos, solo exhiben algunos cambios en la posición de los grupos hidroxilo en el anillo benceno y la sustitución del grupo tertbutilo por isobutilo en la cola de etanolamina (recuadros rojos, Fig. 18). Sin embargo, los sustituyentes en las estructuras del clenbuterol (cloro y amina en el anillo benceno) e ICI (ciclopentano y metilo en el anillo benceno; eter y metilos en la cola etanolamina) difieren del resto de los cambios en los otros compuestos que estudiamos (recuadros azules, Fig. 18), confieréndoles una mayor hidrofobicidad aparente, que posiblemente sea importante para la interacción con el canal TRPV4 y, consecuentemente, para lograr una mayor inhibición de su actividad. Considerando la naturaleza y el mayor efecto del antagonista del β2-AR, el ICI, sobre el canal TRPV4, se realizaron algunos experimentos para caracterizar mejor su papel como antagonista del canal TRPV4. 10) Caracterización de los efectos del ICI sobre el canal TRPV4 Para determinar la dependencia de la inhibición del canal TRPV4 por ICI, se realizó una curva de inhibición en la configuración outside-out y estado cerrado del canal (Fig. 19). El ajuste a la ecuación Fig. 18. Estructuras de los agonistas de corta (SABAs) y larga (LABA) duración así como del antagonista del b2-AR. Cuadros en rojo rodean a los sustituyentes (hidroxilos e isobutilo) que se intercambian entre los agonistas SABA; cuadros azules rodean a los sustituyentes (ciclopentano, metilos, eter e isobutilo) que difieren entre el clenbuterol, ICI y salbutamol. 42 de Hill arrojó una IC50 = 3.9 µM, que inhibió cerca del 40% de la corrientes activadas por GSK y un número de Hill de 0.4. La concentración saturante fue de 500 µM como fue el caso del salbutamol, sin embargo, ICI tuvo un efecto de inhibición mayor (~80%) que el obtenido con salbutamol (~50%). Esto sugiere que se trata de un compuesto cuyas características estructurales (diferentes al resto de compuestos, Fig. 18) le confieren una mayor afinidad aparente por el posible sitio de unión en el canal TRPV4 y, por lo tanto, una mayor efectividad sin la aparente unión cooperativa de varias moléculas (número de Hill < a 1); aunque la presencia de transiciones intermedias entre los estados cerrado y abierto implica detalles específicos que no se consideran en la ecuación de Hill. En ausencia o presencia del agonista GSK, los resultados mostraron una inhibición de las corrientes activadas por GSK después de la aplicación de ICI 500 µM por 5 min de 81.7 ± 6.2% (n=8) para el estado abierto y 77.3 ± 5.3% (n=12) para el estado cerrado (Fig. 20; círculos vacíos y rellenos, respectivamente). A diferencia del salbutamol, el efecto de ICI no presentó ninguna dependencia del estado del canal, inhibiendo de igual manera en presencia o ausencia del agonista GSK. Fig. 19. Curva de inhibición las corrientes del canal TRPV4 ante diferentes concentraciones de ICI. La línea representa el ajuste a la ecuación de Hill: IC50= 3.9 µM y Número de Hill= 0.4 (n=6-13). 43 Sin importar la naturaleza, tanto los agonistas como el único antagonista del β2-AR probado, todas las moléculas presentaron un efecto inhibitorio sobre las corrientes del canal TRPV4 activadas por GSK. Esto sugirió que los efectos de estas moléculas son independientes de la actividad de los β2- AR. 11) Efecto de la co-aplicación de salbutamol e ICI sobre el canal TRPV4. Para sustentar nuestra hipótesis de una posible interacción directa de estas moléculas con el canal TRPV4, se evaluó el efecto del salbutamol a la concentración subsaturante de 100 µM y el efecto de salbutamol [100 µM] después de un pretratamiento de 24 h con el antagonista del receptor β2-AR, ICI [11 µM]. En ausencia del ICI, el porcentaje de las corrientes inhibidas fue de 23.6 ± 4.6% (n=14) mientras que en presencia de ICI fue de 23.4 ± 6.3% (n=9; Fig. 21). Los resultados no mostraron diferencias significativas, sugiriendo que la función del b2-AR no está implicada en el efecto del salbutamol sobre el canal TRPV4. Fig. 20. Efecto de ICI 118,551 sobre las corrientes del canal TRPV4 en estado cerrado y abierto, configuración outside- out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar ICI en estado abierto y cerrado (rosa). Promedio de la corriente inhibida por ICI 118,551: estado abierto = 81.7 ± 6.2% (n=8) y estado cerrado = 77.3 ± 5.3% (n=12). Prueba T de Student no pareada de dos colas, sin diferencias estadísticamente significativas (ns). 44 12) Efecto del salbutamol sobre canales TRPV4 mutantes en la región extracelular de algunos cruces transmembranales Por medio de las simulaciones de dinámica molecular que realizó nuestra colaboradora Ariela Vergara de la Universidad de Talca en Chile y considerando la naturaleza de los residuos con los que se ha reportado que interactúan los broncodilatadores en los receptores β-AR, se produjeron mutaciones individuales con la sustitución de los aminoácidos nativos en los sitios S557, S563, E572, S630, S634, S667, D682, S687, S688 o T689 por alanina. Los resultados demostraron que la sustitución de los aminoácidos en las posiciones 557, 563, 572, 630, 634, 682 o 689 no fueron relevantes para los efectos del salbutamol sobre la actividad del canal TRPV4, al mantenerse sin cambios en la inhibición por el broncodilatador con respecto al canal silvestre (Fig. 22). Lo anterior sugiere que el sitio de unión no se encuentra en los cruces transmembranales TM3 y TM5, ni en la región de unión entre el TM3 y TM4 (Fig. 23). Cabe señalar que las serinas y treoninas son propensas a sufrir fosforilación y glicosilación; sin embargo, no existen reportes hasta la fecha sobre alguna modificación postraduccional de los residuos del canal de humano estudiados en este trabajo. Fig. 21. Efecto de las concentraciones subsaturantes de salbutamol (IC50 = 118 𝜇M) y de ICI (IC50 = 11 𝜇M) sobre las corrientes del canal TRPV4. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol sin y con pretratamiento de ICI (morado). Los valores promedio para las corrientes fueron: sin ICI= 23.6 ± 4.6% (n=14) y con ICI en pretratamiento= 23.4 ± 6.3% (n=9). Prueba T de Student no pareada de dos colas, sin diferencias estadísticamente significativas (ns). 45 . Fig. 22. Efecto del salbutamol sobre las corrientes del canal TRPV4 con mutaciones en los cruces transmembranales 3 y 5 así como del enlazador S3-S4 en estado cerrado y en configuración de outside-out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol (colores). Los valores promedio para la inhibición de las corrientes fueron: S557A = 59.8 ± 4.0 % (n=12); S563A = 63.7 ± 4.8% (n=11); E572A = 55.3 ± 5.5% (n=11); S630A = 62.8 ± 5.7% (n=9); S634A = 53.0 ± 4.4% (n=9); D682A = 53.5 ± 8.1% (n=4) y T689A = 51.4 ± 9.5% (n=5). ANOVA de una vía, sin diferencias estadísticamente significativas (ns). Fig. 23. Modelo del canal TRPV4 de humano hecho por el laboratorio de la Dra. Ariela Vergara a partir de la modificación de la estructura del canal TRPV4 de Xenopus sp. (6BBJ) con quien comparte con un »85% de identidad. En azul una de las subunidades del tetrámero. Las esferas amarillas representan la ubicación de los aminoácidos mutados sin efecto. 46 13) Efecto del salbutamol sobre canales TRPV4 mutantes en la región extracelular del poro Las simulaciones de dinámica molecular también sugirieron posibles sitios en la región extracelular del canal que podrían interaccionar con el salbutamol. Así, se evaluaron los efectos de mutaciones individuales en la región extracelular del poro del canal TRPV4. Los resultados demostraron que los canales TRPV4 mutantes S667A (43.2 ± 3.7%; n=12) y S687A (31.0 ± 3.9%; n=14) fueron menos suceptibles a la inhibición inducida por salbutamol y, el canal doble mutante, S667A-S687A, para estos dos sitios, presentó una inhibición de solo el 10.9 ± 3.9% (n=12). En los tres casos, los resultados son estadísticamente diferentes al compararse con la respuesta promedio del canal WT a salbutamol (60.0 ± 3.0%; n=43; Fig. 24); lo cual nos permitió concluir que ambos residuos son importantes para la unión del salbutamol al canal TRPV4. Es importante mencionar que los residuos D682A y T689A que se encuentran en la torreta del poro (Fig. 25), también podrían influir en la unión del salbutamol al canal, ya que mostraron una tendencia a la baja en el efecto de inhibición por el compuesto aunque no fue estadísticamente significativa. Por lo tanto, la región extracelular del poro se propuso como el posible sitio de unión para el salbutamol en TRPV4. Fig. 24. Efecto del salbutamol sobre el canal TRPV4 con mutaciones en la región del poro en estado cerrado y en configuración outside-out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) y al aplicar salbutamol (colores). Se obtuvieron los siguientes valores promedio para la inhibición de las corrientes: S667A = 43.2 ± 3.7% (n=12); S687A = 31.0 ± 3.9% (n=14); y S667A-S687A = 10.9 ± 3.9% (n=12). ANOVA de una vía, WT vs S667A p=0.0178; WT vs S687A p<0.0001; WT vs S667A-S687A p<0.0001; S667A vs S667A-S687A p<0.0001; S687A vs S667A-S687A p=0.02 (*). 47 Fig. 25. Modelo del canal TRPV4 de humano hecho por el laboratorio de la Dra. Ariela Vergara a partir de la estructura de Xenopus sp. (6BBJ). En azul (marino y turquesa) se muestran dos de las subunidades del tetrámero. Las esferas moradas indican la ubicación de los aminoácidos cuya sustitución representa un cambio en el efecto de salbutamol. Otro de los sitios propuestos por el modelaje in silico fue el residuo S688, también en la región del poro. Bajo nuestras condiciones experimentales, el canal mutante S688A, exhibió un aumento de la inhibición inducida por salbutamol sobre las corrientes activadas por GSK (87.6 ± 3.15%; n=11), en comparación con el canal silvestre (60.2 ± 3.5%; n=30). Sin embargo, al examinar el comportamiento de dicha mutante a detalle, se definió como un efecto independiente del salbutamol, ya que se obtuvo una respuesta similar exponiendo los parches de membrana que expresaban a este canal mutante a solución de registro sin salbutamol (79.2 ± 7.4%; n=7. Fig. 26). Lo anterior sugiere que la mutante exhibe un proceso de rundown constitutivo. Por ende, es probable, que este fenómeno se deba a un cambio en las propiedades de compuerta del canal mutado, por lo tanto, no es posible concluir que sea un sitio de unión al salbutamol. 48 En cuanto a los otros dos residuos identificados como posibles sitios de unión en el TRPV4 para el salbutamol, D613A e Y621A, los canales mutantes no presentaron expresión funcional ya que no se detectó la activación de corrientes en presencia del agonista GSK. Finalmente, se evaluó la respuesta de los canales mutantes con una respuesta reducida a salbutamol que se probaron en este estudio ante el GSK para determinar si también existía un cambio en su respuesta ante el agonista. Así, se construyeron las curvas de activación en respuesta al GSK para las mutantes S667A, S687A y S667A-S687A del TRPV4. Los datos se ajustaron a la ecuación de Hill que arrojó los siguiente valores: EC50= 182.35 nM y número de Hill de 1.53 para la mutante S667A; EC50= 108.9 nM y número de Hill de 2.47 para la mutante S687A y EC50= 91.1 nM y un número de Hill de 2.88 para la doble mutante S667A-S687A (Fig. 27). Estos resultados mostraron que no existen Fig. 26. Canal TRPV4 con la mutación S688A en estado cerrado y en configuración de outside-out. Trazos representativos de la corriente inicial (gris), en presencia de GSK (negro) de salbutamol (morado) y de solución de registro (azul). Promedio de las corrientes inhibidas después de la aplicación en el canal silvestre de 500 µM de salbutamol= 60.2 ± 3.5% (n=30); así como después de la aplicación registro en el canal mutante S688A de solución de registro= 79.2 ± 7.4% (n=7) y de salbutamol a 500 µM = 87.6 ± 3.15% (n=11). ANOVA de una vía, WT vs S688A salb p=0.0002; WT vs S688A(SR) p=0.0338 (*). 49 alteraciones significativas (un orden de magnitud) en la mayoría de los canales mutantes en su respuesta al agonista GSK, solo se obtuvo un ligero corrimiento a la derecha para la curva de activación de S667, sugiriendo una aparente disminución en su sensibilidad al GSK. Fig. 27. Curva de respuesta en función de la dosis de GSK para el canal TRPV4 WT y los canales mutantes S667A, S687A y S667A-S687A en parches escindidos en la configuración de outside-out. Las líneas de colores representan el ajuste a la ecuación de Hill del canal WT (negra) con EC50=112.3 nM y número de Hill = 3.2 (n=5); la mutante S667A (amarillo) con EC50= 182.35 nM y número de Hill de 1.53; la mutante S687A (verde) con EC50= 108.9 nM y número de Hill de 2.47 y la doble mutante S667A-S687A (azul) con EC50= 91.1 nM y un número de Hill de 2.88. 50 Discusión El canal iónico TRPV4 es una proteína que se expresa en distintos tipos celulares del tracto respiratorio, de los pulmones y de la vasculatura pulmonar (Jia y Lee, 2007; Palaniyandi et al., 2020); participando en la remodelación de las vías respiratorias, en la regulación del volumen pulmonar, así como del tono vascular, en el mantenimiento de la integridad de la barrera alveólo-capilar y en la defensa contra organismos patógenos (Jesudason, 2007; Morgan et al., 2018; Yu et al., 2019). Se ha propuesto que la sobreexpresión y/o el aumento de la actividad del canal TRPV4 participan en el desarrollo de patologías como el asma, la COPD, la fibrosis quística y el edema (Li et al., 2011; Balakrishna et al., 2014; Scheraga et al., 2017; Bihari et al., 2017; Palaniyandi et al., 2020). Consecuente con lo anterior, el uso de compuestos antagonistas de la actividad del TRPV4 ayudan a controlar estos síntomas (Thorneloe et al., 2012; Birrell, et al.; 2016; Brnardic et al.; 2018; Goyal et al.; 2019). Durante gran parte del siglo XX, se desarrollaron estrategias para el tratamiento de las enfermedades respiratorias. El descubrimiento de los receptores a y b sensibles a adrenalina (Ahlquist, 1948), cuyo papel es relevante en la mayoría de las enfermedades cardíacas y respiratorias, así como el primer tratamiento efectivo de la angina de pecho utilizando un betabloqueador (propanolol) (Stapleton, 1997), impulsaron la búsqueda y síntesis de moléculas reguladoras de los receptores beta- adrenérgicos dentro de la comunidad científica y la industria farmacéutica. Esta gran oleada de descubrimientos y caracterización de ligandos específicos para los receptores beta (Nelson, 1995), inició con la síntesis de la isoprenalina en 1940 (Gay y Long, 1949) e incluyó a otros compuestos probados en este trabajo como la terbutalina, el metaproterenol y el salbutamol 51 (Emilien y Maloteaux, 1998; Sears y Lötvall, 2005), los cuales mostraron un efecto inhibidor y específico en la actividad del canal TRPV4 en comparación al nulo efecto sobre TRPV1. A pesar de que ambos canales son miembros de la misma subfamilia, la homología de sus secuencias es únicamente del 50% (Nilius y Szallasi 2014). Sumado a esto, se ha descrito que el ordenamiento tridimensional que adopta el canal TRPV4, con especial énfasis en el empaquetamiento y la orientación espacial de los cruces S1–S4 y S5–S6 (donde podría estarse uniendo el salbutamol), es diferente al de los miembros TRPV1, TRPV2 y TRPV6 (Deng et al., 2018), lo cual le confiere al canal TRPV4 características específicas que se reflejan en una respuesta diferente a los SABAs (mayor sensibilidad) en comparación con otros miembros de la familia vaniloide. El salbutamol es un compuesto sintetizado a partir del núcleo de un catecol básico, con un centro quiral en la cola de etanolamina, lo que da lugar a dos enantiómeros (S) y (R), según la posición del grupo hidroxilo (Sears y Lötvall, 2005; Jacobson et al., 2017). Los experimentos que utilizaron la mezcla racémica 50:50 de salbutamol mostraron una dependencia en la dosis del compuesto, en la configuración y en el estado del canal en el que se aplicó, sugiriendo la interacción con alguna parte extracelular del canal y con una mayor accesibilidad/afinidad al mismo cuando el canal se encuentra cerrado. La presencia de los grupos hidroxilo en el anillo benceno y la cola de etanolamina en salbutamol, terbutalina, metaproterenol e isoproterenol les confieren características hidrofílicas que les permiten acceder directamente al bolsillo de unión del b2-AR, donde se unen débilmente, desde el medio extracelular donde están disueltos (Sears y Lötvall, 2005). De ahí que sus características en el alivio (y el nombre que reciben SABAs), sean rápidas pero efímeras en comparación con las de los beta- agonistas LABAs o Ultra-LABAs (Billington et al., 2017). 52 En nuestros experimentos utilizando estos compuestos, además del enantiómero purificado R- salbutamol (levalbuterol), cuya efectividad es mayor a la de la mezcla racémica (Penn et al., 1996; Gawchik et al., 1999); se encontró un efecto de inhibición parcial (~50%) sobre el canal TRPV4 para todos los compuestos SABAs. Lo anterior se podría atribuir a los tres grupos hidroxilos presentes en cada una de las moléculas (figura 19), que estarían entorpeciendo su libre movimiento en un medio hidrofóbico o limitando a interacciones débiles la unión de los SABAs en el posible bolsillo presente en TRPV4. Un estudio previo encontró que el salbutamol fue incapaz de inhibir los canales de sodio del músculo esquelético (hSkM1). Sin embargo, el clenbuterol, un agonista LABA con características estructurales que lo hacen más hidrofóbico, inhibió las corrientes de dichos canales de forma independiente a la vía de los b-ARs (Desaphy et al., 2003). Esto, sumado al reporte en el que ICI, un antagonista del b2-AR también bloqueó las corrientes de canales de potasio de la familia éter-á-go- go (HERG) a través de un mecanismo en el que parece actuar como un bloqueador del poro (Dupuis et al., 2005). Ambos estudios fortalecen la aseveración de la relevancia que tienen las características estructurales conferidas por los grupos químicos que integran a los compuestos en el proceso de unión directa a los canales; comprobado con nuestros experimentos utilizando clenbuterol e ICI sobre el canal TRPV4 donde se obtuvo una inhibición del ~80%, bajo las mismas condiciones en las que se probaron los diferentes SABAs. Además de una inhibición completa y no parcial por parte del antagonista de los b2-AR, el uso de ICI nos permitió descartar la participación del b2-AR y su vía activada por el ligando (Gong et al., 2002). El efecto del antagonista sobre TRPV4 presentó una dependencia a la dosis administrada, con una IC50 menor a la obtenida con salbutamol, reflejando una aparente mayor afinidad de este compuesto al sitio de unión en el canal. Distinto a lo que se observó con el agonista (salbutamol), ICI 53 no tuvo alteraciones en su efecto al ser administrado por la cara intracelular o extracelular del canal, probablemente como consecuencia de la hidrofobicidad que caracteriza a la molécula permitiéndole moverse a través de la membrana al sitio de unión independientemente de su ubicación. Los experimentos para dilucidar el posible sitio de unión en el canal TRPV4 incluyeron tres regiones: el cruce S3, el enlazador S3-S4 y la región del poro. Los canales con mutaciones en los aminoácidos que forman parte de las dos primeras regiones no presentaron ninguna alteración en el efecto de salbutamol descartando regiones cercanas al sitio que se describió para el antagonista HC067047 en el que están involucrados el enlazador S2-S3 (D542), el cruce S4 (M583 e Y587) y el cruce S5 (D609 y F613) (Doñate‐Macian et al., 2022). Por otro lado, cuando se mutaron las serinas nativas en las posiciones 667 y 687 por una alanina de manera individual se produjo una respuesta disminuida al salbutamol, que se acentuó en el canal con la doble sustitución. Estas observaciones sugieren que el asa reentrate del poro y la parte extracelular del S6 son clave para la unión del salbutamol. La posición tan crítica en la que estaría uniéndose el agonista de los receptores beta adrenérgicos con el canal TRPV4 implicaría un mecanismo de bloqueo similar al reportado para la ergtoxina (ErgTx1) en el canal HERG. En este caso, las regiones del cruce S5 (Trp-585, Gly-590 e Ile-593) y el S6 (Pro- 632) en la torreta unen a la toxina e impiden el flujo libre de iones (Pardo-López et al., 2002) sin ser un tapón molecular como la doble nodo toxina (DkTx) en el canal TRPV1 (Bae et al., 2016). Sin embargo, los registros unitarios en los que se expuso el canal TRPV4 a salbutamol solo mostraron una reducción en la probabilidad de apertura (eventos de mediana y larga duración) sin alterar la amplitud de la corriente lo que sugiere un antagonismo alostérico en el que el salbutamol estabiliza la conformación de algún estado cerrado del canal, semejante al efecto de la metanosulfonamida TKDC en el canal de potasio TREK-1 (Luo et al., 2017). 54 En resumen, este trabajo describe por primera vez la interacción de salbutamol, una molécula reportada por mucho tiempo como agonista específico del b2-AR, con un canal TRP; sumándose a los reportes de canales de sodio que responden a clenbuterol (hSkM1; Desaphy et al., 2003) y a ICI (HERG; Dupuis et al., 2005) que también tuvieron actividad sobre el canal TRPV4. Este primer acercamiento reveló todo un grupo de moléculas que tienen la capacidad de regular a la baja la actividad del canal TRPV4 y las diferentes características de la inhibición que provocan en el canal (parcial o total) asociadas a sus características estructurales lo que favorecería su empleo en tratamientos de diferentes patologías, en función, del grado de regulación al que se quiera someter al canal TRPV4. También se dilucidó el mecanismo de modulación alostérica del salbutamol sobre el canal. Al menos dos residuos de la región externa del poro parecen participar, los cuales no necesariamente son el sitio de unión a clenbuterol o ICI. Detallar el mecanismo a través del cual estos compuestos tienen una mayor efectividad en la inhibición del canal TRPV4 sería una importante línea con la que se puede continuar. Conclusiones • El salbutamol, así como otros agonistas del b2-AR de corta duración inhiben parcialmente a la actividad del canal TRPV4. • El salbutamol, la terbutalina y el isoproterenol no alteran a la actividad del canal TRPV1, que es otro canal que se expresa en el sistema respiratorio. • La inhibición del canal TRPV4 por salbutamol es dependiente del tiempo de aplicación y de la dosis del compuesto. • La inhibición del canal TRPV4, inducida por salbutamol es irreversible. • El clenbuterol (agonista del b2-AR de larga duración) y el ICI 118,551 (antagonista del b2- AR) tienen mayor efecto en la inhibición del canal TRPV4, en comparación con salbutamol. 55 • Los efectos del salbutamol sobre el canal TRPV4 parecen ser independientes de la vía de señalización del b2-AR, porque incluso algunos antagonistas de estos receptores inhiben al canal TRPV4. • La inhibición del canal TRPV4 por salbutamol es mayor en el estado cerrado y en la configuración outside-out. Por lo tanto, estas observaciones apoyan nuestros resultados de que el sitio de unión para salbutamol se encuentra en la parte extracelular del canal y tiene un mejor acceso cuando el canal adopta alguna conformación cerrada, estabilizándolo en el estado no conductor. • Los sitios S667 y S687 del asa reentrante en el poro son críticos para la unión del salbutamol con TRPV4. • El salbutamol no altera la amplitud de la corriente del canal TRPV4; sin embargo, reduce alrededor del 50% la probabilidad de apertura en comparación con el efecto obtenido por GSK, por lo que el mecanismo podría ser a través de una inhibición alostérica del canal. Consideraciones finales Los datos presentados arriba fueron publicados en la revista Life Sciene Alliance. Sin embargo, durante mi entrenamiento doctoral en el laboratorio de la Dra. Rosenbaum, también me involucré en otros estudios. En uno de ellos, describí los efectos del ácido lisofosfatídico (LPA) y de la lisofosfatidilcolina (LPC), moléculas de origen endógeno, sobre la activación del canal TRPV4 y soy también primer autor de ese trabajo científico original publicado en el Journal of Physiology (https://doi.org/10.1113/JP284262; se incluye una copia del artículo publicado). Así mismo, participé en la realización de experimentos de colaboración con un laboratorio de Inglaterra, evaluando los efectos de la dapagliflozina, un inhibidor del cotransportador de sodio glucosa (SGLT2) sobre la función del canal TRPV1, mismo que se 56 publicó en Cardiovascular Research (https://doi.org/10.1093/cvr/cvae156; se incluye una copia del artículo publicado). 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(2015) Patch clamping and single-channel analysis. In Handbook of Ion Channels, Zheng J, Trudeau MC (eds). Boca Raton: CRC Press. Research Article Unconventional interactions of the TRPV4 ion channel with beta-adrenergic receptor ligands Miguel Benı́tez-Angeles1, Emmanuel Juárez-González1, Ariela Vergara-Jaque2,3, Itzel Llorente1, Gisela Rangel-Yescas4 , Stéphanie C Thébault5, Marcia Hiriart1, León D Islas4, Tamara Rosenbaum1 The transient receptor potential vanilloid 4 (TRPV4) ion channel is present in different tissues including those of the airways. This channel is activated in response to stimuli such as changes in temperature, hypoosmotic conditions, mechanical stress, and chemicals fromplants, lipids, and others. TRPV4’s overactivity and/ or dysfunction has been associated with several diseases, such as skeletal dysplasias, neuromuscular disorders, and lung patholo- gies such as asthma and cardiogenic lung edema and COVID-19– related respiratory malfunction. TRPV4 antagonists and blockers have been described; nonetheless, the mechanisms involved in achieving inhibition of the channel remain scarce, and the search for safe use of these molecules in humans continues. Here, we show that the widely used bronchodilator salbutamol and other ligands of β-adrenergic receptors inhibit TRPV4’s activation. We also demonstrate that inhibition of TRPV4 by salbutamol is achieved through interaction with two residues located in the outer region of the pore and that salbutamol leads to channel closing, consistent with an allosteric mechanism. Our study pro- vides molecular insights into the mechanisms that regulate the activity of this physiopathologically important ion channel. DOI 10.26508/lsa.202201704 | Received 2 September 2022 | Revised 5 December 2022 | Accepted 8 December 2022 | Published online 22 December 2022 Introduction The transient receptor potential vanilloid 4 (TRPV4) is a Ca2+- permeable non-selective cation channel, which can either be inhibited or potentiated in a Ca2+ concentration-dependent fashion (Strotmann et al, 2000). This protein is found in several cell types and tissues (Rosenbaum et al, 2020), including epidermal kerati- nocyte cells (Chung et al, 2003); vascular endothelium (Fian et al, 2007; Tanaka et al, 2008; Yin et al, 2008); smooth muscle cells in the pulmonary aorta and artery, brain arteries (Earley et al, 2005; Yang et al, 2006), primary afferent sensory neurons that innervate the gastrointestinal tract (Holzer, 2011), and enterocytes and enteroendocrine cells (Boesmans et al, 2011; Bellono et al, 2017); and epithelia of the trachea and lungs (especially in cilia of the bronchial epithelium) (Lorenzo et al, 2008), among others (Tian et al, 2004; Teilmann et al, 2005; Birder et al, 2007; Gevaert et al, 2007; Gradilone et al, 2007; Casas et al, 2008; Pan et al, 2008). TRPV4 is regulated by several stimuli such as temperatures around 27°C (Güler et al, 2002), hypoosmotic conditions, me- chanical stress (Liedtke et al, 2000; Liedtke & Friedman, 2003), plant chemicals such as bisandrographolide A from Andrographis pan- iculata (Smith et al, 2006), phorbol derivatives (i.e., 4α-phorbol 12,13-didecanoate, 4αPDD) (Watanabe et al, 2002), the flavonoid apigenin (Ma et al, 2012), and the synthetic agonist GSK1016790A (GSK) (Jin et al, 2011). Other agonists or activity modulators of TRPV4 include phosphatidylinositol 4,5-bisphosphate (PIP2) (Garcia-Elias et al, 2015), 5,6-epoxyeicosatrienoic acid (5,6-EET) (Watanabe et al, 2003; Berna-Erro et al, 2017), and flavonoids (Ma et al, 2012; Wang et al, 2015). Inhibitors or blockers of TRPV4 are GSK3527497 (Brooks et al, 2019), GSK205 (Phan et al, 2009), and its derivatives (Kanju et al, 2016); ruthenium red (St Pierre et al, 2009) and Gd3+ (Berrier et al, 1992; Phan et al, 2009); RN-1734 (Vincent et al, 2009) and RN-9893 (Vincent & Duncton, 2011); and the biflavone ginkgetin (Alharbi et al, 2021). TRPV4 regulates cellular homeostasis of the intracellular calcium concentration ([Ca2+]i) (White et al, 2016) by participating in maintenance of the integrity of endothelial barriers (Nilius & Droogmans, 2001; Phuong et al, 2017; Pairet et al, 2018), osmoreg- ulation (Liedtke et al, 2000; Liedtke & Friedman, 2003), nociception (Kanju et al, 2016), and control of vascular tone (Sonkusare et al, 2012), bone homeostasis (Masuyama et al, 2008), pulmonary (Bihari et al, 2017; Morgan et al, 2018) and renal (Liedtke & Friedman, 2003) functions, and itch (Tóth et al, 2014; Chen et al, 2021). TRPV4 has also been linked to several human congenital disorders, which have been grouped into skeletal dysplasias and neuromuscular disor- ders (Nilius & Owsianik, 2010; Grace et al, 2017). These diseases encompass progressive degeneration of peripheral nerves and lack of establishment and development of the hard skeletal tissues, and highlight the importance of TRPV4 in human pathophysiology. 1Departamento de Neurociencia Cognitiva, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México (UNAM), México, México 2Center for Bioinformatics, Simulation and Modeling, Faculty of Engineering, Universidad de Talca, Talca, Chile 3Millennium Nucleus of Ion Channel-Associated Diseases, Santiago, Chile 4Departamento de Fisiologı́a, Facultad de Medicina, UNAM, México, México 5Instituto de Neurobiologı́a, UNAM, Campus UNAM-Juriquilla, Querétaro, México Correspondence: trosenba@ifc.unam.mx © 2022 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 1 of 15 on 10 September, 2024life-science-alliance.org Downloaded from http://doi.org/10.26508/lsa.202201704Published Online: 22 December, 2022 | Supp Info: Important roles of TRPV4 in the respiratory system have also been suggested. Smooth muscle cells, fibroblasts, submucosal glands, macrophages, vascular endothelial cells, and bronchial, tracheal, and alveolar epithelia (Liedtke et al, 2000; Delany et al, 2001; Alvarez et al, 2006; Jia & Lee, 2007; Xia et al, 2018; Palaniyandi et al, 2020) express TRPV4, which maintains the homeostasis of osmotic pressure in these tissues and integrates different stimuli that translate into Ca2+ signals regulating the functions of the respiratory system (Garcia-Elias et al, 2014). In the respiratory airway, the overactivation of TRP channels induces airway con- striction, inflammation, sneezing, cough, and mucus secretion, contributing to a defense mechanism of the respiratory system (Parker et al, 1998; Wallace, 2017; Xia et al, 2018). It has been shown that agonists of TRPV4 and hypoosmotic stress result in depolar- ization of the vagal nerves in humans, mice, and guinea pigs. Conversely, the use of TRPV4 antagonists results in a decrease in cough (Bonvini et al, 2016), which has led to the proposal that TRPV4 is a molecular effector of airway protection. Roles of TRPV4 in acute lung injury and in acute respiratory distress syndrome have been also suggested. In these processes, there is an increase in the permeability of pulmonary vascular and endothelial barriers (Catravas et al, 2010) likely involving TRPV4 overactivation, because pharmacological inhibition or genetic deletion of this channel prevents lung damage in animal models of ventilator-induced pulmonary injury by liquids (Hamanaka et al, 2007; Bihari et al, 2017). Moreover, experiments where mice were exposed to chemical compounds (i.e., hydrochloric acid or chloride vapors) (Balakrishna et al, 2014) showed that TRPV4 plays a crucial role in injury to the lungs. In the respiratory system, overactivation of TRPV4 affects excitability of bronchopulmonary sensory neurons (Gu et al, 2016) and TRPV4-deficient mice are protected from airway remodeling that occurs in both large and small airways relevant to miscellaneous respiratory diseases including asthma, a chronic inflammatory disease of the upper airways (Gombedza et al, 2017). This channel has also been linked to the response to contam- inating particles, such as those from diesel exhaust (Li et al, 2011), and it has recently been proposed as a pharmacological target for patients with COVID-19, where inhibition of TRPV4 may reduce lethality by contributing to alveolo-capillary barrier preservation (Goyal et al, 2019). β-Adrenergic receptors (β-ARs) are Gs protein–coupled receptors that elicit smooth muscle relaxation and bronchodilation, through activation of a cAMP-dependent signaling pathway (Barisione et al, 2010). In asthma, agonism of β-ARs by compounds such as sal- butamol results in bronchodilation, allowing for better respiratory function. Although clenbuterol, like salbutamol, is considered a specific β-AR agonist, it has been shown to inhibit sodium channels in rat skeletal muscle fibers, where β-ARs are also expressed (Desaphy et al, 2003). Because TRPV4’s activation influences the function of respiratory airways, here we studied whether salbutamol and other agonists and antagonists of β-ARs could regulate the function of this ion channel in vitro. Our results indicate that not only agonists of β-ARs salbutamol, terbutaline, isoprenaline, metaproterenol, levalbuterol, and clenbuterol inhibit the activation of TRPV4, but also antagonists of these receptors exert inhibitory effects on the channel. Our site-directed mutagenesis data, showing that salbutamol binds to residues located at an extracellular site on the pore turret of TRPV4, suggest that this inhibitory effect is mediated by direct interaction with this tetrameric ion channel. Results Salbutamol inhibits activation of TRPV4, but not of TRPV1 The short-acting bronchodilator (SABD) salbutamol, composed of a racemic mixture of R- and S-salbutamol, which is a sympathomi- metic amine that functions as an agonist of β2-AR, was used to perform experiments in a heterologous expression system of TRPV4. In HEK293 cells transfected with human TRPV4 (hTRPV4), we found that application of 300 nM GSK to outside-out membrane patches resulted in the activation of currents (Fig 1A–D, black traces). Experiments where Ca2+ was added to the external solution showed that after exposure to salbutamol, 64.5% ± 12.3% (Fig 1E, +120 mV, solid circles) of the current magnitude was inhibited, and hence, only a fraction of GSK-activated current remained after re- exposing the patches to the agonist (Fig 1A and E). Because Ca2+ has been shown to exhibit complex modulation properties (potentia- tion and then inhibition) on TRPV4’s activity (Nilius et al, 2004), we performed all electrophysiological experiments, except those shown in Fig 1A and E, in the absence of this ion. When membrane patches were exposed to vehicle only (Tris), there was only a 21.2% ± 9.7% decrease (Fig 1E, +120 mV, empty triangles) in current, as compared to the initial current obtained in the presence of GSK, which could be due to the removal of an intracellular factor re- quired for sustained activity upon membrane patch excision (Garcia-Elias et al, 2013). To determine whether salbutamol inhibited closed or open TRPV4 channels equally, wemeasured currents initially activated by 300 nM GSK and then incubated the membrane patches with salbutamol (500 µM, 5 min) either in the absence (Fig 1B) or in the presence (Fig 1C) of the agonist, and then, we reapplied GSK to determine inhibition by salbutamol (purple traces at −120 and +120 mV). We observed that when salbutamol was applied in the absence of GSK, 54.3% ± 14.5% (closed state, Fig 1E, solid triangles, +120 mV) of TRPV4’s currents were inhibited, as compared to the 32.5% ± 18.6% inhibition when salbutamol was applied in the presence of the agonist (open state, Fig 1E, solid ties, +120 mV), suggesting that there is state-dependent inhibition of TRPV4- mediated current by salbutamol. In contrast, when rundown in the presence of vehicle and GSK101 was assessed, we found that 16.16% ± 8.3% of the current was decreased (Fig 1E, empty ties). Furthermore, we performed experiments using a related TRP channel, TRPV1, to determine whether salbutamol inhibited this channel also. As shown in Fig 1D and E, capsaicin (250 nM) activated the TRPV1 channel, but the addition of salbutamol (500 µM, orange traces at −120 and +120 mV) did not produce a significant current decrease (2.6% ± 16.8%, Fig 1E, orange squares, +120 mV). In another set of experiments, we found that GSK-activated currents were also inhibited by 500 µM salbutamol when it was applied intracellularly (Fig S1A and B). Specifically, in inside-out patches, 500 µM salbutamol inhibited 36% ± 22.4% of the currents, as compared to 12.78% ± 12% inhibition with intracellularly applied Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 2 of 15 vehicle (Fig S1C, +120 mV, solid and empty circles, respectively). Inhibition by salbutamol applied to the extracellular side of the channel was statistically different (54.3% ± 14.5%, Fig S1C, +120 mV, triangles), as compared to when it was applied from the intra- cellular side (36% ± 22.4%, Fig S1C, +120 mV, solid circles). We further examined inhibition of TRPV4 after a 5-min incubation with different concentrations of extracellular salbutamol and found that it is concentration-dependent. The inhibition was quantified by fitting to the Hill equation with an apparent dissociation constant (KD) of 81.3 µM and a Hill coefficient of 1.4 (Fig 2A). It is interesting to note that an inhaler, which can be used three or four times a day, typically contains between 100 and 150 µM/kg salbutamol per dose. Interestingly, nebulized salbutamol has been found to be more effective, as compared to its systemic administration. A possible explanation for this is that there appears to be no direct bio- transformation of salbutamol in the lungs (with a half-life between 2 and 7 h), when applied with an inhaler (Gad, 2014). Finally, we performed experiments to determine the time course of TRPV4 inhibition by 500 µM salbutamol at different time points (as de- tailed in the Materials and Methods section) and found that the current decay could be fitted to a single exponential with a time constant of 225.6 s (Fig 2B). Current decay stabilized after 5 min of exposure of the membrane patches to salbutamol, and it was not measured after 6 min of exposure because current decay, in the presence of only vehicle at 7 min, was already 49.96% ± 25.97% (n = 7). Finally, we also measured recovery from inhibition by measuring initial currents with GSK101 (300 nM), then washing GSK101 off to apply salbutamol (500 µM) for 5 min and reactivating the channels by reapplying GSK101 for up to 10 min. The data in Fig 2C show that right after treatment with salbutamol 500 µM (0–60 s), some current was activated, and then, it was not further recovered in the presence of GSK101, possibly because of a combination of this inhibition being irreversible and rundown of the activity of the channel with excision of the membrane patch. Effects of short- and long-acting bronchodilators on TRPV4 activation It has been shown that the minimal required chemical structure for activation of β-ARs is an aromatic ring system and an aliphatic amino group (Kolb et al, 2009). Structural components such as the chiral β-OH group present in salbutamol and in endogenous ag- onists (i.e., epinephrine and norepinephrine) (Swaminath et al, 2005) also allow for better binding to β-ARs. Different agonists are thought to either partially stabilize or fully activate β-ARs through interactions of different chemical groups with different residues in the receptors (Yao et al, 2006). In the case of salbutamol, these functional groups are the OH groups present in the aromatic ring, which interact with residues S203, S204, S207, D113, and N312 in the β2-AR (Strader et al, 1989; Johnson, 1998). Other SABDs, with structural similarities to salbutamol (Fig 3A–E), include levalbuterol (which is composed only of R-salbutamol), terbutaline, isoprenaline, and metaproterenol. To ascertain whether these compounds could also inhibit TRPV4, we performed exper- iments in which we first activated TRPV4-mediated currents in the presence of GSK (300 nM), then applied each different SABD, and measured the inhibited current after the second application of GSK. We used a concentration of 500 µM for each compound, with which Figure 1. Effects of salbutamol on TRPV4 and TRPV1 channels. (A, B, C, D) Representative traces of currents at +120 and −120 mV for 100ms from a holding potential of 0 mV in the outside-out configuration. Patches were first exposed to recording solution (gray, leak or initial currents), then to agonists (black traces) GSK (300 nM) for TRPV4 channels or capsaicin (200 nM) for TRPV1, washed with recording solution, and then exposed to 500 µM salbutamol. Current inhibition was measured after exposing the patches to the agonists again (purple traces for TRPV4 and orange for TRPV1). (E) Average data for experiments in (A, B, C, D). Data were normalized to the initial value with GSK or capsaicin. *The one-way analysis of variance, followed by Tukey’s post hoc, was used for group comparison. Significant differences between means were considered to exist when the P-value was less than 0.01. After salbutamol treatment in the presence of Ca2+, 64.5% ± 12.3% (n = 6) of currents were inhibited. The percentages of inhibited currents when salbutamol was applied in the closed (absence of agonist) or in the open (presence of agonist) states were 54.3% ± 14.5% (n = 14) and 32.5% ± 18.6% (n = 15), respectively. Application of vehicle only in the closed state produced 21.2% ± 9.7% (empty triangles, n = 13) of the inhibition and 16.16% ± 8.2% (empty ties, n = 15) when vehicle was applied in the presence of GSK101. In the case of TRPV1, 2.6 ± 16.8 (n = 10) of currents were inhibited after treatment with salbutamol (orange squares). *P < 0.0001 for salbutamol + Ca2+ versus vehicle; *P < 0.001 for salbutamol + Ca2+ versus salbutamol + GSK (open); *P < 0.0001 for vehicle versus salbutamol (closed); *P = 0.0005 for salbutamol (closed) versus salbutamol (open); *P < 0.0001 for salbutamol (closed) versus salbutamol in TRPV1; and *P = 0.0002 for salbutamol (open) versus salbutamol in TRPV1, as indicated by brackets. One-way analysis of variance with Tukey’s post hoc test was performed. Source data are available for this figure. Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 3 of 15 we achieved maximal inhibition with salbutamol, and like salbu- tamol 500 µM (Fig 3A and F, 52.12% ± 16.3% current inhibition after salbutamol), the other compounds also produced similar TRPV4 inhibition, evidenced after the second application of GSK. Leval- buterol (Fig 3B and F) resulted in a 42.6% ± 19.4% inhibition of TRPV4 currents, whereas terbutaline (Fig 3C and F), isoprenaline (Fig 3D and F), and metaproterenol (Fig 3E and F) resulted in a 54.4% ± 15%, 51.6% ± 25.9%, and 51.7% ± 28% current inhibition, respectively, at a concentration of 500 µM. Taken together, these data show that several agonists of β-ARs can inhibit to similar levels the activity of the TRPV4 channel. As mentioned before, all the above compounds are considered SABDs, and their chemical structures reveal that they possess certain features such as an aromatic ring, where hydroxymethyl and hydroxyl groups are present in distinct positions (Swaminath et al, 2005). Clenbuterol is a long-acting β-AR agonist, and structurally, it varies from salbutamol in that it does not possess the hydrox- ymethyl group and hydroxyl group at the third and fourth positions of the benzene ring, respectively, but contains chlorine atoms at the third and fifth positions and an amine group at the fourth position of the benzene ring. To determine whether clenbuterol also inhibits TRPV4, we performed electrophysiological experiments where we first activated TRPV4 with GSK (300 nM, black traces), then applied clenbuterol (500 µM, blue traces) for 5 min, and, finally, we mea- sured the inhibited current by reapplying GSK. The results in Fig 4A, B, and E show that clenbuterol (blue traces in Fig 4B) produced a more pronounced inhibition of TRPV4, as compared to salbutamol (purple traces in Fig 4A), because current inhibition after treatment was of 52.12% ± 16.3% for salbutamol versus 71.7% ± 11.2% for Figure 2. Dose-dependent inhibition of TRPV4 channels by salbutamol. (A) Dose–response for inhibition by salbutamol (5 min) of currents activated by GSK 300 nM (+120 mv). Smooth curve is a fit with the Hill equation (KD = 81.3 µM and Hill coefficient = 1.4). A single salbutamol concentration in the absence of GSK was tested per outside-out membrane patch, and the remaining GSK-activated current was normalized to the current obtained with GSK initially (n = 3–12 for each concentration point). (B) Time course of inhibition by 500 µM salbutamol, τ = 225.6 s (n = 8–10 for each time point). Data were obtained at −60 mV and fit to a single exponential. (C) Recovery from inhibition by salbutamol (500 µM). Current inhibition is irreversible, remaining fraction of currents are shown, and the zero time point represents currents in the presence of GSK101 right after application of salbutamol 500 µM for 5 min. Source data are available for this figure. Figure 3. Effects of short-acting bronchodilator on TRPV4 currents. (A, B, C, D, E) Representative traces of currents at +120 and −120 mV, obtained as in Fig 1A for different compounds. Trace colors represent gray for leak or initial currents, black for GSK 300 nM, and different colors for GSK after exposure of outside-out membrane patches to 500 µM of salbutamol, levalbuterol, terbutaline, isoprenaline, or metaproterenol. (F) Average data for experiments in (A, B, C, D, E). Data were normalized to the initial value with GSK. The percentages of inhibited currents after different treatments are as follows: 52.12% ± 16.3% after salbutamol (n = 15), 42.6% ± 19.4% after levalbuterol (n = 13), 54.4% ± 15% after terbutaline (n = 7), 51.6% ± 26% after isoprenaline (n = 5), and 51.7% ± 28% after metaproterenol treatment (n = 6). No statistically significant differences were found with one-way analysis of variance. Source data are available for this figure. Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 4 of 15 clenbuterol (Fig 4E). These data indicate that, like what happens in β-ARs, differences in the chemical structures of agonists of these receptors also yield differences in their effects on TRPV4 activity. Antagonism of β-adrenoreceptors also results in TRPV4 inhibition To gain insight into the mechanism by which bronchodilators can inhibit TRPV4’s activity, we tested an antagonist of β2-ARs, ICI- 118,551 (ICI), which has been described as a potent and selective inhibitor of β2-ARs (Bilski et al, 1983) and for which, to the best of our knowledge, no therapeutic use in humans has been yet assigned. Using our heterologous TRPV4 expression system of HEK293 cells, which endogenously expresses β1- and β2-ARs (Fig 4D), we performed electrophysiology experiments where we tested the hypothesis of whether TRPV4 inhibition was due to the activation of these receptors. In this scenario, we would expect the antagonist not to produce inhibition of the TRPV4 ion channel in response to GSK, as we observed with agonists of β-ARs. Notably, as shown in Fig 4C (pink traces) and Fig 4E, we found that ICI produced even more inhibition of TRPV4’s activation in response to GSK (black traces) than that of salbutamol because 78.3% ± 18% of the current was inhibited after ICI versus 52.12% ± 16.3% with salbutamol. Because ICI was effective in inhibiting TRPV4 activation by GSK, we further characterized its effects on TRPV4 by performing ex- periments to evaluate inhibition when ICI was applied to open channels (in the presence of GSK) or when it was applied to closed channels (in the absence of GSK). ICI inhibited TRPV4 (pink traces) very similarly whether it was applied in the presence or absence of the agonist, because 81.7% ± 17.6% and 77.3% ± 18.5% of the current were inhibited, respectively (Fig 5A and B). Moreover, as evidenced from the dose–response in Fig 5C, ICI produced more inhibition of the channel, as compared to salbutamol, with a KD of inhibition of the channel of 3.9 µM. Together, these data suggest that down-regulation of TRPV4 activity by agonists of β-ARs is independent of these receptors. To further substantiate this conclusion, we performed experiments where we incubated TRPV4-expressing HEK293 cells for 24 h with 11 µM ICI and measured GSK-induced TRPV4 currents from excised HEK293 cell membrane patches in the presence of a concentration near the KD of salbutamol (100 µM), which is expected to inhibit around 25% of the total current because themaximal concentration of 500 µM salbutamol inhibits 50% of current. Fig 5D and E shows that antagonism of β2-ARs did not affect inhibition of TRPV4 by salbutamol (23.6% ± 17.3% with salbutamol versus 23.4% ± 18.96% with ICI preincubation). These results are in accordance with what we would expect if salbutamol was acting directly on TRPV4 and independently of β2-AR–associated signaling pathways. These results prompted us to assess whether, in fact, salbutamol and the other tested compounds could be directly interacting with TRPV4. Salbutamol stabilizes the closed state of TRPV4 We addressed the mechanism of salbutamol inhibition of TRPV4 channels using outside-out membrane patches. Single-channel recordings showed that upon application of 300 nM TRPV4 agonist GSK, the channel stays open (O) (Fig 6A and B, blue traces and symbols). However, co-application of salbutamol and GSK resulted in a decrease in the open probability with the channel transitioning to the closed state (C) more frequently (Fig 6A and B, purple traces and symbols). Single-channel recordings also confirmed that salbutamol did not alter the single-channel current, as the amplitude remains similar before (Fig 6C) and after application of salbutamol (Fig 6D). All the above observations are consistent with the interpretation that Figure 4. Agonists and antagonists of β-adrenergic receptors. (A, B, C) Representative traces of currents at +120 and −120 mV, obtained as in Fig 1A for different compounds. Trace colors represent gray for leak or initial currents, black for GSK 300 nM, and different colors for GSK after exposure of outside-out membrane patches to 500 µM of salbutamol, clenbuterol, or ICI. (D) Total and membrane proteins were analyzed by Western blot for immunodetection of β1- and β2-adrenergic receptors; E-cadherin was used as a positive control of plasma membrane protein. (E) Average data for experiments in (A, B, C). Data were normalized to the initial value with GSK. The percentages of inhibited currents after different treatments are as follows: 52.12% ± 16.3% after salbutamol (n = 15), 71.7% ± 11.2% after clenbuterol (n = 7), and 78.3%± 18%after ICI (n = 13). *P = 0.0328 for salbutamol versus clenbuterol and *P = 0.0005 for salbutamol versus ICI, as indicated by brackets. One-way analysis of variance with Tukey’s post hoc test was performed. Source data are available for this figure. Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 5 of 15 salbutamol allosterically stabilizes TRPV4 channel in the closed state or produces a very slow open-channel block. Interaction sites of salbutamol with TRPV4 In β2-ARs, R-salbutamol binds in the catecholamine pocket, with its secondary and β-hydroxyl groups forming hydrogen bonds with D, N, and S residues and/or van der Waals contacts with F, W, T, S, and V residues (Katritch et al, 2009). Molecular docking simulations were performed to gain insight into the structural mechanism that governs the association between TRPV4 and salbutamol. Exploring the entire channel surface, we identified most of the salbutamol (R-isomer) conformations inter- acting with the extracellular region of TRPV4. The search conforma- tional space was then delimited to this area, finding four clusters, totalizing 1,000 TRPV4–salbutamol-docked conformations (Fig 7A). The lowest scoring (lowest energy) conformation of salbutamol in clusters 1, 2, 3, and 4 interacted with residues E514-S563-Y567, S667-V693-I696- I697, S687-S688-D682, and A489-P493-S634-N637, respectively (Fig 7B). Serine residues were demonstrated to be particularly relevant to coordinate the hydroxyl groups in the salbutamol molecule. To verify the functional relevance of TRPV4 residues that could potentially interact with salbutamol, based onwhat is known for β-ARs and thedataweobtained from the in silico experiments, we performed electrophysiological experiments to assess inhibition of TRPV4 by salbutamol in the following mutant channels: S557A and S563A in S3, E572A in the S3-S4 linker, S630A and S634A in S5, and S667A, D682A, S687A, S688A, and T689A in the pore, as compared to WT channels. As shown in Fig S2, TRPV4 mutant channels with single-residue substitutions: S557A, S563A, E572A, S630A, S634A, D682A, and T689A, produced proteins that responded similar toWT TRPV4 after addition of 500 µM salbutamol for 5 min and reactivation of currents with 300 nM GSK (different color traces). Hence, we concluded that these sites were not responsible for interactions of salbutamol with TRPV4. We also tested the mutation S688A, which was identified by our MD simulations as a possible candidate for interaction of salbutamol with TRPV4. However, this mutation gave rise to channels with en- hanced rundown that could not be recovered with GSK101 after 5 min in the presence of recording solution only (Fig S3); consequently, effects of salbutamol on this mutant could not be evaluated. However, substitution of residues S667A and S687A, located extracellularly at the entrance of the pore, produced ion channels that were less inhibited by salbutamol (Fig 8A and B), as compared to the WT TRPV4 channels. The TRPV4-S667A mutant channels exhibited 43.2% ± 12.9% inhibition (squares, Fig 8B) of currents activated with GSK after treatment with salbutamol, whereas TRPV4-S687A channels exhibited a 31% ± 14.5% inhibition of cur- rents activated with 300 nM GSK (triangles, Fig 8B) after salbutamol, as compared to WT channels (60.1% ± 19.6%; circles, Fig 8B). Finally, mutant channels containingmutations of S667A and S687A together were tested for inhibition with salbutamol and the results show that the double mutant only exhibits 10.9% ± 13.4% inhibition (bow ties, Fig 8B). WT TRPV4 channels and S667A, S687A, and S667A-S687A mutants all activated in a dose-dependent fashion in response to GSK (Fig 8C). These data indicate that serine residues at positions 667 and 687 are important for salbutamol interaction with TRPV4. Discussion The activity of the TRPV4 ion channel has been widely linked to several physiological and pathophysiological processes. This ion Figure 5. Salbutamol inhibits TRPV4 independently of β-adrenergic receptors. (A) Representative traces of currents at +120 and −120 mV. Gray traces are leak or initial currents, and black traces are with GSK 300 nM before treatment with ICI and after ICI (500 µM) applied in the presence of GSK (open state, pink traces) and absence of GSK (closed state, pink traces). (B) Average data for experiments in (A). Data were normalized to the initial value with GSK. The percentages of inhibited currents after different treatments are as follows: 81.7% ± 17.6% after ICI + GSK (n = 8), and 77.3% ± 18.5% after ICI only (n = 12). (C) Dose–response for inhibition by ICI (5 min) of currents activated by GSK 300 nM (+120 mv). Smooth curve is a fit with the Hill equation (KD = 3.9 µM and Hill coefficient = 0.36). A single ICI concentration was tested per outside-out membrane patch, and the remaining GSK-activated current was normalized to the current obtained with GSK initially (n = 6–13 for each concentration point). (D) Representative traces of experiments where patches were exposed to salbutamol or salbutamol in cells previously incubated with ICI for 24 h. Both compounds were used at concentrations near their KD values. (E) Average data for experiments in (D). Data were normalized to the initial value with GSK. The percentages of inhibited currents after different treatments are as follows: 23.6% ± 17.3% after salbutamol (n = 14) and 23.4% ± 18.96% after preincubation with ICI (n = 9). (B, E) Data were not statistically significant with an unpaired t test for data in (B, E). Source data are available for this figure. Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 6 of 15 channel is expressed in various organs and contributes to their function (Nilius & Owsianik, 2010; White et al, 2016; Rosenbaum et al, 2020). Specifically, of interest to the present study is that over- expression or increased activity of TRPV4 leads to changes in the alveolo-capillary barrier (Gombedza et al, 2017). Moreover, it has been suggested that TRPV4 plays a role in the remodeling and obstruction of airways through influencing the proliferation of cells in the lungs (Zhao et al, 2014) and, hence, contributing to the presence of chronic asthmatic conditions. Activation of TRPV4 has been shown to result in increased entrance of Ca2+, leading to the proliferation of smooth muscle cells and influencing chronic asthmatic conditions (Zhao et al, 2014). In addition, inhibition of the channel with GSK3491943 and GSK3527497 can mitigate TRPV4- induced pulmonary edema (Cheung et al, 2017; Brnardic et al, 2018). Thus, it stems that inhibition of this channel in the air- ways (Balakrishna et al, 2014), as has also been proposed for patients with SARS-CoV-2, which are subject to lung barrier damage because of mechanical overstimulation by respirators, may be beneficial under certain scenarios (Kuebler et al, 2020). Inhibitors of TRPV4’s activity have been described and tested in animals. Also, assays in humans have determined their safe use (Mizuno et al, 2003; Everaerts et al, 2010; Vincent & Duncton, 2011; Thorneloe et al, 2012; Kanju et al, 2016; Yin et al, 2016; Cheung et al, 2017; Pero et al, 2018; Brooks et al, 2019; Goyal et al, 2019; Achanta & Jordt, 2020; Kuebler et al, 2020; Yang et al, 2022). Here, we report for the first time that commonly used compounds to treat broncho- spasms inhibit TRPV4 currents. Although it is established that these compounds interact with β-ARs (Nelson, 1995), our data show that TRPV4 channels respond to salbutamol and other related chem- icals, albeit at higher concentrations than those to which β-ARs are sensitive. Salbutamol is a chiral compound usually found as a 50:50 ra- cemic mixture of two enantiomers (stereoisomers), which are molecules with non-superimposable mirror images. It has one chiral center in the ethanolamine tail, resulting in the two enan- tiomers (S)- and (R)- for the hydroxyl-group position. Our experi- ments using the racemic mixture of salbutamol show partial inhibition of TRPV4 (Figs 1 and 2) and the R-salbutamol enantiomer (levalbuterol; Fig 3) produces a similar inhibitory effect to that observed with the racemic mixture. We do not know whether the S- enantiomer inhibits TRPV4, but it is noteworthy that S-salbutamol alone is not clinically used because it has been reported either to not have any kind of effect on asthmatic patients or to produce bronchoconstriction (Templeton et al, 1998; Gumbhir-Shah et al, 1999). It is also important to mention that most of the experiments in this study were performed in the absence of Ca2+ because, as stated above, this ion can produce inhibition of TRPV4. In this sense, it is hard to hypothesize how the compounds tested here would affect the function of TRPV4 under physiological conditions because several scenarios are viable with the channel possibly exhibiting smaller currents because of the presence of Ca2+, but also being subject to potentiation of its activity by changes in its phosphorylation state (Wegierski et al, 2009), changes in PIP2 concentration (Garcia-Elias et al, 2013; Harraz et al, 2018), and other changes in the cellular environment. One previous study suggested that clenbuterol but not salbu- tamol inhibits skeletal muscle sodium (hSkM1) channels and that such inhibition is independent of β-ARs (Desaphy et al, 2003). To our knowledge, no other ion channel has been shown to be directly modulated by agonists of β-ARs, but an antagonist (ICI) of these receptors that is not for human use has been shown to inhibit the human ether-a-go-go-related gene potassium ion channels, through a mechanism that seems to involve pore block (Dupuis et al, 2005). Our data show that TRPV4, but not the related TRPV1 channel, is partially inhibited by salbutamol in a dose- and time-dependent fashion, with some preference for the extracellular region of the channel. We demonstrate that other SABDs, which display a higher affinity for β2-ARs (Barisione et al, 2010), such as levalbuterol, terbutaline, isoprenaline, and metaproterenol, and a long-acting β-AR agonist, clenbuterol, are inhibitors of TRPV4. This suggests that chemical structural features shared by these compounds allow for TRPV4 inhibition. Importantly, antagonism of β2-ARs with ICI Figure 6. Salbutamol produces a decrease in the open probability of TRPV4. (A) Representative single-channel recordings in outside-out membrane patches show that in the presence of GSK (300 nM), TRPV4 is mostly in the open (O) state (blue traces). When salbutamol (500 µM) is added in the presence of 300 nM GSK, the channel starts to close (C), leading to a decrease in the open probability (Po, purple traces). (B) Po of TRPV4 calculated for the representative trace in (A). Blue symbols correspond to the Po calculated for each sweep (+60mV) in the presence of only GSK, and the purple symbols are for sweeps in the co- application of GSK + salbutamol. (C, D) Co-application of salbutamol decreases Po without significantly changing the unitary channel current, as shown for GSK alone ((C), blue) and GSK + salbutamol ((D), purple). Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 7 of 15 does not preclude inhibition of TRPV4 by salbutamol, supporting a direct action of this compound on TRPV4. In fact, inhibition of TRPV4 by ICI is more efficient, as compared to salbutamol, and TRPV4 exhibits inhibition of the channel at lower concentrations than those shown to affect human ether-a-go-go-related gene channels (Dupuis et al, 2005). These observations led us to screen possible interaction sites for salbutamol with TRPV4. Computational modeling and Figure 7. Computational modeling of TRPV4–salbutamol association. (A) Binding conformations of salbutamol (R-isomer), derived from docking simulations, into the human TRPV4 channel (gray cartoon representation). The salbutamol molecule is represented as sticks colored by atom types—C in green, O in red, N in blue, and H in white. Four clusters of docked conformations were obtained. (B) Lowest score docking pose in each cluster. TRPV4 residues at 5 Å of the salbutamol molecule are shown as sticks (C in gray). Figure 8. Two residues in the pore region mediate TRPV4 inhibition by salbutamol. (A) Representative traces of currents at +120 and −120 mV, obtained as in Fig 1A. Trace colors represent gray for leak or initial currents, black for GSK 1 µM, and purple for GSK after exposure of outside-out membrane patches to 500 µM of salbutamol for WT and mutant TRPV4 channels. (B) After salbutamol treatment, WT TRPV4 (n = 43) channels displayed a 60.1% ± 19.6% inhibition of currents, whereas 43.2% ± 12.9%, 31% ± 14.5%, and 10.9% ± 13.4% inhibition was observed for the S667A (n = 12), S687A (n = 14), and S667A-S687A (n = 12) mutants, respectively. Mutated residues are displayed as spheres in each subunit in a side and top view. Only the transmembrane region of the human TRPV4 ion channel (PDB code: 7AA5) is exhibited. *P = 0.0178 for WT TRPV4 versus TRPV4-S667A; *P < 0.0001 for WT TRPV4 versus TRPV4-S687A; *P < 0.0001 for WT TRPV4 versus TRPV4-S667A-S687A; *P < 0.0001 for TRPV4-S667A versus TRPV4-S667A-S687A; and *P < 0.02 for TRPV4-S687A versus TRPV4-S667A-S687A, as indicated by brackets. One-way analysis of variance with Tukey’s post hoc test was performed. (C) Dose–response for activation of WT, S667A, and S687A TRPV4 channels by GSK (+120 mv). Smooth curve is a fit with the Hill equation. The fits yield KD = 101 nM and Hill coefficient = 2.5 for WT channels, KD = 182 nM and Hill coefficient = 1.5 for the S667A mutant channels, KD = 108 nM and Hill coefficient = 2.4 for S687Amutant channels, and KD = 91 nM and Hill coefficient = 2.9 for S667A-S687A mutant channels. A GSK concentration was tested per outside-out membrane patch (n = 4–11 for each concentration point). Source data are available for this figure. Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 8 of 15 electrophysiological experiments revealed the importance of residues S667 and S687 for inhibition of TRPV4 by salbutamol. Moreover, the mutation S688A renders the channel more sensitive to rundown, as demonstrated by our data where GSK-activated currents could not be recovered after 5 min in the presence of recording solution only (Fig S3). Further studies will be required to determine whether this residue undergoes posttranslational modification that is important for channel function, which is eliminated by the inserted point mutation to alanine, or whether the mutation just renders the channel energetically unstable. We also identified that residues in the S3, S3-S4 linker, S5, and pore could mediate salbutamol’s binding to TRPV4. By testing in- hibition of S557A, E572A, S630A, S563A, S634A, and D682A by sal- butamol, no significant differences among inhibition of these mutants and the WT TRPV4 channels were found. It is noteworthy that molecular mechanisms underlying the inhibition of TRPV4 by its antagonists have only been described for some compounds. For example, it has been described that the antagonist HC067047 binds to residues in the S2–S3 linker (D542), in S4 (M583 and Y587), and in S5 (D609 and F613) (Doñate-Macian et al, 2022). By further testing sites identified through our molecular docking experiments, we found for salbutamol that mutation of residues S667 and S687 to alanine produces TRPV4 channels with a diminished response to this compound. These residues are located near the mouth of the pore of the channel; hence, the possibility of salbutamol acting as a pore blocker arises. One would expect a blocker of the pore to produce a decrease in the single-channel amplitude (or conductance). Nonetheless, our single-channel ex- periments show that the single-channel amplitude is not de- creased by exposure of TRPV4 channels to salbutamol, but that the channels transition to the closed states in the presence of the bronchodilator, which is consistent with an allosteric mechanism rather than that of pore block. 80 µM of salbutamol is equivalent to 19 µg/ml (salbutamol molecular weight = 239.311 g/mol). Several studies have reported plasma levels of salbutamol after its intake (inhaled or oral) in the treatment of acute asthma that range between 1.75 and 18.77 ng/ml in adults (Elers et al, 2010) and between 2.77 and 18.22 ng/ml in infants (Rotta et al, 2010), but in lung edema fluid from patients treated with salbutamol, the mean concentration reaches 700 ng/ ml (Atabai et al, 2002). Moreover, levels of salbutamol in the urine during asthma treatment were shown to increase up to 2,422.2 ng/ ml (Elers et al, 2010). Thus, the micromolar concentrations that we found inhibit TRPV4 suggest that some tissues receive doses of salbutamol at which TRPV4 is inhibited. In summary, here we show that TRPV4, a relevant ion channel for airway function, is partially inhibited by several widely used bronchodilators, which have been classically associated with their actions through β-ARs, and that, for inhibition of TRPV4 by sal- butamol, the mechanism involves allosteric modulation of the ion channel through interactions by, at least, two residues located in the outer pore region of the protein. The present study provides insight into the molecular mechanisms that down-regulate TRPV4 activity, a physiologically important ion channel. β-Adrenergic medicines such as salbutamol have been contemplated and studied for treatment of spinal muscular atrophy (SMA), with en- couraging results, albeit the mechanism by which they function remains unknown. Future studies will be required to assess whether salbutamol could be of importance in the treatment of disease in which TRPV4 gain-of-function mutations can be present, such as SMA, Charcot–Marie–Tooth disease, and other pathologies. For example, it has been reported that in children with SMA, which can present defects in respiratory muscle strength, treatment with oral salbutamol benefits respiratory function (Kinali et al, 2002; Pane et al, 2008; Khirani et al, 2017). Materials and Methods Cell cultures HEK293 cells (ATCC CRL-1573) were cultured in a complete growth medium containing Dulbecco’s modified Eagle’s medium with high glucose (DMEM; Gibco) complemented with 10% fetal bovine serum (HyClone) and 100 U/ml of penicillin–streptomycin (Gibco). Cell cultures were maintained in a humidified incubator at 37°C with an atmosphere of 95% of air and 5% of CO2. Cells were subcultured every 3 d using 0.25% (w/V) trypsin–EDTA solution (Gibco). Western blot Total andmembrane protein extracts of HEK293 cells were obtained using the Pierce Cell Surface Protein Isolation Kit (89881; Thermo Fisher Scientific). Then, 40 µg of protein (total and membrane) was separated by electrophoresis on a 7% SDS–polyacrylamide gel and transferred to a PVDF membrane. Blots were preincubated with 5% low-fat milk in TBS–0.1% Tween for 1 h at room temperature and incubated overnight at 4°C with β1- AR (ab3442) (Sun et al, 2021) or β2-AR (ab182136) (Cellini et al, 2021) antibodies from Abcam or E-cadherin antibody (3195) (Ye et al, 2022) from Cell Signaling Technology (as a positive control for membrane proteins) diluted 1:1,000 for each case. After washing of the primary antibodies, binding was visualized using a secondary horseradish peroxidase (Thermo Fisher Scientific)–labeled anti-rabbit antibody (for adrenergic receptors; 1:10,000 dilution) and anti-mouse anti- body (for E-cadherin; 1:10,000 dilution) and enhanced with dia- minobenzidine at 100 µg/ml in TBS with 30% H2O2. Transient cell transfection and patch-clamp experiments For patch-clamp experiments, the cells were grown on coverslips and transfected with the pEGFP-N3 plasmid together with either the human WT or mutant TRPV4 channels for identification of suc- cessfully transfected cells. The jetPEI Polyplus-transfection reagent was used to transfect HEK293 with human TRPV4 channels (500 ng of plasmid for excised patch-clamp or 20 ng for single-channel experiments), per the manufacturer’s instructions as previously described (Chen et al, 2021). TRPV4 currents from transiently transfected HEK293 cells were recorded using the patch-clamp technique in the inside-out and outside-out configurations, as indicated for different experiments (Hamill et al, 1981). Solutions were changed with an RSC-200 rapid solution changer (Molecular Kinetics). The recording solutions Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 9 of 15 contained the following: 130 mM NaCl, 3 mM Hepes (pH 7.2), and 1 mM EDTA for the bath and pipette, and 130 mM NaCl, 3 mM Hepes, and 2 mM CaCl2+ (pH 7.2) when experiments were performed in the presence of Ca2+, as indicated. Because the channel is inhibited by Ca2+, all experiments (except those shown in Fig 1E) were obtained in the absence of Ca2+ to avoid a current decrease (Voets et al, 2002). GSK1016790A (GSK; Sigma-Aldrich) was prepared in DMSO to a 15.25 mM concentration for the stock, which was kept at −20°C. Salbutamol or albuterol (α1-[(tert-butylamino)methyl]-4- hydroxy-1,3-benzenedimethanol hemisulfate salt), terbutaline (5-[2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-1,3-benzenediol hemisulfate salt), levalbuterol ([R]-salbutamol hydrochloride), isoprenaline (1-[39,49-dihydroxyphenyl]-2-isopropylaminoethanol hydrochloride), metaproterenol (α-[(isopropylamino)methyl]-3,5- dihydroxybenzyl alcohol hemisulfate salt), clenbuterol (4-amino- α-[t-butylaminomethyl]-3,5-dichlorobenzyl alcohol hydrochloride), and ICI 118,551 ([2R,3R]-rel-3-isopropylamino-1-[7-methylindan-4- yloxy]-butan-2-ol hydrochloride) were all purchased from Sigma- Aldrich, and stock solutions were prepared in water and then diluted in recording solutions immediately before use in experi- ments. pHwasmeasured after addition of compounds to ensure that it was not modified in the recording solutions to which membrane patches containing TRPV4 channels were exposed. Experiments were performed at room temperature (24°C). Mean current values were measured after channel activation had reached the steady state (~2 min). Currents were obtained using voltage protocols, where the holding potential was 0 and 10 mV steps from −120 to 120 mV for 100 ms, as indicated for each ex- periment in the figure legends. Borosilicate glass was used for pipette fabrication (5 MΩ). Currents were low-pass-filtered at 2 kHz and sampled at 10 kHz with an EPC 10 amplifier (HEKA Elektronik) and were plotted and analyzed with IGOR Pro (WaveMetrics, Inc.). Initial or leak currents were obtained in the absence of agonist, at a given voltage, and GSK was added for 90 s to activate TRPV4 channels. Then, GSK was washed off the membrane patches and salbutamol (or other chemicals, as indicated) was applied for 5 min either in the presence or in the absence of GSK. Finally, currents were remeasured after 90 s of GSK, to assess inhibition of currents. Dose–response curves for the inhibition of 300 nM GSK-induced TRPV4 currents were performed by applying a given concentration of salbutamol or ICI in the absence of GSK to outside-out excised membrane patches of HEK293 cells expressing hTRPV4 channels. A single concentration of the compoundswas tested in eachmembrane patch, and the data of several patches at a given concentration were pooled. All currents weremeasured at a voltage of +120mV. Data were normalized to the currents initially obtained in the presence of only 1 µM GSK. The Hill equation (Equation (1)) was fitted to the data as previously described (Morales-Lázaro et al, 2016) to estimate the steepness of the curve, n, and the apparent dissociation constant, KD. I Imax = 0 B B @ 1 1 + ½X ½KD 1 C C A n (1) Time courses of inhibition by 500 µM salbutamol were obtained using continuous pulses at −60mV. Each time point was obtained by averaging several patches, first exposed to 300 nM GSK, washed, and then exposed to salbutamol in the absence of GSK, and then, inhibition of currents was measured by exposing the patches to 300 nM GSK again. Results are expressed as the fraction of inhibited currents, which was obtained by dividing the currents obtained at −60 mV after treatment with salbutamol by the initial currents obtained in the presence of GSK. The decay in the current was fitted to a single exponential to obtain the time course of inhibition (τ). Recovery from inhibition was determined by activating TRPV4 channels with GSK101 (300 nM), then adding salbutamol (500 µM) for 5 min, and then exposing the patches to GSK101 again. Data were obtained by averaging the results of several patches for each time point. For single-channel recordings, borosilicate glass (30 MΩ) pi- pettes were used. Recordings were obtained at +60 mV by acquiring several traces of 1- to 3-s duration. The effect of salbutamol on single TRPV4 channels activated by GSK was studied in the outside- out configuration. Currents were low-pass-filtered at 3 kHz and sampled at 50 kHz. Patches containing only one channel activated by different compounds were identified as those that did not contain overlapping opening events. The single-channel current (i) in each condition was determined by building all point histograms from traces with clear closings and openings. The resulting histograms were fitted to a Gaussian function, where the peak corresponded to single-channel openings and closures were identified with the half-threshold crossing technique, to compile a list of durations of all open events in a single sweep (Islas, 2015). Channel open probability (Po) was cal- culated as the sum of the total open time divided by the sweep duration. Recordings were performed in the absence of Ca2+ to avoid block/desensitization of the channel. Mutagenesis Mutations in the human TRPV4 channels were constructed by a two- step PCRmethod, as previously described (Salazar et al, 2008, 2009). Mutations in various regions of the TRPV4 channels were con- structed using a method involving oligonucleotides synthesized to contain a mutation in combination with WT oligonucleotides in PCR amplifications of fragments of the complementary DNA. The product of the PCR was then cut with two different restriction enzymes to generate a cassette containing the mutation. The cassette was ligated into the channel complementary DNA cut with the same two restriction enzymes. The entire region of the amplified cassette was sequenced to confirm the mutation and ensure against second-site mutations. Computational modeling To evaluate the interaction of salbutamol with the TRPV4 channel, molecular docking simulations were carried out. The structure of the human channel was built using Xenopus tropicalis TRPV4 as a template (≈85% of sequence identity). A total of 2,000 hTRPV4 models were built with the software Modeller 9.25 (Eswar et al, 2007). The best model was selected as that with the lowest Molpdf energy value of Modeller and the highest Procheck score (Laskowski et al, 1993). TRPV4 and salbutamol molecules were Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 10 of 15 prepared for docking with the software AutoDockTools 1.5.7 (Morris et al, 2009), assigning Gasteiger partial charges. Salbutamol is formulated as a racemic mixture of the R- and S-isomers (com- pound CID in PubChem is 2083). The R-isomer of salbutamol was used in this study because it has at least 100 times greater affinity for the ß2-receptor (Penn et al, 1996; Ameredes & Calhoun, 2009) than the S-isomer and the S-isomer has been associated with toxicity (Mitra et al, 1998; Volcheck et al, 2005). Search space for docking was initially demarcated through a grid box large enough to cover the entire surface of the channel. The grid parameters were generated with AutoGrid4.2.6 (Morris et al, 1998). The Lamarckian genetic algorithm of AutoDock4.2.6 (Morris et al, 1998, 2009) was used to model the TRPV4–salbutamol binding conformations. Docking refinement was performed, gradually reducing the con- formational searching area. A total of 1,000 TRPV4 channel– salbutamol conformations were generated, which were clustered and analyzed with VMD 1.9.3 software (Humphrey et al, 1996). Statistical analysis Group data are reported as the mean ± SD. The two-tailed t test and one-way analysis of variance, followed by Tukey’s post hoc test, were used for group comparison and calculated with Prism soft- ware (Dotmatics). Significant differences between means were considered to exist when the P-value was less than 0.01 or 0.05, as indicated. Supplementary Information Supplementary Information is available at https://doi.org/10.26508/lsa. 202201704 Acknowledgements We thank the following personnel from Instituto de Fisiologı́a Celular, UNAM: Ana Marı́a Escalante Gonzalbo, Francisco Pérez Eugenio, Juan Manuel Bar- bosa Castillo, and Gerardo Coello Coutiño for technical assistance with computer software and hardware; Laura Kawasaki for help with technical assistance with experiments; and Ana E López-Romero for preliminary control experiments. We thank Drs. Sidney A Simon and Roberto Coria for helpful discussion of our article. M Benı́tez-Angeles is a doctoral student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and received a fellowship from the Consejo Nacional de Humanidades, Ciencias y Tecnologı́as (CONAHCyT; 1002182), and E Juárez-González received a fellowship 902482 from CON- AHCyT. This work is in fulfillment of the requirements for a doctoral degree of the Programa de Doctorado en Ciencias Biomédicas for M. Benı́tez-Angeles at the Universidad Nacional Autónoma de México. This research was funded by the Dirección General de Asuntos del Personal Académico (DGAPA)- Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológ- ica (PAPIIT) (grant number IN200720 to T Rosenbaum and grant number IN215621 to LD Islas), and CONACyT (grant number A1-S-8760) and Secretarı́a de Educación, Ciencia, Tecnologı́a e Innovación del Gobierno de la Ciudad de México (grant number SECTEI/208/2019) to T Rosenbaum. A Vergara-Jaque was funded by Fondo Nacional de Desarrollo Cientı́fico y Tecnológico (FONDECYT; grant number 1220110). The Millennium Nucleus of Ion Channel- Associated Diseases is a Millennium Nucleus supported by the National Agency of Research and Development (ANID), Chile. Author Contributions M Benı́tez-Angeles: conceptualization, data curation, formal anal- ysis, supervision, validation, investigation, methodology, and wri- ting—original draft, review, and editing. E Juárez-González: conceptualization, data curation, formal anal- ysis, validation, investigation, methodology, and writing—original draft, review, and editing. A Vergara-Jaque: conceptualization, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, and writing—original draft, review, and editing. I Llorente: conceptualization, data curation, formal analysis, vali- dation, methodology, and writing—original draft, review, and editing. G Rangel-Yescas: conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, and wri- ting—original draft, review, and editing. SC Thébault: conceptualization, supervision, validation, investiga- tion, methodology, and writing—original draft, review, and editing. M Hiriart: conceptualization, supervision, validation, investigation, visualization, and writing—original draft, review, and editing. LD Islas: data curation, formal analysis, supervision, funding ac- quisition, validation, investigation, visualization, methodology, and writing—original draft, review, and editing. T Rosenbaum: conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, project administration, and writing—original draft, review, and editing. Conflict of Interest Statement The authors declare that they have no conflict of interest. References Achanta S, Jordt S (2020) Transient receptor potential channels in pulmonary chemical injuries and as countermeasure targets. 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Am J Respir Cell Mol Biol 50: 1064–1075. doi:10.1165/rcmb.2013-0416OC License: This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/ licenses/by/4.0/). Beta-adrenergic ligands inhibit TRPV4 Benı́tez-Angeles et al. https://doi.org/10.26508/lsa.202201704 vol 6 | no 3 | e202201704 15 of 15 J Physiol 601.9 (2023) pp 1655–1673 1655 T h e Jo u rn a l o f P h y si o lo g y Modes of action of lysophospholipids as endogenous activators of the TRPV4 ion channel Miguel Benítez-Angeles1, Ana E. López Romero1, Itzel Llorente1, Ileana Hernández-Araiza1, Ariela Vergara-Jaque2,3, Fernando H. Real4, Óscar Eduardo Gutiérrez Castañeda5 , Marcelino Arciniega5 , Luis E. Morales-Buenrostro6, Francisco Torres-Quiroz5, Refugio García-Villegas7 , Luis B. Tovar-y-Romo4, Wolfgang B. Liedtke8,9, León D. Islas10 and Tamara Rosenbaum1 1Departamento de Neurociencia Cognitiva, Instituto de Fisiología Celular, Universidad Nacional Autónoma deMéxico (UNAM), Mexico City, Mexico 2Center for Bioinformatics, Simulation and Modelling (CBSM), Faculty of Engineering, Universidad de Talca, Talca, Chile 3Millennium Nucleus of Ion Channel-Associated Diseases (MiNICAD), Santiago, Chile 4Departamento de Neuropatología Molecular, Instituto de Fisiología Celular, UNAM, Mexico City, Mexico 5Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, UNAM, Mexico City, Mexico 6Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico 7Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico 8Department of Neurology, Duke University, Durham, North Carolina, USA 9Department of Molecular Pathobiology, New York University College of Dentistry, New York, New York, USA 10Departamento de Fisiología, Facultad de Medicina, UNAM, Mexico City, Mexico Handling Editors: Peying Fong & Helle Praetorius The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP284262#support-information-section). Abstract The Transient Receptor Potential Vanilloid 4 (TRPV4) channel has been shown to function in many physiological and pathophysiological processes. Despite abundant information on its importance in physiology, very few endogenous agonists for this channel have been described, and very few underlying mechanisms for its activation have been clarified. TRPV4 is expressed by several types of cells, such as vascular endothelial, and skin and lung epithelial cells, where it plays pivotal roles in their function. In the present study, we show that TRPV4 is activated by lysophosphatidic acid (LPA) in both endogenous and heterologous expression systems, © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. DOI: 10.1113/JP284262 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 1656 M. Benítez-Angeles and others J Physiol 601.9 pinpointing this molecule as one of the few known endogenous agonists for TRPV4. Importantly, LPA is a bioactive glycerophospholipid, relevant in several physiological conditions, including inflammation and vascular function, where TRPV4 has also been found to be essential. Here we also provide mechanistic details of the activation of TRPV4 by LPA and another glycerophospholipid, lysophosphatidylcholine (LPC), and show that LPA directly interacts with both the N- and C-terminal regions of TRPV4 to activate this channel. Moreover, we show that LPC activates TRPV4 by producing an open state with a different single-channel conductance to that observed with LPA. Our data suggest that the activation of TRPV4 can be finely tuned in response to different end- ogenous lipids, highlighting this phenomenon as a regulator of cell and organismal physiology. (Received 13 December 2022; accepted after revision 5 January 2023; first published online 10 January 2023) Corresponding author Tamara Rosenbaum, Departamento de Neurociencia Cognitiva, Instituto de Fisiología Celular Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico. Email: trosenba@ifc.unam.mx Abstract figure legend TRPV4 is differentially activated by distinct ligands. Lysophosphatidic acid 18:1 (LPA18:1) activates TRPV4 with a larger conductance as compared to lysophosphatidylcholine 18:1 (LPC18:1). These different modes of activation can lead to differences in cell excitability. Key points  The Transient Receptor Potential Vaniloid (TRPV) 4 ion channel is a widely distributed protein with important roles in normal and disease physiology for which few endogenous ligands are known.  TRPV4 is activated by a bioactive lipid, lysophosphatidic acid (LPA) 18:1, in a dose-dependent manner, in both a primary and a heterologous expression system.  Activation of TRPV4 by LPA18:1 requires residues in the N- and C-termini of the ion channel.  Single-channel recordings show that TRPV4 is activated with a decreased current amplitude (conductance) in the presence of lysophosphatidylcholine (LPC) 18:1, while LPA18:1 andGSK101 activate the channel with a larger single-channel amplitude.  Distinct single-channel amplitudes produced by LPA18:1 and LPC18:1 could differentially modulate the responses of the cells expressing TRPV4 under different physiological conditions. Introduction The Transient Receptor Potential (TRP) family of ion channels comprises seven subfamilies in various organisms, ranging from yeasts to vertebrates. TRP channels are polymodal, non-selective cationic channels, and their importance is evidenced by their diverse roles in physiology, including sensory transduction, vascular function, barrier function and cation homeostasis (Nilius & Owsianik, 2011). 0 Miguel Benítez-Angeles is a biologist and currently a candidate for theDoctorate degree in Biomedical Sciences under the super- vision of Dr Tamara Rosenbaum within the Division for Neurosciences at the Institute for Cellular Physiology of the National Autonomous University of Mexico (UNAM). He is trained and has worked on several aspects of the physiology and biophysics of Transient Receptor Potential (TRP) ion channels and hopes to pursue a career in basic research directed towards understanding the molecular mechanisms underlying ion channel function. One of the most widely studied TRPs, for which a variety of agonists or modulators have been described, is TRPV1 (Benítez-Angeles et al., 2020; Caterina et al., 1997; Klein et al., 2008; Morales-Lázaro et al., 2014). Among these modulators, phospholipids have been shown to affect TRPV1’s activity (Klein et al., 2008; Rohacs, 2015). Previous work from our group showed that lysophosphatidic acid (LPA) 18:1 activates TRPV1 channels by binding to a residue located in the TRP box (Nieto-Posadas et al., 2011). However, © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1657 lysophosphatidylcholine (LPC), the metabolic pre- cursor of LPA, does not activate TRPV1 (Morales-Lázaro et al., 2014). Furthermore, compared to capsaicin, its canonical agonist, LPA18:1 elicits larger macroscopic and single-channel currents through TRPV1 by inducing a distinct conformational change (Morales-Lázaro et al., 2014; Nieto-Posadas et al., 2011). Unlike TRPV1, TRPV4 channels are much less understood in terms of their activation by endogenous agonists. TRPV4 channels are expressed prominently in mammalian epithelial tissues and vascular cells, such as endothelia, and are activated by hypoosmotic conditions and mechanical stress (Liedtke et al., 2000; Matthews et al., 2010; O’Conor et al., 2014; Servin-Vences et al., 2017, 2018), temperatures around 27°C (Güler et al., 2002; Shibasaki, 2016) and UVB radiation (Moore et al., 2013). Known exogenous ligands include chemicals found in plants such as phorbol derivatives (Watanabe et al., 2002), bis-andrographolide A from Andrographis paniculata (Smith et al., 2006), the flavonoid apigenin (Ma et al., 2012) and the synthetic molecule GSK1016790A (here referred to as GSK101) (Jin et al., 2011a). The effects of lipid molecules such as phosphatidylinositol-4,5-biphosphate (PIP2), which has been suggested to bind to the N-terminus, on TRPV4 channel modulation are the subject of an ongoing investigation (Garcia-Elias et al., 2015; Takahashi et al., 2014). Aproduct of arachidonic acidmetabolismvia P450, called 5,6-epoxyeicosatrienoic acid (5,6-EET), binds to the S2–S3 linker activating the channel (Berna-Erro et al., 2017; Watanabe et al., 2003), while polyunsaturated fatty acids affect TRPV4 activity by altering membrane fluidity (Caires et al., 2017). Due to the importance of TRPV4 ion channels in physiology and pathophysiology (Everaerts et al., 2010; Liedtke, 2005; Rosenbaum et al., 2020), here we assessed if the bioactive glycerophospholipid LPA18:1, for which circulating levels in serum can increase considerably in various forms of inflammation and other pathological conditions (Lin et al., 2010; Tigyi, 2001), could regulate the activity of TRPV4. The present study shows that LPA18:1 can activate TRPV4 ion channels expressed endogenously in brain microvascular endothelial cells (BMECs), and in the heterologous expression system of HEK293 cells. LPA18:1 increases the open probability of TRPV4 and interacts with residues in the protein’s N- and/or C-termini. We find that while GSK101 and LPA18:1 produce similar single-channel current amplitudes, LPC18:1 leads to a decrease in the single-channel current amplitude of TRPV4. This occurs possibly by promoting a distinct open state through an interaction with different residues in the TRPV4 channel. Regarding LPA18:1, we show that activation of TRPV4 occurs through a different mechanism to what we had previously described for TRPV1 (Canul-Sánchez et al., 2018). These results expand the notion that TRPV channels are proteins with finely tuned activity in their response to endogenous agonists. Methods Ethical approval This study used primary BMECs harvested from 6-week-old (270–290 g) wild-type Wistar rats. Animals were bred at the Animal Facility of IFC-UNAM certified by the Secretariat of Agriculture and Rural Development (SADER–Mexico) and housed in individual cages with food and water available ad libitum. All experimental procedures were conducted under the current Mexican law for the use and care of laboratory animals (NOM-062-ZOO-1999) with the Institutional Animal Care and Use Committee approval (CICUAL-IFC-LTR212-22). Cell cultures HEK293 cells (ATCC CRL-1573, Manassas, VA, USA) were cultured in a complete growth medium containing Dulbecco’s modified Eagle’s medium with high-glucose (DMEM; Gibco, Waltham, MA, USA) complemented with 10% fetal bovine serum (HyClone, Waltham, MA, USA) and 100 U/ml of penicillin-streptomycin (Gibco). Cell cultures were maintained in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2. Cells were sub-cultured every 3 days using 0.25% (w/v) Trypsin-EDTA solution (Gibco). BMECs were obtained from the brain cortex of adult Wistar male rats. Briefly, rats were administered 100 mg/kg sodium pentobarbital (Pisa, Guadalajara, Mexico) i.p. before being transcardially perfused with ice-cold Hanks’ balanced salt solution (HBSS 1×; Gibco). Afterwards, the brain cortices were collected and minced. The tissue was digested with 1 U/ml dispase, 2.5 U/ml papain and 250 U/ml DNAse I (all from Sigma-Aldrich, St Louis, MO, USA) at 37°C for 15 min. Then, the homogenate was centrifuged at 1000 × g for 3 min, and the pellet was resuspended in 15% dextran (>500 kDa; Sigma-Aldrich) and ultracentrifuged at 10 000 g for 15 min at 4°C. The pellet was further digested with 0.1% dispase and 0.1% papain for 1 h at 37°C with regular shaking. Then, cells were collected by centrifugation at 1000 g for 5 min and washed twice with HBSS. The cells were plated on culture dishes pre- viously coated with attachment factor (AF; Gibco) in M131 medium supplemented with microvascular growth supplement (MVSG; Gibco). Cell identity and culture purity above 95% were assessed by CD31 immuno- labeling (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1658 M. Benítez-Angeles and others J Physiol 601.9 Transient cell transfection and patch-clamp For patch-clamp experiments, the cells were grown on coverslips and cotransfected with the pcDNA3.1 plasmid (Invitrogen, Waltham, MA, USA) with either the human wild-type or mutant human TRPV4 channels and with the pIRES-hr-GFP (green fluorescent protein, Agilent Technologies, Santa Clara, CA, USA) to identify successfully transfected cells. The jetPEI Polyplus trans- fection reagent (Illkirch-Graffenstaden, France) was used to transfect HEK293 cells with human TRPV4 channels (500 ng TRPV4-containing plasmid for excised patch clamp or 5–20 ng for single-channel experiments and 400 ng of pIRES-hr-GFP), according to themanufacturer’s instructions as previously described (Chen et al., 2021). For control experiments, some cells were only trans- fected with the ‘empty’ pcDNA3.1 plasmid (not coding for TRPV4) and with the pIRES-hr-GFP plasmid. TRPV4 currents from transiently transfected HEK-293 cells were recorded using the patch-clamp technique in the inside-out and outside-out configurations, as indicated for different experiments (Hamill et al., 1981). Solutions were changed with an RSC-200 rapid solution changer (Molecular Kinetics Inc., Indianapolis, IN, USA). Currents were low-pass filtered at 2 kHz and sampled at 10 kHz with an EPC 10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) and were plotted and analysed with Igor Pro (Wavemetrics Inc., Portland, OR, USA). The recording solutions contained (in mM): 130 NaCl, 3 HEPES (pH 7.2) and 1 EDTA for the bath and 130 NaCl, 3 HEPES (pH 7.2) and 1 CaCl2 in the pipette only for representative traces and current–voltage relationship obtained in Fig. 1A and B. Since Ca2+ inhibits the channel, other experiments were obtained without Ca2+ to avoid current decrease (Voets et al., 2002). GSK1016790A (Sigma-Aldrich) was prepared in DMSO (Sigma-Aldrich) to a concentration of 15.25 mM for the stock, which was kept at−20°C and diluted to 1µM in the bath solution. LPA18:1, LPA18:0, LPC18:1, LPC18:0 and LPC16:0, all from Avanti Polar Lipids (Birmingham, AL, USA), were prepared in DMEM-BSA1%, as previously described (Nieto-Posadas et al., 2011) at 10 mM for stock solutions, kept at −70°C and diluted to 5 µM in the bath solution. A 10 mM BrP-LPA (Echelon Biosciences, Salt Lake City, UT, USA) stock was diluted in water and then in recording solution before use, as previously described (Nieto-Posadas et al., 2011). TRPV4 antagonist, RN1734 (Sigma-Aldrich), was prepared in DMSO as 15 mM stocks and diluted (20 or 30µM) in recording solution for experiments with BMECs andHEK293 cells. Experiments were performed at room temperature (24°C). Mean current values were measured after channel activation had reached the steady-state (∼3 min). Since this is an outwardly rectifying ion channel that displays low open probability at negative voltages, currents were obtained using voltage protocols where the holding potential was 0 mV and 10 mV steps from −120 to +120 mV for 100 ms to 0 mV, as indicated for each experiment in the figure legends. Pipettes were fabricated with borosilicate glass (Sutter Instrument Company, Novato, CA, USA) and had a resistance of 5 M. Dose responses to agonists were obtained by applying a given concentration to an inside-out membrane patch and normalizing by the value of the magnitude of the current obtained with the highest agonist concentration at +120 mV, a voltage at which currents are larger and can be more accurately measured. The curves were constructed by applying a single concentration of agonist to each membrane patch and then averaging several patches at this concentration. For comparison of activation by different lipids to activation with GSK1016790A, currents obtained in the presence of the different compounds were measured at +120 mV and normalized to the maximal current at the same voltage after activation with GSK1016790A. BMECs were recorded in the inside-out configuration using isoosmolar 130 mM NaCl, 10 mM HEPES and 1 mM EDTA (pH 7.2) solutions, using the same voltage protocol described above for HEK293 cells. The insertion of different residues in mutagenesis experiments can lead to protein expression differences, but comparison of the responses obtained in the presence of the different lipids to activation by GSK101 allowed us to determine whether activation by the glycerophospholipid was altered. For single-channel recordings, borosilicate glass pipettes of 30 M resistance were used. Recordings were obtained at +60 mV by acquiring several traces of 1–3 s duration. The effect of different agonists on single TRPV4 channels was studied in an inside-out configuration. Currents were low-pass filtered at 3 kHz and sampled at 50 kHz. Patches containing only one channel activated by different compounds were identified as those that did not contain overlapping opening events. The single-channel current (i) in each condition was determined by building all point-histograms from traces with clear closings and openings. The resulting histograms were fitted to a Gaussian function where the peak corresponded with i. The half-threshold crossing technique identified single-channel openings and closures (Islas, 2015). Channel open probability (Po) was calculated as the sum of the total open time divided by the sweep duration. Dwell times and amplitude histograms in the closed or open states were collected in logarithmic time histograms according to the Sine–Sigworth trans- formation (Sigworth & Sine, 1987). Sums of three or two exponential components were fitted to histograms using a least-squares algorithm. Recordings were performed in the absence of Ca2+ to avoid block/desensitization of the channel. © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1659 Mutagenesis Mutations in the human TRPV4 channels were constructed by a two-step PCR method, as pre- viously described (Salazar et al., 2008, 2009). Briefly, mutations in TRPV4 channels were introduced using a method involving oligonucleotides synthesized to contain a mutation in combination with wild-type (WT) oligonucleotides for PCR amplification of fragments of the complementary DNA. The product of the PCR was then cut with two restriction enzymes to generate a cassette containing the mutation. The cassette was ligated into the complementary channel DNA cut with the same two restriction enzymes. The entire region of the amplified cassette was sequenced to confirm the mutation and ensure against second-site mutations. Molecular docking (MD) and MD simulations The association of LPA18:1 with TRPV4 was modelled through molecular docking simulation employing the recently elucidated cryogenic electron micro- scopy (cryo-EM) structure of the human channel (PDB code 7AA5). Missing fragments in the TRPV4 structure [i.e. residues 533–548 and 648–658 were added according to the AlphaFold model (Code AF-Q9HBA0-F1)]. The LPA structure was built with the Figure 1. TRPV4 channels are activated by LPA in endogenous and heterologous expression systems A, representative currents for BMECs at −120 to +120 mV. Grey current traces were obtained initially in the absence of agonist, blue traces were obtained in the presence of 5 µM LPA18:1 and purple traces in the presence of 5 µM LPA18:1 and 30 µM RN1734. B, fraction of remaining currents after 5 µM LPA18:1 and 30 µM RN1734 in BMECs (32.8 ± 16.6%; n = 11), calculated at +120 mV. C, representative traces of leak or initial (grey traces), LPA18:1-activated (blue traces) currents and after inhibition with RN1734 (purple traces) of TRPV4 channels from transiently transfected HEK293 cells, in inside-out patches, obtained by stepping the voltage from −120 mV to +120 mV in the presence of 1 mM Ca2+. Membrane patches were exposed to 5 µM LPA18:1 for approximately 3 min. D, fraction of remaining currents after 5 µM LPA18:1 and 30 µM RN1734 in HEK293 cells (9.35 ± 10.4%; n = 7), calculated at +120 mV. E, current–voltage relationships for conditions in C for the initial currents (grey trace, n = 4) and in the presence of 5 µM LPA18:1 (blue trace, n = 4). F, fraction of the current activated with 5 µM LPA18:1 (53.9 ± 14.1%; n = 26), as normalized to the current activated with 1 µM GSK101 (+120 mV). G, dose–response curve for activation with LPA18:1 at +120 mV in the inside-out configuration obtained in the absence of Ca2+. Smooth curve is a fit with the Hill equation (EC50 = 4.12 µM and Hill coefficient = 3.9). Due to seal instability a single LPA18:1 concentration was tested per membrane patch and the current was normalized to the current activated with 1 µMGSK101 in the same patch (n = 3–12). Group data are represented as mean ± SD. [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1660 M. Benítez-Angeles and others J Physiol 601.9 software ACD/ChemSketch15.01 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The TRPV4 channel and LPA molecule were prepared for docking with the software AutoDockTools 1.5.7 (Morris et al., 2009), assigning Gasteiger partial charges. Search space for docking was demarcated through a grid box (34 × 34 × 38 Å3) surrounding the residue R746 of TRPV4. The grid parameters were generated using AutoGrid 4.2.6 (Morris et al., 1998; Vanommeslaeghe et al., 2009) and the Lamarckian genetic algorithm of AutoDock 4.2.6 (Morris et al., 1998, 2009) was used to generate 1000 channel–LPA binding conformations. The resulting conformers were analysed with the software PyMOL2.3 (Schrödinger, LLC, New York, NY, USA). The TRPV4-LPA18:1 lowest-scoring docked conformation was used as starting point for MD simulations. The channel–LPA complex was embedded in a pre-equilibrated palmitoyl-oleyl-phosphatidyl-choline (POPC) bilayer solvated with explicit TIP3P water molecules. Sodium and chloride ions (0.15 M NaCl) were added to the aqueous phase to mimic physiological conditions and to ensure charge neutrality. The calcium ion included in the cryo-EM TRPV4 structure was kept within the selectivity filter. Similar systems were built to evaluate the interaction of LPA with the mutants TRPV4-R746D and TRPV4-R446D-R746D. The initial configuration of each system was optimized bymeans of 30 000 steps of energyminimization, followed by equilibration and relaxation in a 100 nsMD simulation at 310 K in the isobaric-isothermal ensemble. Three replicas were simulated in each case, totalling 300 ns. The parameters for the LPA molecule were obtained from the ParamChem server using theCGenFF force field. Soft harmonic constraints were applied to the wild-type and mutant channel backbone and LPA during the first 10 ns of simulations, whichwere decreased gradually from 10 to 0 kcal mol−1 Å−2 over this period. The constant temperature was enforced using a Langevin thermostat with a damping coefficient of 1 ps−1. The Langevin piston method (Feller et al., 1995) was used to maintain constant pressure (101.325 kPa). Long-range electro- static interactions were computed using the particle-mesh Ewald summation method (Essmann et al., 1995), with a smooth real-space cutoff applied between 8 and 9 Å. The Verlet-I/r-RESPA multiple time-step integrator (Tuckerman et al., 1992) was used with a time step of 2 fs. All MD simulations (≈305 500 atoms) were performed using the software NAMD3.0 (Phillips et al., 2020) with the standard ions, lipids and water molecules of the CHARMM36 (MacKerell et al., 1998) force field. Structural analyses of the systems were performed using the software VMD1.9.3 (Humphrey et al., 1996). Contact frequency between the polar region of LPA and polar residues of the wild-type and mutated channel were computed as the ratio between the number of frames where a contact was present and the total number of frames in the equilibrated MD trajectories. A contact was considered present if, in a given frame, a distance of 7 Å was found between the centres of mass of the polar region of LPA and the polar residues in the channel. Statistical analysis Group data are reported as themean± standard deviation (SD). The two-tailed t test and one-way or two-way ANOVA, followed by Tukey’s post hoc test, were used for group comparison and calculated with Prism software (Graphstats Technologies, Karnataka, India). Significant differences between means were considered to exist when the P-value was less than 0.05. Results Activation of TRPV4 by LPA We have recently described that LPC18:1 activates the TRPV4 ion channel (Chen et al., 2021). Here we investigated whether LPA18:1, an endogenously produced bioactive glycerophospholipid whose precursor is LPC18:1 (Pagès et al., 2001), acts as a modulator of the activity of TRPV4, by testing the role of this molecule on channel function. We first tested whether cells that endogenously express TRPV4, such as BMECs, which are a key component of the blood–brain barrier and whose function is regulated by this ion channel (Kumar et al., 2020), could be activated by LPA. Thus, we recorded currents in the absence of agonist (Fig. 1A, grey traces), and then we applied 5 µM LPA18:1 to excised inside-out membrane patches from rat primary BMECs (Fig. 1A, blue traces). The results in Fig. 1 show that currents, obtained at voltages ranging from −120 to +120 mV, can be induced by LPA and that 67.2± 16.6% specific of these currents could be inhibited by the specific antagonist of TRPV4, RN1734 (Fig. 1A, violet traces, and Fig. 1B), indicating that brain microvascular endothelia respond to the glycerophospholipid and that TRPV4’s activity partly accounts for this response. Interestingly, in the brain, it has been suggested that LPA can modulate intracranial pressure through actions on TRPV4 (Toft-Bertelsen et al., 2022). To better study the activation of TRPV4 by LPA, we expressed this channel in HEK293 cells and recorded currents in the absence (Fig. 1C, grey traces) and in the presence of LPA (Fig. 1C, blue traces) and in the presence of 30µMRN1734 (Fig. 1C, purple traces), which inhibited 90.6 ± 10.4% of LPA-sensitive current in these cells (Fig. 1D). Currents were activated with LPA at voltages ranging from −120 to +120 mV, displaying an outwardly rectifying behaviour (Fig. 1E). © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1661 Next, we determined activation of TRPV4 by 5 µM LPA18:1, a concentration that we could apply to inside-out membrane patches without most of these breaking, and compared it to activation of TRPV4 in the presence of the well-established synthetic agonist GSK101 (1 µM, at +120 mV). Exposure of TRPV4 to 5 µM LPA18:1 applied from the intracellular face of the channel produced activation of currents that were, on average, 53.9 ± 14.1% of the magnitude obtained in the presence of 1 µM GSK101 (Fig. 1F). The dose–response to LPA18:1, using the inside-out patches, shows that the EC50 is 4.12µMand the Hill coefficient (n) is 3.9, as estimated from fits to the Hill equation (Fig. 1G). Specificity of activation of TRPV4 by LPA and LPC Other studies on TRPV4 channels have used lower concentrations of GSK101 to activate TRPV4 currents; however, most of these were not performed in excised membrane patches, in contrast to the present work and, in these other works, the steady-state for current activation was achieved after several minutes of using GSK101 at nanomolar or evenmicromolar concentrations (Cao et al., 2018; Chen et al., 2021; Jin et al., 2011b; Kumar et al., 2020; Mihara et al., 2018). In the present study, we used the saturating concentration of 1 µM GSK101 to be able to achieve steady-state of current activation through TRPV4 faster and completely. We conclude that currents generated by the application of 1 µM GSK101 are due to activation of TRPV4 since, in cells transfected only with the empty pcDNA3.1 and the pIRES-hr-GFP plasmids, initial currents (in the absence of GSK101, grey trace, Fig. 2A) are similar in magnitude to those obtained by exposure to 1 µM GSK101 (Fig. 2A and B; 16.8 ± 7 pA for initial currents vs. 16.02± 6.8 pA after 1µMGSK101). Moreover, as shown in Fig. 2C andD, activation of TRPV4 by 1 µM GSK101 in membrane patches of HEK293 cells transiently transfected with the ion channel was inhibited in 82.4 ± 10.7% by the antagonist RN1734 (20 µM). Finally, under our experimental conditions, GSK101 activated TRPV4 with an EC50 = 46.3 nM (Fig. 2E) and maximal activation was achieved at concentrations above 100 nM of GSK101. To determine whether the vehicle in which LPA18:1 was diluted could activate TRPV4 by modifying the membrane properties of HEK293 cells, we first performed experiments in which we exposed the patches to this vehicle (DMEM + 1% BSA). The results show that while TRPV4 could be activated in the presence of GSK101 (black trace, Fig. 2F), no significant activation was observed in the presence of the vehicle [purple trace and symbols; 0.8 ± 1.4%, as normalized to GSK101 and compared to initial currents (grey trace); Fig. 2F and I]. TRPV4 is a channel that has been shown to respond to mechanical stimuli (Berrout et al., 2012; Liedtke et al., 2000, 2003; Matthews et al., 2010; Poole, 2022; Servin-Vences et al., 2017, 2018; Sianati et al., 2021; Thodeti et al., 2009). The insertion of lipids in the membrane can influence its properties (Lundbaek & Andersen, 1994; Maingret et al., 2000; Matthews et al., 2010; O’Conor et al., 2014; Servin-Vences et al., 2017, 2018) and hence the effect of LPA on TRPV4 could be indirect. We next tested whether LPA18:0, a glycerophospholipid very similar in structure to LPA18:1, which is also inserted into the membrane causing changes in its properties and adding charges through its phosphate groups, could activate TRPV4. To this end, we performed experiments where we applied 5 µM LPA18:0 to membrane patches expressing TRPV4 and found that, similarly to what was observed with the vehicle, LPA18:0 failed to activate currents efficiently (orange trace and symbols Fig. 2G and I, 9.7 ± 15.3%), as normalized to GSK101 and also compared to initial currents (black and grey traces, Fig. 2G). These data are in contrast with activation of TRPV4 currents in the presence of LPA18:1 (blue trace, Fig. 2H), which activated 53.9 ± 14.1% of the currents, as compared to GSK101 (black trace and blue symbols, Fig. 2H and I). Previously, we showed that LPC activates TRPV4 channels and that this activation leads to the entry of Ca2+ into keratinocytes, resulting in itch in the whole organism via cellular signalling from epithelial cell to skin-innervating pruriceptor sensory neurons (Chen et al., 2021). LPC18:1 activated TRPV4 in inside-out patches of HEK293 cells. Similar to the experiments shown in Fig. 2, using the heterologous cellular expression system, we tested a couple of species of LPC to assess the activation of TRPV4 by these lipids (Fig. 3A–C). Our results show that while LPC18:1 activates TRPV4 (orange for LPC18:1 and black for GSK101, Fig. 3A, D and G; 48.7± 18%, normalized current to activation byGSK101), as previously shown (Chen et al., 2021), LPC18:0 (green for LPC18:0 and black for GSK101, Fig. 3B, E and G; 8.9 ± 6.3%) and LPC16:0 (yellow for LPC16:0 and black for GSK101, Fig. 3C, F and G; 2.5 ± 2.8%), which are also found circulating in human serum (Song et al., 2012), fail to activate heterologously expressed TRPV4 channels in inside-out patches. While future experiments using several different lipids will determine the details of the structural determinants required for activation of TRPV4 by LPC, the experiments shown here indicate that the presence of a negatively charged head group is not enough for ion channel activation by LPC either. Interactions of LPA with the C-terminus of TRPV4 LPA18:1 and structurally similar molecules have been shown to interact with TRPV1 through a residue (K710) located in its TRP box in the C-terminus (Morales-Lázaro © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1662 M. Benítez-Angeles and others J Physiol 601.9 et al., 2014; Nieto-Posadas et al., 2011). Hence, we tested whether the equivalent R746 residue in the C-terminus of TRPV4 determined activation of this channel by LPA18:1. To assess this possibility, we generated the charge-reversal TRPV4-R746D mutant and compared its activation by LPA18:1 to that of the wild-type channel. By applying 5 µM LPA18:1 and then 1 µMGSK101 to the membrane patches expressing TRPV4-R746D channels, we observed that LPA18:1 only activated 16.1 ± 13.2% of the current, as compared to 53.9 ± 14.1% for wild-type channels (blue traces represent activation by LPA18:1 and black traces by GSK101; Fig. 4A, B and G). To test whether the TRPV4-R746D mutant exhibited modifications in its response to GSK101 that could indicate changes in gating properties due to the mutation, we obtained dose–response curves for this agonist from wild-type- and TRPV4-R746D-expressing membrane patches. As shown in Fig. 4C, the response of these two types of channels to GSK101 was similar [EC50 = 37 nM and Hill coefficient = 1.6 for wild-type TRPV4 channel (black symbols) vs. EC50 = 48 nM and Hill coefficient = 1.9 for TRPV4-R746D channel (blue symbols)], supporting the idea that R746 is only important for gating of TRPV4 by LPA18:1. Interestingly, single-nucleotide polymorphisms (SNPs) have been reported for R746 in humans Figure 2. Effects of three different phosphated lipids on TRPV4 activation A, representative traces from currents in the absence (grey) and presence (black) of 1 µM GSK101 from cells transfected only with pcDNA3.1 and pIRES-hr-GFP. B, current values for membrane patches before (initial, turquoise symbols) and after 1 µM GSK101 (grey symbols). Initial currents are paired by colour-coded lines to the value of the currents after 1 µM GSK101 (n = 4; 16.8 ± 7 pA for initial currents vs. 16.02 ± 6.8 pA after 1 µM GSK101). C, representative current traces from membrane patches of HEK293 cells transiently transfected with TRPV4 in the absence (grey, initial) or presence of 1 µM GSK101 (black) and after 20 µM RN1734 with GSK101 (1 µM, blue). D, fraction of remaining currents after treatment of membrane patches with 20 µM RN1734 + 1 µM GSK101 18:1 (17.6 ± 10.7%; n = 6), calculated at +120 mV. E, dose–response of inside-out membrane patches (n = 4-6) from HEK293 cells transiently expressing TRPV4 to different concentrations of GSK101, calculated at +120 mV. F, representative traces from currents with the vehicle (DMEM + 1% BSA) where LPA18:1 was diluted. The initial currents (grey), in the presence of the vehicle (purple) and in the presence of 1 µM GSK101 (black) in the same membrane patch of TRPV4-expressing HEK293 cells. G, representative traces from currents with LPA18:0. The initial currents (grey), in the presence of LPA18:0 (orange) and in the presence of 1 µMGSK101 (black) in the same membrane patch. H, representative traces from currents with LPA18:1. The initial currents (grey), in the presence of LPA18:1 (blue) and in the presence of 1 µM GSK101 (black) in the same membrane–patch. Currents shown in F–H were obtained at −120 and +120 mV in the same membrane patch. Structures for each lipid are shown to the right of the representative currents. I, fraction of currents represented in (F) for DMEM + 1% BSA (0.8 ± 1.4%; n = 5, purple symbols), (G) LPA18:0 (9.7 ± 15.3%; n = 10, orange symbols) and in (H) LPA18:1 (53.9 ± 14.1%; n = 26; blue symbols). The currents activated by the vehicle, DMEM + 1% fatty acid free-albumin (F) or by LPA18:0 (G) were normalized to the current with 1 µM GSK101. No statistical differences were found between DMEM + 1% BSA and LPA18:0. Both experimental groups were statistically different from LPA18:1 (P < 0.0001; one-way ANOVA with Tukey’s post hoc test). [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1663 (R746G, https://www.ncbi.nlm.nih.gov/clinvar/variation/ VCV000307123.2; and R746C, https://www.ncbi.nlm.nih. gov/clinvar/variation/VCV000450199.2), although no clinical phenotypes have yet been associated. Thus, we tested whether these reported changes in this position altered the activation of TRPV4 by LPA18:1. Figures 4D, E and G show that, while TRPV4-R746C and R746G mutant channels respond to GSK101 (black traces), their response to 5 µM LPA18:1 is lower (14.6 ± 7.4% for TRPV4-R746C and 7.3 ± 10.8 % for TRPV4-R746G, yellow and blue traces), as compared to the response to 1 µM GSK101 (black traces). In contrast, when R746 is substituted for lysine (K), as the residue present in the Figure 3. LPC species-dependent activation of TRPV4 Representative traces obtained with different species of LPC18:1 (A), 18:0 (B) and 16:0 (C) (at −120 and +120 mV) in inside-out patches of HEK293 cells expressing TRPV4 channels. Structures for each lipid are shown above representative currents. The presence of a charged phosphate group is not sufficient for activation of currents through TRPV4. While GSK101 and LPC18:1 activate outwardly rectifying currents (−120 to +120 mV; D, black and orange symbols, n = 4), LPC18:0 (E, green) and LPC16:0 (F, yellow) exhibit currents similar to those obtained initially without any agonist (E and F, grey). G, on average, currents activated by LPC18:1 (n = 9), at +120 mV, were 48.75 ± 18% of activation obtained with 1 µM GSK101, while LPC18:0 (n = 7) and LPC16:0 activated 8.9 ± 6.3% and 2.5 ± 2.8% (n = 5), respectively (∗P < 0.0001 to LPC18:1 vs. LPC18:0 and LPC18:1 vs. LPC16:0; one-way ANOVA with Tukey’s post hoc test). [Colour figure can be viewed at wileyonlinelibrary.com] homologous site in TRPV1 (Nieto-Posadas et al., 2011), these TRPV4 channels respond to LPA18:1 similarly to the wild-type (R746) channel (62 ± 24.3% in R746K vs. 53.9 ± 14.1% in the wild-type TRPV4; purple trace for LPA18:1 and black for GSK101, Fig. 4F andG), suggesting that the nature of the charge at this site is important for activation of the channel by LPA18:1. However, as will be discussed later, the mechanism of activation of TRPV4 by LPA displays differences, as compared to what we had previously shown for TRPV1 (Nieto-Posadas et al., 2011). BrP-LPA, is a soluble analogue of LPA (Fig. 5A andB), which inhibits several G protein-coupled receptors (GPCRs) associated with the signalling pathways of LPA and also inhibits autotaxin (Jiang et al., 2007), the enzyme that produces LPA. Here we tested the effects of BrP-LPA on TRPV4, and the results in Fig. 5B (crimson trace for BrP-LPA and black for GSK101) show that this chemical also activates currents through this channel (49.7 ± 21%, with respect to GSK101; Fig. 5E, circles). These data support that activation of TRPV4 by BrP-LPA occurs independently of GPCR-associated signalling pathways. Consistent with what we observed with LPA18:1 and the TRPV4-R746D mutant (Fig. 4B and G), activation of this mutant by BrP-LPA (Fig. 5C, crimson traces for BrP-LPA and black for GSK101) is also diminished as compared to the wild-type channel (23 ± 16.15%; Fig. 5D, squares). Single TRPV4 channels exhibit distinct conductance in response to LPA and LPC For TRPV1, we have previously shown that two agonists can produce different conductance states (Canul-Sánchez et al., 2018). This observation implies that TRP channels exhibit conformational flexibility that allows them to fine-tune their responses to different stimuli. This phenomenon may be physiologically relevant depending on the overall context (type of cell or tissue, presence of disease, etc.) under which they are activated. Hence, we decided to perform single-channel experiments using TRPV4 and further evaluate the properties of its activation in the presence of LPA18:1 and LPC18:1. Our experiments show that in the presence of 100 nM GSK101 the open probability (Po) for wild-type TRPV4 channels is 0.76 ± 0.13 and the single-channel amplitude is 6.45 ± 1.11 pA (black traces, Fig. 6A and B), while with 5 µM LPA18:1 the Po was 0.47 ± 0.26 and the single-channel amplitude was 6.47± 0.75 pA (blue traces, Fig. 6C and D). Thus, a lower Po with LPA18:1 in comparison to that exhibited with GSK101, was consistent with the data obtained for activation of macroscopic TRPV4 currents by LPA18:1 (Fig. 1). We have reported that 5 µM LPC18:1 produced a Po of 0.77 ± 0.09 but TRPV4’s single-channel amplitude was 3.27 ± 0.83 pA at +60 mV (Chen et al., 2021). © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1664 M. Benítez-Angeles and others J Physiol 601.9 Figures 6E and F show a representative trace (purple) and its histogram obtained with 5 µM LPC18:1, which is in accordance with our previous data (Chen et al., 2021). In summary, GSK101 and LPA18:1 produce similar single-channel amplitudes, albeit with different Po values, whereas LPC18:1 opens TRPV4 channels with a higher Po than LPA18:1, butwith opening events that exhibit smaller single-channel amplitudes, as can be clearly discerned in Fig. 6G, where we have quantified the amplitude of each detected opening event and plotted it as a function of the duration of that event. Together, all these experiments show that LPC18:1 evoked a different open state than that of GSK101 and/or LPA18:1. Since the opening of TRPV4 by LPC18:1 involves its interaction with residue R746, we assessed whether the TRPV4-R746Dmutant exhibited a single-channel amplitude that was now similar to that of LPA18:1. We performed single-channel recordings using the TRPV4-R746D channel and applied either GSK101 or LPC18:1 to the intracellular side of the channels. Several traces are shown in Fig. 7 to illustrate better that mutation of this site renders a channel that responds both to GSK101 (left, crimson trace) and LPC18:1 (left, blue trace) by opening with a similar single-channel amplitude (right panels, 6.91 ± 0.12 pA vs. 6.16 ± 0.2 pA, respectively). LPC18:1 is a negatively charged conical lipid, which could alter the shape, fluidity and/or add a negative charge to the membrane (i.e. change in surface charge), influencing ion channel activity. However, our data support the interpretation that the change in single-channel current amplitude induced by LPC18:1 is not due to changes in the membrane’s physical properties caused by the accumulation of the lipid. Consequently, we conclude that this glycerophospholipid may cause a different open conformation through a mechanism that requires the R746 residue. LPA interacts with a complex site in TRPV4 Although our experimental data show that residue R746 is important for activating TRPV4 by LPA18:1, themutation Figure 4. Residue R746 participates in TRPV4 activation by LPA18:1 A and B, representative inside-out currents for (A) WT-TRPV4 (n = 26, also shown in Fig. 1F) and (B) TRPV4-R746D (n = 11) channels in transiently transfected HEK293 cells. Currents were obtained initially in the absence of agonist (grey), in the presence of 5 µM LPA18:1 (light blue and cyan, respectively) and in the presence of 1 µM GSK101 (black). C, dose–response curve for activation with GSK101 at +120 mV in the inside-out configuration for WT-TRPV4 (black symbols) and TRPV4-R746D (blue symbols). Smooth curve is a fit with the Hill equation [EC50 = 37 nM and Hill coefficient = 1.6 (n = 5–6) for WT-TRPV4 and EC50 = 48 nM and Hill coefficient = 1.9 (n= 3–6) for TRPV4-R746D]. Due to seal instability, a single GSK101 concentration was tested per membrane-patch and the current was normalized to the current activated with 1 µM GSK101 in the same patch. Group data are represented as mean ± SD D– F, representative currents for TRPV4-R746C (yellow, n = 6), TRPV4-R746G (dark blue, n = 9) and TRPV4-R746K (purple, n = 7) channels. Currents were obtained in the same conditions as in A and B. G, average activation of WT-TRPV4 (53.9 ± 14.1%, n = 26), TRPV4-R746D (16.1 ± 13.2%, n = 11), TRPV4-R746C (14.6 ± 7.4%, n = 6), TRPV4-R746G (7.3 ± 10.8%, n = 9) and TRPV4-R746K (62 ± 24.3%, n = 7) by 5 µM LPA18:1. Data were normalized to activation obtained with 1 µM GSK101. A one-way ANOVA with Tukey’s post hoc test was performed between groups in G. Statistical analysis resulted in ∗P < 0.0001 between groups as compared by the brackets. [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1665 R746D still shows some activation by LPA18:1, suggesting that more than one residue is involved in this inter- action. This prompted us to examine whether interaction of LPA18:1 with TRPV4 required other residues besides R746. Protein–ligand docking and MD simulations were performed to gain insight into the structural mechanism that governs the TRPV4–LPA18:1 interaction. A structural model of LPC18:1 interacting with Xenopus tropicalis TRPV4 was previously reported (Chen et al., 2021). Here we take advantage of the recently elucidated cryo-EM structure of human TRPV4 to characterize the interaction with LPA18:1. Missing residues in the TRPV4 structure were modelled Figure 5. An antagonist of LPA receptors, BrP-LPA, activates TRPV4 A, representation of the chemical structures of LPA and BrP-LPA, respectively. B and C, traces shown are representative from the activation of (B) WT-TRPV4 channels and (C) TRPV4-R746D mutant channels with 5 µM BrP-LPA, respectively. Currents from excised inside-out membrane patches were obtained initially in the absence of agonist (grey), in the presence of 5 µM BrP-LP (crimson) and in the presence of 1 µM GSK101 (black) at −120 and +120 mV. D, fraction of the currents (at +120 mV) obtained with 5 µM BrP-LPA in the WT-TRPV4 (circles, 49.7 ± 21%; n = 18) and TRPV4-R746D (squares, 23 ± 16.15%; n = 10) channels as normalized to the current in the presence of 1 µM GSK101; two-tailed Student’s t test, ∗P = 0.0018 between groups. [Colour figure can be viewed at wileyonlinelibrary.com] (see Methods), particularly those surrounding residue R746. From docking studies on the refined TRPV4, we identified an LPA18:1 binding pocket comprising five positively charged residues (i.e. K442, R446 in the N-terminus, K535 in the S2–S3 linker, R594 in the S4–S5 linker, and R746 in the TRP box) (Fig. 8A). The lowest energy score docking conformation of LPA18:1 inter- acting with those residues in the wild-type channel was then subjected to MD simulation (Fig. 8B) to identify the most prevalent interactions. A representative binding mode of LPA18:1 with TRPV4 during the simulation is illustrated in Fig. 8C, showing that LPA’s 18:1 phosphate group can interact with either of residues K442, R446 or Figure 6. Effects of LPA and LPC in the single-channel properties Single-channel recordings and all point histograms of WT-TRPV4 activated by 100 nM GSK101 (A and B), 5 µM LPA18:1 (C and D) and 5 µM LPC18:1 (E and F), at +60 mV. Letters O and C represent the open and closed current levels, respectively, for the inside-out membrane patches. The average for the open level amplitudes was 6.45 ± 1.11 pA (n = 4) with an open probability (Po) of 0.76 ± 0.13 (n = 4) for GSK101, 6.47 ± 0.75 pA (n = 4) with Po of 0.47 ± 0.26 (n = 5) for LPA18:1, and 3.27 ± 0.83 pA (n = 4) with Po of 0.77 ± 0.09 (n = 3) for LPC18:1. G, the amplitude of each open event is plotted as a function of its duration. GSK101 openings (black crosses) have a mean amplitude of 6.2 pA, LPA18:1 openings (blue crosses) of 6.5 pA and LPC18:1 openings (purple crosses) of 3.99 pA, at +60 mV. [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1666 M. Benítez-Angeles and others J Physiol 601.9 K535 through electrostatic interactions. These residues create an overall positive potential modulating binding of LPA18:1 through its polar headgroup. Ester groups of LPA18:1 interact with residues R746 and R594, whereas the alkyl chain of the phospholipid is stabilized by other residues in helices S1 and S2 of the channel. Through an analysis of contact frequencies during the MD simulation for the wild-type TRPV4–LPA18:1 complex (Fig. 8D), four residues predominantly stabilizing phospholipid binding were identified (i.e. Y439, R594, T739 and D743). For residues K442, R446, K535 and R746, interchangeable contacts were observed, and, notably, mutation of R746 by aspartic acid exhibited an increased interaction of LPA18:1 with R446 (Fig. 8E). Meanwhile, a reduction of contacts with residue D746 was observed. In the case of the R446D-R746D mutation (Fig. 8F), LPA18:1 contacts with residues K442 and D446 were significantly reduced, while some interactions with D746 were detected. Together these results support that the residues identified by docking and MD simulations stabilize the bound state of LPA18:1 to TRPV4, constituting a different binding site than that previously described for LPC18:1 (Chen et al., 2021). Importantly, distinct to what we had described for LPA18:1 and TRPV1 (Nieto-Posadas et al., 2011) and for LPC18:1 and TRPV4 (Chen et al., 2021), our data suggest that the binding site for LPA18:1 in TRPV4 requires also residues from the N-terminus. The cryo-EM structure of TRPV4 shows that, after the N-terminal ankyrin-like repeat domain (ARD), there is a linker domain before the S1–S4 domain. In this channel, the linker domain, two β-strands, β1 and β2, together with a C-terminal β-strand, β3, form a three-stranded β-sheet, which ties theN-terminal ARD to the C terminus in each subunit and also interacts with the ARD from an adjacent subunit, creating a subunit–subunit assembly interface (Deng et al., 2018). After the β-strands, there is a helix–turn–helix motif and a pre-S1 helix that tightly lodges the C-terminal TRP box, which runs parallel to the membrane and is located close to the S4–S5 linker, which connects the S1–S4 and pore domains. These particular structural features are thought to provide a privileged location of the TRP helix between the cytosolic and transmembrane domains that may influence the gating of TRPV4 (Cao, 2020; Deng et al., 2018). Since we had already observed that R746 in the TRP box influences activation of TRPV4 by LPA18:1, we wondered whether two of the residues in theN-terminus of TRPV4, identified with the MD simulations, participate in activation of the channel by LPA18:1. To this end, we performed electro- physiological experiments using TRPV4-K442D and TRPV4-R446D mutant channels and evaluated their activation by LPA18:1. As shown in Fig. 9A and B, the TRPV4-K442D mutant produces channels that are not statistically different in their activation by LPA18:1 (violet traces), although a tendency to a decrease is observed (48 ± 23% activation relative to GSK101; Fig. 9B, empty circles), as compared to wild-type TRPV4 (56.8 ± 22.3% activation relative to GSK101, black traces; Fig. 9B, filled circles). However, TRPV4-R446D channels (Fig. 9A, blue traces) display a statistically significant decrease in their activation by LPA18:1 (28 ± 16.6% activation relative to GSK101; Fig. 9B, filled squares), in comparison to wild-type channels (56.8 ± 22.3% relative to activation by GSK101, Fig. 9A and B). We also performed electro- physiological experiments to attempt to determine if the double mutant TRPV4-K442D-R446D completely suppressed TRPV4’s activation by LPA18:1, and the results show that activation of these mutant channels by LPA18:1 (Fig. 9A, violet traces) is almost completely abrogated (6.3± 8.2%), as is activation of K535A channels (Fig. 9A andB, filled triangles, 12.2± 12.3%), as compared to wild-type TRPV4 (Fig. 9B, empty squares). We also studied whether BrP-LPA required residues K442, R446 Figure 7. Single-channel activation of TRPV4 R746D by GSK or LPC18:1 Representative traces of single-channel currents elicited by GSK101 (crimson traces) or LPC18:1 (blue traces) at +60 mV in the same inside-out membrane patch. The open and closed states are indicated with dashed lines and the letters O and C indicate the open and closed state, respectively. The single-channel current elicited by 100 nM GSK was 6.91 ± 0.12 pA, while the current elicited 5 µM LPC18:1 was 6.16 ± 0.20 pA, as shown in the corresponding all-points amplitude histograms of the currents (left); n = 5 for each condition. The histogram is shown by the continuous line while the fit to two Gaussian functions is shown by the dotted curves. [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1667 and K535 to activate TRPV4 channels and found that, in the double mutant K442-R446D channels and K535A mutant channels, activation of TRPV4 was diminished, as compared to WT channels (3.5 ± 5.8% activation relative to GSK101 for K442D-R446D and 8.7 ± 8.2% for K535A; Fig. 9B, filled and empty pink diamonds, respectively). Moreover, the results in Fig. 9C demonstrate that these mutations do not affect activation of the channel by other agonists since the dose–responses for GSK101 remain very similar among wild-type (EC50 = 112.3 nM, black symbols), TRPV4-K442D (EC50 = 96.7 nM, blue symbols), TRPV4-R446D (EC50 = 111.4 nM, orange symbols), TRPV4-K442D-R446D (EC50 = 64.8 nM, purple symbols) and TRPV4-K535A (EC50 = 93.1 nM, pink symbols) channels. These results further support the hypothesis that residues K442, R446, K535 and R746 are required for activation of TRPV4 by LPA18:1. Finally, we performed control experiments testing whether TRPV4-K442D-R746D channels modified activation by LPA18:1 and, as expected, Fig. 9D and E show that the channels were activated by GSK101 (black traces) but not LPA18:1 (blue traces). However, Fig. 9D also shows that TRPV4-R446D-R746D channels were not functionally expressed in the plasma membrane of trans- fected HEK293 cells, since they could not be activated by GSK101 (black traces), possibly due to increased instability from the inserted amino acid substitutions. All the above data suggest that R746 in the C-terminal region of TRPV4 is possibly necessary to drive the channel into opening in the presence of LPA18:1. Our data show that the interaction site for LPA18:1 also involves residues K442 and R446 in the N-terminus. Figure 8. Computational modelling of the TRPV4-LPA association A, binding conformations of LPA18:1 (yellow molecules), derived from docking calculation, into the region surrounding the R746 residue of the human TRPV4 channel (grey cartoon representation). B, all-atom system of LPA18:1 bound to TRPV4 used for MD simulations. C, representative conformation of LPA18:1 interacting with wild-type TRPV4 during the equilibrated MD trajectory. Positively charged residues and LPA18:1 are shown as sticks coloured by atom type – carbon in green (residues) and yellow (LPA18:1), oxygen in red, nitrogen in blue, and phosphorus in dark yellow. D, contact frequency plot of the LPA18:1 polar region interacting with polar residues of TRPV4 wild-type and the mutants (E) TRPV4-R746D and (F) TRPV4-R446D-R746D. The plots were generated by averaging the contacts over the three replica simulations in each case. [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1668 M. Benítez-Angeles and others J Physiol 601.9 Figure 9. The N-terminus of TRPV4 participates in activation of the channel by LPA and BrP-LPA A, representative inside-out currents for WT-TRPV4, TRPV4-K442D, TRPV4-R446D, TRPV4-K442D-R446D and TRPV4-K535A channels from transiently transfected HEK293 cells. Currents were obtained initially in the absence of agonist (grey), in the presence of 5 µM LPA18:1 (lilac) and in the presence of 1 µMGSK101 (black) at −120 and +120 mV. B, fraction of the currents obtained with 5 µM LPA18:1 (lilac symbols) in the WT-TRPV4 (56.2 ± 22.3%; n = 26), TRPV4-K442D (48.6 ± 23.4%; n = 9), TRPV4-R446D (28 ± 16.6%; n = 6), TRPV4-K442D-R446D (6.3 ± 8.2%; n = 7) and TRPV4-K535A (12.2 ± 12.3%; n = 11) channels. Fraction of currents obtained with 5 µM BrP-LPA (pink diamonds) for WT (49.7 ± 21%; n = 18), TRPV4-K442D-R446D (3.5 ± 5.8%; n = 5) and TRPV4-K535A (8.7 ± 8.2%; n = 4) channels as normalized to the current (+120 mV) in the presence of 1 µM GSK101. Group data are represented as mean ± SD A one-way ANOVA with Tukey’s post hoc test was performed between groups. For LPA18:1 activation of TRPV4, statistical analysis resulted in ∗P = 0.0176 for WT vs. R446D; ∗P < 0.0001 for WT vs. K442D-R446D and ∗P < 0.0001 for WT vs. K535A. For BrP-LPA activation of TRPV4, statistical analysis resulted in ∗P = 0.0001 for WT vs. K442D-R446D and ∗P = 0.0011 for WT vs. K535A. C, dose–response curve for activation with GSK101 at +120 mV in the inside-out configuration for WT-TRPV4, TRPV4-K442D, TRPV4-R446D and TRPV4-K442D-R446D and K535A. Smooth curve is a fit with the Hill equation (WT-TRPV4 EC50 = 112.3 nM and Hill coefficient = 3.2, n = 8–10; TRPV4-K442D EC50 = 96.7 nM and Hill coefficient = 2.4, n = 3–5; TRPV4-R446D EC50 = 111.4 nM and Hill coefficient = 2.5, n = 3–6; TRPV4-K442D-R446D EC50 = 64.8 nM, Hill coefficient = 3, n = 4–6; and TRPV4-K535A EC50 = 93.1 nM and Hill coefficient = 2.7, n = 4–8). A single GSK101 concentration was tested per membrane patch and the current was normalized to the current activated with 1 µM GSK101 in the same patch. Group data are represented as mean ± SD. D, representative currents for WT-TRPV4, TRPV4-K442D-R746D and TRPV4-R446D-R746D channels. Currents were obtained initially in the absence of agonist (grey), in the presence of 5µMLPA18:1 (blue) and in the presence of 1µMGSK101 (black) at −120 and +120 mV. E, fraction of the currents obtained with 5 µM LPA18:1 in the WT-TRPV4 (62.1 ± 27.1%; n = 9, empty symbols) and TRPV4-K442D-R746D (3.7 ± 4.2%; n = 9, filled symbols) channels as normalized to the current (+120 mV) in the presence of 1 µM GSK101A. Group data are represented as mean ± SD. Two-tailed Student’s t test, ∗P < 0.0001 between groups. [Colour figure can be viewed at wileyonlinelibrary.com] © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1669 Discussion LPA is present systemically. It is synthesized in serum and plasma from LPC mainly by autotaxin, an enzyme also found in serum (Okudaira et al., 2010), and can be detected in significant concentrations. Although the flip-flop rate of LPA is unknown, activation by extracellular LPA of the nuclear PPARγ receptor has been shown to be physiologically relevant, suggesting that efficient mechanisms of transbilayer movement of LPA exist. McIntyre et al. (2003) have shown that LPA is a transcellular PPARγ agonist that displaces the drug rosiglitazone from the ligand pocket of PPARγ and that it is able to do this evenwhen the LPA source is extracellular. It has also been shown that cytoplasmic LPA can be produced de novo and activate ion channels (Kittaka et al., 2017). Only a few endogenous agonists have been elucidated for TRPV4 channels. The experimental evidence pre- sented here supports the following concepts: (1) LPA and also, as previously shown, LPC, activate TRPV4 channels; (2) an antagonist of LPA receptors structurally similar to LPA, BrP-LPA, promotes TRPV4 activation through a mechanism that does not depend on GPCRs; (3) LPA18:0 does not activate TRPV4, whereas LPA18:1 does, showing that structural differences among LPA species are important; (4) mutations of positively charged residues in the N- and C-termini of TRPV4 eliminate effects of LPA18:1 on the channel and encompass different regions for activation than those described for TRPV1; (5) TRPV4 and TRPV1 activation by LPA18:1 is different since the glycerophospholipid does not produce changes in the single-channel amplitude of TRPV4, as compared to its canonical agonist, GSK101; and (6) LPC18:1, which we had previously shown to bind and activate TRPV4, probably produces a different conformational open state than the one(s) achieved by GSK101 and LPA18:1. Our data show that BrP-LPA, an inhibitor of several of the GPCR LPA receptors, activates the channel by inter- acting with the same residues identified here for LPA18:1. Moreover, the lack of activation of TRPV4 by LPA16:0 or 18:0 or LPC16:0 or 18:0 (Strawn et al., 2012) suggests that the mere presence of lipids with hydrophobic acyl chains, the insertion of negative charges (phosphate headgroup) in the membrane, which could lead to ion accumulation near the pore, or changes in its curvature due to the insertion of conical lipids are not sufficient for activation of TRPV4. Further studies are needed to establish the structural determinants required for activation of TRPV4 by lipids. Regrading BrP-LPA, we show that it interacts with the same residues in TRPV4 tested here for activation of the channel by LPA18:1, suggesting that it may have acquire a similar three-dimensional arrangement to that of LPA18:1. Residue R746, in the TRP box of TRPV4, is important for LPC18:1 binding and activation of the channel (Chen et al., 2021). However, in the case of TRPV4’s activation by LPA18:1, R746 participates in opening of the channel by the glycerophospholipid but, as shown by ourmutagenesis and functional data, it does not necessarily constitute the only or the most important residue in the binding site for the glycerophospholipid. Our data also show that LPA’s activation of TRPV4 requires additional positively charges residues such as K442 and R446 in the N-terminus of the channel. Studies pertaining to the interaction of other phospholipids, such as PIP2, with TRPV4 have shown that the pre- sence of positively charged residues in the N-terminus of the protein is pivotal to either potentiating or inhibiting the channel (Garcia-Elias et al., 2013; Takahashi et al., 2014). Moreover, activation of TRPV4 by 5,6-EET seems to depend on the interaction with a positively charged residue (K535), identified in the present study (Berna-Erro et al., 2017; Watanabe et al., 2003), further highlighting the importance of interactions of lipids with critical subdomains of TRP channels for their gating functions. R746 is located in the TRP helix, and is advantageously placed within the C-terminus and transmembrane domains to coordinate conformational changes between these two regions and the N-terminus, potentially influencing the gating of the channel (Deng et al., 2018). Hence, it is possible that, as shown by our computational modelling and our results using the N-terminal mutants (K442D and R446D), LPA18:1 promotes the open state of TRPV4 by driving conformational changes that are dependent upon the interactions of the N- and C-termini. Regarding LPC18:1, this agonist of TRPV4 displays a two-pronged effect by which it increases the open probability of the channel, as compared to LPA18:1, but it also features a decrease in the single-channel conductance that is dependent upon the R746 residue. This result suggests that LPC18:1 can produce a different open conformation to that obtained in the presence of GSK101 or LPA18:1. We have previously observed for TRPV1 that capsaicin and LPA18:1 lead to opening events with different single-channel amplitudes (of a larger conductance for activation with LPA18:1), a phenomenon that probably reflects transitions to different open conformational states (Canul-Sánchez et al., 2018). Physiologically, the ability of TRP channels to exhibit such a unique behaviour could represent a mechanism of fine-tuning their responses to different endogenous lipid activators/modulators in the context of a given cell or tissue. In summary, the data shown here demonstrate that LPA18:1 binds to a pocket between the voltage sensing-like domain (VSLD) and the pore domain and © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se 1670 M. Benítez-Angeles and others J Physiol 601.9 forms interactions with specific residues, distinct from those described for LPC18:1 (Chen et al., 2021). These different binding modes lead to activation of the channel with different characteristics. LPA18:1 produces openings with larger conductance but decreased open probability, while activation by LPC18:1 produces channels with small conductance but increased open probability. Our present data suggest that residue R746 might be important for channel opening by LPA18:1 and that a reduced electro- static ‘pulling’ on R746 might account for the reduced single-channel conductance. It is worth considering that mutations at the R746 site have been found in humans, although no clinical significance has been yet clearly determined for this mutation in TRPV4; hence, it will be interesting to determine whether a lack of activation of the channel by LPA could contribute to the development or severity of disease in particular organ contexts. Finally, TRPV4 effectively maintains the integrity of physiological barriers, such as in the brain and lungs (Simmons et al., 2019), as it is expressed in respiratory epithelia, smooth muscle, macrophages, sub- mucosal glands, fibroblasts, alveolar epithelia and vascular endothelial cells. In the future, it will also be interesting to evaluate whether there is a connection between the activation of TRPV4 by LPA18:1 and respiratory function. References Benítez-Angeles, M., Morales-Lázaro, S. L., Juárez-González, E., & Rosenbaum, T. (2020). TRPV1: Structure, end- ogenous agonists, and mechanisms. International Journal of Molecular Sciences, 21(10), 3421. Berna-Erro, A., Izquierdo-Serra, M., Sepúlveda, R. V., Rubio-Moscardo, F., Doñate-Macián, P., Serra, S. A., Carrillo-Garcia, J., Perálvarez-Marín, A., González-Nilo, F., Fernández-Fernández, J. M., & Valverde, M. A. (2017). Structural determinants of 5′,6′-epoxyeicosatrienoic acid binding to and activation of TRPV4 channel. 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S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se J Physiol 601.9 The TRPV4 ion channel is activated by lysophosphatidic acid 1673 Additional information Data availability statement All data for the summary figures are provided in the Supporting Information section. Competing interests Senior co-authorW.B.L. has been a full-time executive employee of Regeneron Pharmaceuticals (Tarrytown NY) since 2021. He co-founded biotechnology start-up company TRPblue Inc. (Durham, NC, USA) in 2017. These relationships are not related to this study, and there is no additional conflict. All other authors declare that they have no competing interests. Author contributions M.B.-A., A.E.L.-R., I.H.-A. and T.R. performed electro- physiological experiments, analysed data and wrote the paper; I.L. performed mutagenesis, analysed data and edited the paper; A.V.-J., O.E.G.-C., M.A. and F.T.-Q. performed in silico modelling and analysis and wrote and edited the paper; F.H.R. prepared BMEC cultures and analysed data; F.T.-Q., L.E.M.-B., W.B.L., R.G.-V.-V., L.D.I., L.B., T.yR. and T.R. conceptualized the project, supervised experiments, analysed data and wrote the manuscript. Funding This research was funded by the Dirección General de Asuntos del Personal Académico (DGAPA)-Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) IN200720 to T.R.; IN215621 to L.D.I., IN213320 to Marcelino Arciniega; Consejo Nacional de Ciencia y Tecnología (CONACyT) A1-S-8760 and Secretaría de Educación, Ciencia, Tecnología e Innovación del Gobierno de la Ciudad de México SECTEI/208/2019 to T.R. A.V-J. was funded by FONDECYT grant no. 1220110. The Millennium Nucleus of Ion Channels-Associated Diseases is a Millennium Nucleus supported by theNationalAgency of Research andDevelopment (ANID), Chile. Acknowledgements We thank the following personnel from Instituto de Fisiología Celular, UNAM: Ana María Escalante Gonzalbo, Francisco Pérez Eugenio, Juan Manuel Barbosa Castillo and Gerardo Coello Coutiño for technical assistance with computer software and hardware, Alan Medina and Félix Sierra for help with some control electrophysiology experiments and Cristina Aranda Fraustro for technical assistance with BMEC cultures. Keywords ion channel, lysophosphatidic acid, lysophosphatidylcholine, physiology, TRPV4 Supporting information Additional supporting information can be found online in the Supporting Information section at the end of the HTML view of the article. Supporting information files available: Statistical Summary Document Peer Review History © 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 1 4 6 9 7 7 9 3 , 2 0 2 3 , 9 , D o w n lo ad ed fro m h ttp s://p h y so c.o n lin elib rary .w iley .co m /d o i/1 0 .1 1 1 3 /JP 2 8 4 2 6 2 b y U n iv ersid ad N acio n al A u to n o m a D e M ex ico , W iley O n lin e L ib rary o n [1 0 /0 9 /2 0 2 4 ]. S ee th e T erm s an d C o n d itio n s (h ttp s://o n lin elib rary .w iley .co m /term s-an d -co n d itio n s) o n W iley O n lin e L ib rary fo r ru les o f u se; O A articles are g o v ern ed b y th e ap p licab le C reativ e C o m m o n s L icen se . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crucial role for sensory nerves and Na/H exchanger inhibition in dapagliflozin- and empagliflozin-induced arterial relaxation Elizabeth A. Forrester1, Miguel Benítez-Angeles2, Kaitlyn E. Redford3, Tamara Rosenbaum2, Geoffrey W. Abbott3, Vincenzo Barrese 4, Kim Dora5, Anthony P. Albert1, Johs Dannesboe6, Isabelle Salles-Crawley1, Thomas A. Jepps6, and Iain A. Greenwood 1* 1Vascular Biology Section, Molecular & Clinical Sciences Research Institute, St George’s University, Cranmer Terrace, London SW17 ORE, UK; 2I Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico; 3Bioelectricity Lab, Department of Physiology & Biophysics, School of Medicine, University of California, Irvine, USA; 4Department of Neuroscience, Reproductive Sciences and Dentistry, University of Naples Federico II, Naples, Italy; 5Department of Pharmacology, Oxford University, Oxford, UK; and 6Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark Received 19 March 2024; revised 6 June 2024; accepted 5 July 2024; online publish-ahead-of-print 26 July 2024 Time of primary review: 37 days Aims Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart fail- ure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries, but the underlying mechan- isms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) released from sensory nerves independent of glucose transport. Methods and results The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, and empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using wire myography. SGLT2, CGRP, TRPV1, and NHE1 expression was determined by western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin. All SGLT inhibitors (1–100 µM) and cariporide (30 µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mes- enteric arteries, relaxations to dapagliflozin, empagliflozin, and cariporide were attenuated by the CGRP receptor antagonist BIBN- 4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors. Conclusion SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner. * Corresponding author. Tel: +442077252857, E-mail: grenwood@sgul.ac.uk © The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Cardiovascular Research (2024) 00, 1–14 https://doi.org/10.1093/cvr/cvae156 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Graphical Abstract Pathway of SGLT2 inhibitor-induced relaxation in mesenteric arteries. SGLT2 inhibitors act on the sodium/hydrogen exchanger (NHE) to induce vasor- elaxation. H, hydrogen; Na, sodium; Ca2+, calcium; CGRP, calcitonin gene-related peptide; TRPV1, transient receptor potential vanilloid 1; CLR, calcitonin receptor-like receptor; RAMP1, receptor activity-modifying protein 1; cGMP, cyclic guanosine monophosphate; PKA, protein kinase A; Kv7, voltage-gated potassium channel. Keywords Sodium/glucose transporter 2 • Sodium/hydrogen exchanger • Calcitonin-gene related peptide • Sensory nerves • Vasodilatation 1. Introduction Inhibitors of sodium-dependent glucose transporter 2 (SGLT2 encoded by SLC5A2), such as dapagliflozin or empagliflozin,1,2 lower blood glucose levels through increased urinary excretion of glucose.3 The UK National Institute for Health and Care Excellence also recommend SGLT2 inhibitors for treat- ment of heart failure with reduced ejection fraction and chronic kidney dis- ease independent of raised blood glucose.3–5 In addition to decreased cardiac fluid retention, reduced reactive oxygen species generation, and les- sened fibrosis (summarized in Preda et al.3), the cardioprotective effect of SGLT2 inhibitors has been linked to a reduction in blood pressure (∼4 mmHg). Still, the mechanism underlying this effect is not known.6 Some of the initial effects may be due to a decrease in circulatory volume due to increased natriuresis, but SGLT2 inhibitors still lower blood pressure at low glomerular filtration rates.6 The main non-natriuretic mechanism is the relaxation of arterial smooth muscle and the associated reduction in per- ipheral resistance. Previous ex vivo studies showed that empagliflozin and da- pagliflozin relaxed rabbit aortic rings, rat mesenteric arteries, and rat left anterior descending coronary artery, but the underlying mechanisms were ill defined and often contradictory. In rabbit aortic rings, dapagliflozin- and empagliflozin-mediated relaxations were impaired by protein kinase G inhi- bitors and the non-specific Kv channel blocker 4-aminopyridine (4-AP),7,8 but specific blockers of Kv subfamilies (Kv1.5, Kv2.1, and Kv7s) did not im- pair relaxations. In contrast, SGLT2 inhibitors relaxed mesenteric resistance arteries in an endothelium-independent manner that was sensitive to block- ers of Kv1.5 and Kv7 potassium channels9–11 without an effect on protein kinase G. In the left anterior descending coronary arteries, a significant relax- ation was only observed with a high concentration of dapagliflozin (500 µM) and the relaxation was not affected by a range of potassium channel block- ers.12 In this study, we aimed to investigate whether SGLT2 inhibitors were acting via a shared upstream mechanism that could explain the vascular- dependent effects observed in many of the trials, as well as the artery- specific differences that have been reported. Resistance arteries that dictate blood pressure are richly innervated with peptidergic sensory nerves.13,14 Release of calcitonin gene-related peptide (CGRP) from sensory nerves has a pronounced vasodilatory effect in many arteries.15–17 CGRP has been shown to have beneficial effects in hyperten- sive and heart failure patients. Animal studies suggest that CGRP has vascular-dependent and vascular-independent processes by which it could protect the vasculature and myocardium against cardiovascular 2 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 dysfunction, particularly heart failure,18 to a similar extent as the SGLT2 in- hibitors. The artery-specific effects of SGLT2 inhibitors reported previous- ly are somewhat in line with CGRP-induced relaxation. Kv7 channels have been shown to be downstream mediators of CGRP signaling,19–21 and CGRP can increase NO production. The present study aimed to ascertain whether SGLT2 inhibitors promoted CGRP release from perivascular sensory nerves, which would explain not only the vascular-dependent car- diovascular protective effects of SGLT2 inhibitors but also the vascular- independent protective effects. 2. Methods 2.1 Animals Experiments were performed on second-order mesenteric and cardiac septal resistance arteries as well as conduit renal arteries from male rats aged 11–14 weeks and weighing 175–300 g. Animals were maintained un- der an institutional site licence and sacrificed by a Schedule 1 method (cer- vical dislocation) in accordance with the UK Animal (Scientific Procedures) Act 1986; therefore, no approval from a local or university ethics review board was required. This investigation conforms to Directive 2010/63/ EU of the European Parliament on the protection of animals used for sci- entific purposes. 2.2 Myography Arteries were cut into ∼2 mm segments and mounted on 40 µm stainless steel wires in a myograph (DMT, Aarhus, Denmark). The myograph chambers contained physiological salt solution (composition in Supplementary material online, Section S1.9) that was bubbled with 95% oxygen and 5% carbon dioxide at 37°C. Tension in each segment was recorded using LabChart Pro Software (ADInstruments, Oxford, UK). All vessels were subject to a normalization procedure22 to stand- ardize the experimental conditions, and arteries were set to an internal cir- cumference 90% of the diameter at in vivo transmural pressure (13.3 or 10.3 kPa for septal arteries). Endothelial integrity was estimated by the re- sponse to 10 µM carbachol applied to arteries constricted with 10 µM of the α1-adrenoreceptor agonist, methoxamine. The endothelium was de- nuded by mechanical abrasion with an eyebrow hair, and effectiveness of re- moval was ascertained by a carbachol challenge in all experiments with septal arteries. Arterial segments were pre-contracted with 10 µM methoxamine and dapagliflozin, empagliflozin, or mizagliflozin applied cumulatively (1–100 µM). The Ki for these agents to inhibit SGLT2 is 1.2, 3.1, and 8170 nM, respectively.1,23 Similar experiments were performed with the NHE1 in- hibitor cariporide (1 and 30 µM), CGRP (10 pM–10 nM), and capsaicin (10 µM). To identify possible underlying mechanisms, arteries were pre- incubated with a variety of agents for 15 min including the following: solv- ent control dimethyl sulfoxide (DMSO), linopirdine (pan-Kv7 channel blocker, 10 µM), BIBN-4096 (CGRP receptor blocker, 1 µM), capsaicin (TRPV1 agonist, 10 µM), AMG-517 (TRPV1 blocker, 1 µM), AM0902 (TRPA1 channel blocker, 10 µM), HMR-1556 (Kv7.1 channel blocker, 10 µM), iberiotoxin (BKCa channel blocker, 100 nM), 4-AP (1 mM), tet- raethylammonium (TEA, 1 mM), and glibenclamide (KATP channel blocker, 1 and 3 µM). To confirm that sensory nerve-derived mediators were involved with SGLT inhibitor responses, we used repeated chal- lenges of the TRPV1 agonist capsaicin (10 µM) to deplete CGRP con- tent in sensory nerves.14,16 Capsaicin (10 µM) was applied to relaxed arteries for 5 min followed by washout to remove any released CGRP. Two further challenges of capsaicin were applied followed by extensive washout over 10 min before contraction with methoxamine (depletion protocol). 2.3 Immunohistochemistry After completion of functional myography experiments, arterial segments were fixed in situ in myograph chambers with 4% paraformaldehyde (J61899, Thermo Scientific) for 1 h at room temperature. Arteries were then incubated for 90 min at room temperature with blocking buffer con- taining permeabilization agents [1% bovine serum albumin (BSA), 0.5% Triton X-100, 0.05% Tween 20 in phosphate buffered saline (PBS), com- position in Supplementary material online, Section S1.9] and incubated overnight at 4°C with guinea pig anti-TRPV1 (Ab10295, 1:1000, Abcam), goat anti-CGRP (Ab36001, 1:1000, Abcam), rabbit anti-NHE1 (PA5115917, 1:1000, Invitrogen), mouse anti-SGLT2 (sc-393350, 1:200, Santa Cruz), rabbit anti-smooth muscle myosin heavy chain 11 (ab125884, 1:500, Abcam), and goat anti-mouse CD31/PECAM-1 (AF3628-SP, 1:150, R&D Systems) diluted in blocking buffer. This was fol- lowed by a secondary antibody incubation with either goat anti-guinea pig (Alexa Fluor 488, A11073, Life Technologies), donkey anti-goat (Alexa Fluor 633, A21082, Life Technologies), donkey anti-rabbit (Alexa Fluor 488, A21206, Life Technologies), or donkey anti-mouse (Alexa Fluor 594, A21203, Invitrogen) diluted in blocking buffer for 90 min at room tem- perature. Arteries were then placed in mounting medium (Vectashield Plus Antifade, Vector Laboratories) and laid flat between two glass coverslips. Arteries were excited at 405, 488, 536, and 635 nm, and fluorescence was acquired through a water immersion objective (1024 × 1024 pixels; ×40, 1.15 NA objective, Olympus) using a FV1000 laser scanning confocal microscope (Olympus, Southend-on-Sea, UK). Z-stacks were acquired through each artery wall in 1 µm increments using Fluoview (version 4.1, Olympus) software and analysed offline using Imaris (version 8.0.2, Bitplane) software. 2.4 Western blot and immunocytochemistry SGLT2 expression was identified by western blot and immunocytochem- istry (ICC). Protein lysates were prepared from whole mesenteric arcade, right and left renal arteries, and a combination of septal and left anterior des- cending artery using Triton buffer (Fisher Scientific) supplemented with pro- tease and phosphatase inhibitors (cOmplete, mini, and PhosSTOP from Roche). Protein concentrations were determined via the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Loughborough, UK). Ten micro- grams of each artery sample was run under reducing conditions with 4–12% Bolt™ Bis-Tris Plus pre-cast gels (Invitrogen), and proteins were transferred to a nitrocellulose membrane. Membranes were blocked for at least 0.5 h in 3% BSA–PBS and incubated overnight at 4°C with the primary SGLT2 mouse monoclonal antibody (D-6, sc-393350, 1/200 dilution, Santa Cruz). The membranes were incubated with highly adsorbed horseradish peroxidase-conjugated goat anti-mouse IgG (A16078, Fisher Scientific) for 1 h at room temperature and developed using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). Full quantification protocol is in the Supplementary material. For ICC, vascular smooth muscle cells (VSMCs) were isolated from six mesenteric branches, right and left main re- nal arteries, and whole septal arteries (protocol and solution composition in Supplementary material online, Section S1.9), and SGLT2 localization was identified using a mouse anti-SGLT2 antibody (dilution 1:200, Santa Cruz, TX, USA) with a donkey anti-mouse secondary antibody conjugated to Alexa Fluor 488 (dilution 1:100, Thermo Fisher, Paisley, UK). Wheat Germ Agglutinin (WGA) Texas Red was used as a membrane stain before permeabilization. 2.5 Electrophysiology A possible direct effect of dapagliflozin and empagliflozin was assessed on currents generated by the over-expression of Kv7 and TRPV1 genes in Xenopus laevis oocytes and HEK293 cells, respectively. Cell culture and chan- nel expression methods are described fully in the Supplementary material. Potassium currents generated by the expression of KCNQ4, KCNQ5, and KCNE4 (accepted molecular combination in arterial smooth muscle24) were recorded using two-electrode voltage clamp at room temperature using an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) and pClamp10 software (Molecular Devices, Sunnyvale, CA, USA) 2–5 days after cRNA injection. TRPV1 currents were recorded from transi- ently transfected HEK293 cells using the patch-clamp technique in the outside-out configuration. TRPV1-dependent currents were activated using a sub-saturating concentration (250 nM) of capsaicin (see Ortíz-Rentería SGLT2 inhibitors and arterial sensory nerves 3 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 et al.25). Currents were low-pass filtered at 2 kHz and sampled at 10 kHz with an EPC 10 amplifier (HEKA Elektronik) and were plotted and analysed with Igor Pro (WaveMetrics Inc.). All internal and external solutions are shown in the Supplementary material online, Section S1.9. 2.6 Morpholino studies As NHE1 knockout mice have a short lifespan,26 studies were performed ex vivo using translation-stopping morpholinos to reduce NHE1 expression with the assumption that local translation of mRNA occurred in the sen- sory neurites like in motor nerves.27 Knockdown of NHE1 in mesenteric arteries was performed by transfection with morpholino oligonucleotides targeting NHE1 or a scrambled control as described previously.28 All mor- pholino oligonucleotides (5 µM; Gene Tools, Oregon, USA) were mixed in Opti-MEM and transfected using Lipofectamine 2000 (Thermo Fisher, Paisley, UK). Arteries were then incubated in DMEM/F-12 with 1% penicil- lin/streptomycin for 48 h. Transfected arteries tended to lose tone in the continued presence of methoxamine so a modified protocol was used to study the effect of SGLT2 inhibitors in these conditions. Methoxamine (10 µM) was applied for 4 min followed by washout for 20 min. This was repeated, and then 30 µM empagliflozin or dapagliflozin was applied 5 min before the third application of methoxamine. Arteries were fixed in situ and permeabilized, and NHE1 staining was detected to ascertain NHE1 knockdown. 2.7 Data and statistical analysis All values from functional experiments are expressed as mean ± standard error of the mean (SEM) with no less than five individual data points, each representing a biological repeat. Measurements of total cell florescence dur- ing ICC involved five biological repeats with a minimum of five cells to be re- corded per sample. For quantification of SGLT2 protein via western blot, a minimum of three biological repeats were obtained. For functional experi- ments, cumulative concentration effect curves were produced, whereby the contraction produced by 10 µM methoxamine at stable tone was taken as the maximal contraction of 100%. The tone of the artery was recorded after each subsequent addition of the pharmacological agent, and the values were formulated as a percentage of the maximum contraction. Using GraphPad Prism (RRID:SCR_002798, Version 9.0.0), a transformed data set of mean values was generated using X = Log(X ) to reduce representative skew. A four-parametric linear regression analysis was then performed to produce a concentration effect curve on a log(x) graph with the SEM. When comparing multiple groups, a two-way analysis of variance (ANOVA) was performed followed by a post hoc Bonferroni or Dunnett’s test. For data comparing two groups, an unpaired parametric t-test was per- formed. Significance values are represented as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, and data sets subject to statistical analysis con- tained at least five animals per group, where N = number of independent values. 3. Results 3.1 Expression of SGLT2 in mesenteric and renal arteries from male Wister rats The expression of SGLT2 within the vasculature is ill defined; hence, we characterized its expression in different vascular beds. A band at the expected molecular weight for SGLT2 (∼75 kDa) was detected in protein lysates from kidney (positive control), mesenteric, renal, and coronary arteries (Figure 1A and B). Additional bands of ∼49and ∼33 kDa were also detected corresponding to the presence of the other known isoform for SGLT2 and/or products of degradation. Immunocytochemical studies on VSMCs showed a strong co- localization of SGLT2 with the plasma membrane marker WGA, sug- gesting a robust expression in the membrane of VSMCs (Figure 1C–E). We performed immunohistochemistry experiments with well- validated CGRP and TRPV1 antibodies to delineate sensory nerves in the different arteries. Figure 1F shows robust TRPV1 and CGRP staining in the adventitia of mesenteric arteries with comparatively little staining in the smooth muscle or endothelial layers (see Supplementary material online, Figure S1). In contrast, negligible TRPV1 or CGRP staining was identified in the adventitial layer of the renal artery (Figure 1G; Supplementary material online, Figure S1), and none was de- tected in a septal artery (Figure 1H). Thus, mesenteric arteries exhibit ro- bust sensory nerve networks that were not present in renal or cardiac septal arteries. 3.2 SGLT2 inhibitors relax mesenteric arteries In rat mesenteric artery segments, dapagliflozin and empagliflozin, as well as a SGLT1 inhibitor, mizagliflozin, produced concentration-dependent re- laxations (1–100 µM), with approximate IC50 values of 9.5, 7.3 and 5.5 µM, respectively (Figure 2A and B; n = 5–6). The relaxation elicited by each SGLT inhibitor was not affected by the size of the induced contraction and not dependent upon a functional endothelium (see Supplementary material online, Figures S2 and S3). In contrast to their effect on mesenteric arteries, dapagliflozin and empagliflozin were significantly poorer relaxants of pre-contracted renal arteries and were ineffective relaxants of septal ar- teries (up to 100 µM; Figure 2C and D). 3.3 Role of Kv7 channels in SGLT2 inhibitor-induced relaxations Previous studies implicated Kv channels encoded by KCNQ genes (Kv7 channels) in the relaxant response to different SGLT2 inhibitors.9–11 In the present study, the relaxation of mesenteric arteries produced by da- pagliflozin, empagliflozin, and mizagliflozin was significantly attenuated by pre-incubation with the pan-Kv7 channel inhibitor linopirdine (10 µM) when compared to DMSO control (Figure 3A and B; n = 5–6). Specific blockers of Kv7.1 (HMR1556), BKCa (iberiotoxin), KATP (glibenclamide), and the non-selective K channel blockers 4-AP and TEA had no effect on relaxations produced by SGLT2 inhibitors (Figure 3C and D; Supplementary material online, Figure S5). However, electrophysiological experiments on oocytes co-expressing KCNQ4/KCNQ5 and KCNE4—a molecular combination found in most arterial smooth muscle24 showed that neither dapagliflozin nor empagliflozin at 100 µM had any effect on the current amplitude nor the voltage dependence of activation of the Kv currents and neither agent affected the resting membrane potential (Figure 3E–J). Thus, dapagliflozin and empagliflozin do not activate Kv7 channels directly. 3.4 Sensory nerve contribution in mesenteric and renal arteries Kv7 blockers attenuate arterial relaxations produced by several agonists of Gs-linked receptors.19–21 As Kv7 channels are not directly activated by SGLT2 inhibitors, a logical conclusion is that these agents promoted the re- lease of a mediator that recruited Kv7 channels. As dapagliflozin- and empagliflozin-mediated relaxations were not endothelium dependent (see Supplementary material online, Figure S3), we focused on CGRP released from the sensory nerves and the different effects of the SGLT2 inhibitors be- tween mesenteric and renal or septal arcades were due to a differential abundance of sensory nerves (see Figure 1). CGRP can be released from sen- sory neurones upon TRPV1 channel activation.14 Applying the TRPV1 acti- vator capsaicin fully relaxed pre-contracted mesenteric arteries (Figure 4A), which was abrogated by the CGRP receptor blocker BIBN-4096 (see Supplementary material online, Figure S4A). Consistent with the lack of sen- sory nerves (Figure 1F and G), capsaicin had no effect in pre-contracted renal or septal arteries (Figure 4A). Linopirdine inhibited mesenteric artery relaxa- tions produced by exogenous CGRP or SGLT2 inhibitors to a similar extent (Figure 4B), consistent with our hypothesis that SGLT2 inhibitor-induced re- laxations are mediated by CGRP. 4 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 Figure 1 SGLT2 expression in mesenteric, renal, and coronary arteries. (A) Western blot quantification of SGLT2 protein in mesenteric, renal, and coronary arteries with whole kidney as a positive control (N = 3). Quantification of the western blot in mesenteric (left), renal (centre), and coronary (right) arteries shown in (B). (C ) Representative staining of SGLT2 (middle row) and membrane stain WGA (top row) in isolated mesenteric, renal, and septal coronary VSMCs (N = 5, n = 25) with total cell fluorescence in (D) and membrane-to-cytosol ratio of SGLT2 expression in each artery in (E). All values were shown as mean ± SEM denoted by error bars, and a one-way ANOVA was used to calculate significance where *P < 0.05 and **P < 0.01. (F–H ) show representative labelling for TRPV1 (green, left column) and CGRP (magenta, middle column) indicative of sensory nerve presence in the adventitia of whole mesenteric (F), renal arteries (G), and septal arteries (H). Nuclei were labelled in blue. Similar images seen in arteries from 4 animals). Non-significance is shown by ns. SGLT2 inhibitors and arterial sensory nerves 5 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 A e K~Y Men nterlc; ~Oo - , ~~ , ~~ , . ~ - ------ ..... B /' ... .1" . ' F G H -- LO •• , " , . t n 3.5 Dapagliflozin- and empagliflozin-evoked relaxations are sensitive to CGRP and TRPV1 blockade To identify a role for CGRP in SGLT2 inhibitor-induced relaxations, we ap- plied dapagliflozin, empagliflozin, and mizagliflozin to mesenteric arteries pre-incubated with either DMSO (control) or BIBN-4096 (1 µM). The re- laxation to all three agents was significantly attenuated by 1 µM BIBN-4096 (Figure 4C–E). To confirm that SGLT2 inhibitors relaxed mesenteric arter- ies through provoking CGRP release from sensory nerves, we depleted CGRP stores through treatment with three 5-min applications of capsaicin (10 µM) followed by washout of the bathing solution or directly blocked TRPV1 with AMG-517 (1 µM). Both treatments prevented the relaxation produced by 1 µM capsaicin (see Supplementary material online, Figure S4B and C). The relaxations produced by both dapagliflozin and empagliflozin were significantly attenuated after treatment with capsaicin or incubation with the TRPV1 blocker, AMG-517 (Figure 4F and G, N = 6–10). The mizagliflozin-induced relaxation was also sensitive to AMG-517 (Figure 4H). The relaxation to dapagliflozin was not affected by pre- application of the TRPA1 blocker AM0902 (1 or 10 µM, Supplementary material online, Figure S5). Neither the TRP blockers nor capsaicin pre- treatment affected methoxamine-induced contraction amplitude (see Supplementary material online, Figure S6). Hence, relaxations induced by SGLT2 inhibitors were dependent upon activation of the TRPV1 channel on perivascular sensory nerves and subsequent CGRP release. 3.6 Effect of SGLT2 inhibitors on heterologously expressed TRPV1 currents To determine whether dapagliflozin could activate TRPV1 channels direct- ly, we performed patch-clamp experiments using outside-out excised membrane patches of HEK293 cells expressing rat TRPV1 (rTRPV1). As shown in Figure 5, we first obtained the leak currents (in the absence of agonist, grey traces) elicited by a square voltage pulse to −120 mV fol- lowed by a pulse to 120 mV, then used the same voltage protocol to as- sess currents after exposing the patches to either 30 μM (Figure 5A) or 100 μM dapagliflozin (Figure 5B) for 5 min and, finally, to 250 nM capsa- icin alone (black traces). All currents were leak subtracted and normal- ized to the current obtained at +120 mV (Figure 5C). Currents after exposure to 30 μM dapagliflozin were 12.9 ± 2.2 and 7.6 ± 3.7% after 100 μM dapagliflozin of the currents elicited by the TRPV1 agonist, cap- saicin (Figure 5C, n = 6). These data indicate that TRPV1 is not directly activated by dapagliflozin. Next, we studied whether dapagliflozin could potentiate capsaicin- or low pH-activated TRPV1 currents. For this set of experiments, we first recorded leak currents (grey traces), then activated TRPV1 in outside- out excised membrane patches of HEK293 cells with either a sub- saturating concentration (250 nM) of capsaicin or with a solution at pH 6, then washed the membrane patches to close the channels and exposed the patches to 30 μM dapagliflozin for 5 min and remeasured the currents in the presence of 250 nM capsaicin or solution with low Figure 2 The effect of SGLT2 and SGLT1 inhibitors on mesenteric, renal, and septal arterial tones. (A) A representative trace of the effect of dapagliflozin in mesenteric arteries pre-contracted with 10 µM methoxamine. (B) Mean effect of dapagliflozin (blue), empagliflozin (green), and mizagliflozin (purple), with mean vehicle control in grey, N= 5–6. All values are shown as mean ± SEM denoted by the error bars (N = 6–8). (C and D) show the relaxation of dapagliflozin and empagliflozin in renal mesenteric (left hand data set), renal (middle data set), and septal (right hand data set) arteries. All data are individual experiments and a two-way ANOVA with a post hoc Sidak test was used to calculate significance values where **, ***, and **** denote P < 0.01, 0.001 and 0.0001 respectively. Non-significance is shown by ns. 6 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 extracellular pH (Figure 5D–G). The results from these experiments in- dicate that 30 μM dapagliflozin did not potentiate TRPV1 currents ac- tivated by 250 nM capsaicin (Figure 5D and E; 84.1 ± 13.5%, n = 5) or by low pH (Figure 5F and G; 94 ± 6.5%, n = 5). Thus, SGLT2 inhibitors nei- ther activate TRPV1 directly nor sensitize the channel to known mediators. 3.7 NHE1 co-localizes with TRPV1 and cariporide prevents SGLT2 inhibitor-induced responses Various studies have shown that SGLT2 inhibitors block NHE iso- forms29–34 with in silico studies predicting a binding site in the extracellular Figure 3 SGLT2 inhibitors and Kv7 channels. (A) Representative trace of the effect of dapagliflozin on pre-contracted mesenteric arteries in the presence (upper trace/blue) and absence (lower trace) of 10 µM linopirdine. (B) Mean data for relaxations to dapagliflozin, empagliflozin, and mizagliflozin (30 µM) in solvent control (left data set) and when pre-incubated with 10 µM linopirdine (right data set/blue) (N = 5–6). All values are individual experiments with mean ± SEM denoted by the error bars. A two-way statistical ANOVA with a post hoc Sidak test was used to generate significant values (*P < 0.05, **P < 0.01, and ****P < 0.0001). The effect of dapagliflozin (C ) and empagliflozin (D) in the absence and presence of HMR1556, iberiotoxin, 4-AP, TEA, and glibenclamide (data sets left to right respectively). All data values are shown as mean ± SEM (N = 5–6). (E–G) show currents produced by the co-expression of Kv7.4, Kv7.5, and KCNE4 in the absence and presence of 100 µM dapagliflozin. Representative traces in (E), mean current–voltage relationship in (F ), and mean membrane potential in (G). The effect of 100 µM empagliflozin on currents produced by co-expression of Kv7.4, 7.5, and KCNE4 is shown in (H–J ). Representative traces in (H ), mean current–voltage relationship in (I), and mean membrane potential in (J ). Data are the mean of N oocytes with error bars denoting the SD. SGLT2 inhibitors and arterial sensory nerves 7 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 sodium-binding pocket of NHE1.29 We postulated that SGLT2 inhibitors promoted release of CGRP from sensory nerves by inhibiting NHE and pro- ducing a localized pH change sufficient to activate TRPV1. Functional experiments revealed that the NHE1 inhibitor cariporide29 relaxed mesen- teric arteries, which were markedly impaired by BIBN-4096 or AMG-517 pre-treatment (Figure 6A and C). Cariporide was ineffective at relaxing Figure 4 Dapagliflozin-, empagliflozin-, and mizagliflozin-induced relaxations are blocked by CGRP receptor antagonist and TRPV1 blockade. (A) shows the percentage relaxation to 10 µM capsaicin in mesenteric (left hand data set), renal (middle data set), and septal arteries (right hand data set) (N = 6–8). (B) shows the mean relaxation to CGRP in the absence and presence of 10 µM linopirdine (blue, N = 5–8). All data points are represented as mean ± SEM denoted by the error bars. A two-way statistical ANOVA with a post hoc Bonferroni test was used to generate significant values (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). The mean effect of dapagliflozin (C, N = 6), empagliflozin (D, N = 7), and mizagliflozin (E, N = 5) on pre-contracted mesenteric arteries in the presence of DMSO (left data set) and 1 µM BIBN (right data set). The mean relaxations produced by 1 and 30 µM dapagliflozin (F, N = 7–10) and empagli- flozin (G, N = 6–10) in mesenteric arteries pre-incubated in DMSO (solvent control, black), 1 µM AMG-517 (right hand data set), or after sensory nerve de- pletion with 10 µM capsaicin (centre data set). The relaxation to mizagliflozin in theabsence and presence of TRPV1 blocker AMG-517 (right hand data set) is shown in (H ) (N = 5). All values are expressed as mean ± SEM. A two-way statistical ANOVA with a post hoc Sidak test was used to generate significant values (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). 8 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 pre-contracted renal or septal arteries (Figure 6B). We stained mesenteric arteries with SGLT2 and NHE1 antibodies and used TRPV1 to delineate the sensory nerves. As shown in Figure 6D, prominent NHE1 staining was observed in the adventitia of mesenteric arteries co-localized with TRPV1 and some staining in the smooth muscle and endothelial layers (see Supplementary material online, Figure S7). In contrast, negligible staining of SGLT2 was observed in the adventitial and endothelial layers, but traces of staining were identified in the smooth muscle layer of mesenteric arteries (see Supplementary material online, Figure S7), consistent with the staining identified in dispersed single smooth muscle cells (Figure 1C–E). No staining Figure 5 SGLT2 inhibitors do not activate TRPV1 directly. (A and B) Representative traces of currents at +120 and −120 mV from outside-out membrane patches of HEK293 cells expressing TRPV1. Leak currents were obtained in the absence of any agonist and after 5-min application of dapagliflozin (DAPA) 30 μM (blue traces, A) and 100 μM (green traces, B). The top and bottom traces shows the subsequent effect of 250 nM capsaicin. (C ) Average data for ex- periments in (A and B). Currents were leak subtracted, and data were normalized to activation by capsaicin 250 nM in the steady state at +120 mV (N = 6 and N = 5 for DAPA 30 and 100 μM, respectively). (D) Representative traces of currents at +120 and −120 mV from outside-out membrane patches of HEK293 cells expressing TRPV1 in control conditions (grey), after application with 250 nM capsaicin (black traces) and after application of 250 nM capsaicin + 30 μM DAPA for 5 min (lilac traces). (E) The data in (D) were normalized by dividing the currents obtained at +120 mV in response to 250 nM capsaicin + 30 μM DAPA by the currents in response to 250 nM capsaicin alone; (N = 5). (F ) Representative traces of currents at +120 and −120 mV from outside-out membrane patches of HEK293 cells expressing TRPV1 under control conditions (grey), after activation of TRPV1 by pH 6 (black) and after 5-min application of 30 μM DAPA to pH 6 conditions (blue). (G) The data in (F ) were normalized by dividing the currents obtained at +120 mV in response to pH 6 + 30 μM DAPA by the currents in response to pH 6 alone (N = 6). Group data are reported as the mean ± SEM. SGLT2 inhibitors and arterial sensory nerves 9 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 for NHE1 or SGLT2 was detected in renal artery adventitia (Figure 6E; Supplementary material online, Figure S8). Therefore, NHE1 co-localized with TRPV1 in sensory nerves in the mesenteric artery, but SGLT2 was only found in the smooth muscle layer. In mesenteric arteries, pre-treatment with 30 µM cariporide reduced the relaxations to 30 µM dapagliflozin or 30 µM em- pagliflozin significantly (P < 0.001; Figure 6F–H). These experiments suggest that vasodilatory effects of SGLT2 inhibitors were mediated by NHE1 inhibition. To confirm this, we used morpholino-based molecular interference to reduce NHE1 protein expression in mesenteric arteries. In control arteries trans- fected with non-targeting scrambled morpholinos, 5-min application of either 30 µM empagliflozin or dapagliflozin reduced contractions produced by the subsequent application of 10 µM methoxamine by >70% (Figure 7A and B; Supplementary material online, Figure S9). However, in arteries transfected with translation-blocking morpholinos targeted against NHE1, empagliflozin or dapagliflozin had a negligible effect on methoxamine-induced contractions (Figure 7A and B). Immunohistochemical studies on the same arteries revealed that targeting morpholinos reduced NHE1 staining in the adventitia consider- ably compared to scrambled morpholinos (Figure 7C). These studies corrob- orate our hypothesis that SGLT2 inhibitors relax mesenteric arteries through NHE1 inhibition-induced release of CGRP. 4. Discussion This study investigated the underlying mechanism of SGLT2 inhibitor- induced vasorelaxation and identified a role for CGRP release from sensory nerves secondary to NHE1 inhibition. We show that structurally different SGLT inhibitors relaxed mesenteric arteries equipotently, divergent from their ability to block SGLT2 (or SGLT1)-mediated glucose transport.1,23 Relaxations produced by SGLT inhibitors were sensitive to TRPV1 and Kv7 channel blockade; however, electrophysiology recordings showed that SGLT2 inhibitors did not activate vascular Kv7 or TRPV1 channels directly. In addition, relaxations to dapagliflozin, empagliflozin, and the SGLT1 inhibi- tor mizagliflozin were attenuated by CGRP receptor blockade or by deple- tion of sensory nerve transmitters through capsaicin challenge. In contrast, SGLT2 inhibitors were poor relaxants of pre-contracted conduit renal and ineffective in septal resistance arteries where sensory nerves, evinced by staining for CGRP and TRPV1 in the adventitia, were absent compared to mesenteric arteries. The NHE1 blocker cariporide also relaxed mesenter- ic arteries, which was prevented by CGRP receptor and TRPV1 blockade, but had no effect in renal or septal arteries. Strikingly, NHE1 but not SGLT2 proteins co-localized with TRPV1 in the sensory nerves, and pre- application of cariporide or transfection with NHE1-targeted morpholino attenuated the inhibitory effects of empagliflozin and dapagliflozin. The study provides strong evidence that SGLT2 inhibitors influence arter- ial reactivity by promoting the release of CGRP from sensory nerves. As the density of sensory nerve innervation varies across the vasculature and within an artery (see staining in Figure 1), this seminal finding explains much of the variability in data seen in previous publications.9–11 Interestingly, the effect- iveness of zinc pyrithione, which also relaxes arteries through a release of CGRP from sensory nerves, was far greater in mesenteric arteries compared to renal and coronary arteries35 consistent with our observations with SGLT inhibitors. While SGLT2 is present in arterial smooth muscles, our pharma- cological studies and comparative imaging suggest that the effects of the gli- flozins are not mediated by an action on SGLT2 per se but via an additional effect of these agents on NHE1, as reported previously.29–31 This hypothesis was corroborated here by molecular knockdown of NHE1. Interestingly, a previous study in human visceral adipose arteries speculated NHE1 may be involved in the moderate relaxation produced by canaglifozin.31 4.1 SGLT2 inhibitors induce arterial relaxations The present study shows that SGLT2 inhibitors relaxed pre-contracted mesenteric arteries at concentrations between 10 and 100 µM, in general agreement with previous work in mesenteric arteries and aortic rings.7–11 This is slightly higher than the therapeutic plasma concentrations in humans, which for empagliflozin is about 0.3 µM for the commonly pre- scribed dose of 10 mg and 8 µM for the maximum dose of 800 mg.36 But in an ex vivo setting, small changes in tension are hard to relate to the physiological impact of resistance and flow. It is common to use higher concentrations of drugs in ex vivo studies to delineate cellular processes. The present study shows that the mesenteric artery relaxations produced by dapagliflozin and empagliflozin, as well as mizagliflozin, were not sensi- tive to a range of K channel blockers (HMR-1556, iberiotoxin, 4-AP, TEA, and glibenclamide) but were sensitive to the pan-Kv7 channel blocker linopirdine, in agreement with earlier work in mesenteric arteries.9–11 However, our electrophysiological recordings showed that neither dapagli- flozin nor empagliflozin directly enhanced potassium currents in oocytes expressing Kv7.4, Kv7.5, and KCNE4 (the combination in arterial smooth muscle24), suggesting that Kv7 channel activation is secondary to the release of a chemical intermediate. We have previously found that Kv7 channel-specific blockers impaired CGRP-induced relaxations in cerebral and mesenteric arteries19–21 so we propose that these chan- nels are a functional endpoint of a relaxant cascade that involves CGRP release from sensory nerves. 4.2 The role of CGRP in arterial effects of SGLT2 inhibitors Our hypothesis is that the arterial relaxation produced by the structurally dissimilar SGLT2 and SGLT1 inhibitors was mediated predominantly by CGRP release from perivascular sensory nerves.14 Thus, empagliflozin, da- pagliflozin, or mizagliflozin relaxed mesenteric arteries that were impaired by blocking the CGRP receptor with BIBN-4096, although the lack of com- plete inhibition suggests other neuropeptides such as substance P may also be released. Immunohistochemistry with validated antibodies for TRPV1 or CGRP showed that mesenteric arteries had dense sensory nerve net- works in the adventitia. In contrast, SGLT2 inhibitors were poor or inef- fective relaxants in renal and septal arteries, respectively, that correlated with negligible or no TRPV1 or CGRP staining in the adventitia. The SGLT2 inhibitor-induced relaxations of mesenteric arteries were equally prevented by the application of the TRPV1 blocker AMG-517 or by deple- tion of CGRP through capsaicin treatment but not by the TRPA1 inhibitor AM0902. This suggests that the recruitment of TRPV1 rather than TRPA1 channels in sensory nerves is a key step in SGLT2 inhibitor-induced relaxa- tions in the mesenteric artery. TRPV1 is a polymodal cation channel regulated by various exogenous and endogenous activators. These include noxious chemicals (capsaicin or vanilloids), low pH (<6.0), high temperatures >43°C,37 lipid mediators (i.e. anandamide), lipoxygenase products (e.g. LTB4),38 and several signal- ling molecules (NGF, ATP, and PAR-2 agonists).39,40 The subsequent influx of cations through TRPV1 is sufficient to promote fusion of synaptic vesi- cles containing CGRP and other neuropeptides. Our data are consistent with SGLT2 inhibitors relaxing mesenteric arteries by promoting CGRP release in a manner dependent upon TRPV1 channels. However, in over- expression systems, dapagliflozin and empagliflozin failed to either activate TRPV1 currents or enhance the effect of low pH or capsaicin, suggesting that these agents do not work directly on the channel. Therefore, TRPV1 activation is a consequence of an additional mechanism. 4.3 NHE1 and arterial relaxation NHE1 plays a primary role in cardiomyocytes and VSMCs to maintain cel- lular pH levels at ∼7.2.41,42 Altered NHE expression and activity have been linked to severe cardiac events, where during ischaemia, the pH change activates NHE, leading to cardiac injury.42 Many of the cardiopro- tective effects of SGLT2 inhibitors have been linked to reduced Na+ load and pH development in cardiomyocytes through an effect on NHE1,29–31 although this has been disputed.43 However, an arterial role for NHE1 suppression in the clinical benefit of SGLT2 inhibitors has not been de- monstrated, although a role for NHE1 inhibition in relaxations of human visceral arteries by canagliflozin was speculated.31 NHE1 activation is linked to vasoconstriction and enhances the myogenic response in mouse 10 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 Figure 6 Effect of cariporide and NHE1 localization in mesenteric arteries. (A) A representative trace of the relaxation to 1 and 30 µM cariporide in mes- enteric arteries pre-contracted with 10 µM methoxamine. (B) Mean percentage relaxation to 1 and 30 µM cariporide in mesenteric (left hand data), renal (middle data set), and septal arteries (right hand data set) (N = 6–10). (C ) shows the mean percentage relaxation to cariporide (1–30 µM) in pre-contracted mesenteric arteries when incubated with DMSO (left) and 1 µM BIBN (right) (N = 5–6). (D and E) show representative labelling for TRPV1 alone (green, top) and with NHE1 (magenta, bottom) in the adventitia of whole mesenteric (D, N = 3) and renal (E, N = 4) arteries. Nuclei were labelled in blue. (F ) Representative trace of the relaxation to empagliflozin (1–30 µM) in mesenteric arteries pre-contracted with 10 µM methoxamine in the presence (orange) and absence (black) of cariporide. The mean data for the response to 1 and 30 µM dapagliflozin (N = 5) and empagliflozin (N = 5) in the presence (top trace) and absence (bottom trace) of 10 µM cariporide are shown in (G and H ). All data are individual experiments with the mean ± SEM denoted by the error bars. A two-way statistical ANOVA with a post hoc Sidak test was used to generate significant values (*P < 0.05, **P < 0.01, and ****P < 0.0001). SGLT2 inhibitors and arterial sensory nerves 11 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 resistance arteries.44 Increased NHE1 activity is also implicated in pulmon- ary artery hypertension, proliferation, and remodelling,45 with the protein regulating pH or acting as a protein anchor. We propose that NHE1 lo- cated in the sensory nerves also has a profound effect in arteries because they influence TRPV1 activity and the subsequent cation influx precipitates vesicular release of potent vasodilators including CGRP. Thus, in the pre- sent study, relaxations to SGLT2 inhibitors and the NHE1 blocker caripor- ide30 were prevented by a CGRP receptor antagonist and TRPV1 blocker. Moreover, pre-application of cariporide and, crucially, morpholino-based knockdown of NHE1 abrogated the response to dapagliflozin and empagli- flozin. Interestingly, only NHE1 was identified in sensory nerves, unlike in the proximal convoluted tubule, where SGLT2 and NHE1 co-habit in the same microdomain.46 Inhibition of NHE1 in the smooth muscle cells would also influence the contractile state to some degree (see Boedtkjer et al.26), but our work provides robust evidence that SGLT2 inhibitors relax mesenteric arteries via TRPV1-dependent release of CGRP following NHE1 inhibition. Future studies will ascertain the precise mechanisms link- ing NHE1 inhibition and CGRP release. Figure 7 Knockdown of NHE1 impairs the relaxation of dapagliflozin and empagliflozin in mesenteric arteries. (A) A representative trace of the contraction to 10 µM methoxamine in the presence of 30 µM empagliflozin in scrambled control morpholino (black) and knockdown NHE1 morpholino (green/lighter line) arteries. (B) shows the mean data of the contraction to 10 µM methoxamine in scrambled control morpholino and knockdown NHE1 morpholino arteries when pre-incubated with 30 µM dapagliflozin or 30 µM empagliflozin. All data are represented as mean ± SEM denoted by the error bars. A two-way statistical ANOVA with a post hoc Sidak test was used to generate significant values (***P < 0.001 and ****P < 0.0001). (C ) shows representative labelling for NHE1 alone (top image) and with TRPV1 (lower image) in the adventitia of whole mesenteric arteries transfected with scrambled morpholino (Ctrl) and NHE1- targeted morpholino (right hand column). Representative of 4 such experiments. 12 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 Translational perspective SGLT2 inhibitors like empagliflozin are effective hypoglycaemic agents but are also recommended treatments for heart failure and chronic kidney dis- ease independent of circulating glucose. However, the mechanisms underlying the cardiovascular benefit are ill defined. The present study reveals that SGLT2 inhibitors relax arteries ex vivo via the release of calcitonin gene related peptide (CGRP) from sensory nerves, which is mediated by inhibition of sodium/hydrogen exchangers. The ensuing vasodilatation allied to anti-inflammatory, anti-fibrotic, and pro-inotropic actions of CGRP will ameliorate cardiovascular stress. This understanding of the mechanisms that underlie the arterial vasodilatation by these agents may inform future use. 5. Conclusion CGRP is a potent vasodilator of many vascular beds,14 is a safeguard against cardiac ischaemia, and promotes cardiac contractility in failing hearts.18 Our data reveal that the beneficial effects of SGLT2 inhibitors likely stem from an ability to release cardioprotective CGRP into stressed circulations. The ensuing vasodilatation allied to anti-inflammatory, anti-fibrotic, and pro-inotropic actions of CGRP will support effective circulation and help to ameliorate cardiovascular stress. Supplementary material Supplementary material is available at Cardiovascular Research online. Authors’ contributions E.A.F. generated and analysed data, and wrote the manuscript. M.B.-A. and K.E.R. generated and analysed electrophysiological data. K.D. supervised generation of whole artery staining. V.B. and I.S.-C. supervised the PCR, WB, and ICC data and edited the manuscript. A.P.A., T.A.J., G.W.A., and T.R. edited the manuscript. I.A.G. supervised the project, edited the manu- script, and provided the funding for the project. Acknowledgements Dr Lillian Wallis at the Department of Pharmacology, Oxford University, provided expertise in whole artery immunohistochemistry and Itzel Llorente from the Instituto de Fisiología Celular at UNAM performed cell culture and transfection of HEK293 cells with TRPV1. Thanks to Professor Christian Aalkjaer, University of Arhus, for his insightful com- ments about sensory nerves and sodium/hydrogen exchangers. Conflict of interest: none declared. Funding This work was supported by a PhD studentship for E.A.F. (FS/PhD/21/2912) from the British Heart Foundation awarded to I.A.G. G.W.A. was supported by the National Institute of General Medical Sciences (GM130377). K.E.R. was supported by the National Institute of Neurological Disorders and Stroke (T32NS045540). T.A.J. was funded by the Lundbeck Foundation (grant R323-2018-3674), and T.R. was funded by Dirección General de Asuntos del Personal Académico (DGAPA)-Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) (IN200423). Data availability The data underlying this article will be shared on reasonable request to the corresponding author. References 1. Grempler R, Thomas L, Eckhardt M, Himmelsbach F, Sauer A, Sharp DE, Bakker RA, Mark M, Klein T, Eickelmann P. Empagliflozin, a novel selective sodium glucose cotransporter-2 (SGLT-2) inhibitor: characterisation and comparison with other SGLT-2 inhibitors. Diabetes Obes Metab 2012;14:83–90. 2. Wright EM. SGLT2 inhibitors: physiology and pharmacology. Kidney360 2021;2:2027–2037. 3. Preda A, Montecucco F, Carbone F, Camici GG, Lüscher TF, Kraler S, Liberale L. SGLT2 in- hibitors: from glucose-lowering to cardiovascular benefits. Cardiovasc Res 2024;120:443–460. 4. 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Pessoa TD, Campos LCG, Carraro-Lacroix L, Girardi ACC, Malnic G. Functional role of glu- cose metabolism, osmotic stress, and sodium-glucose cotransporter isoform-mediated transport on Na+/H+ exchanger isoform 3 activity in the renal proximal tubule. J Am Soc Nephrol 2014;25:2028–2039. 14 E.A. Forrester et al. D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /c a rd io v a s c re s /a d v a n c e -a rtic le /d o i/1 0 .1 0 9 3 /c v r/c v a e 1 5 6 /7 7 2 1 2 3 6 b y U N A M u s e r o n 1 0 S e p te m b e r 2 0 2 4 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kchl20 Channels ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kchl20 Recent advances on the structure and the function relationships of the TRPV4 ion channel Raúl Sánchez-Hernández, Miguel Benítez-Angeles, Ana M. Hernández-Vega & Tamara Rosenbaum To cite this article: Raúl Sánchez-Hernández, Miguel Benítez-Angeles, Ana M. Hernández-Vega & Tamara Rosenbaum (2024) Recent advances on the structure and the function relationships of the TRPV4 ion channel, Channels, 18:1, 2313323, DOI: 10.1080/19336950.2024.2313323 To link to this article: https://doi.org/10.1080/19336950.2024.2313323 © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 14 Feb 2024. Submit your article to this journal Article views: 1668 View related articles View Crossmark data Recent advances on the structure and the function relationships of the TRPV4 ion channel Raúl Sánchez-Hernández, Miguel Benítez-Angeles, Ana M. Hernández-Vega, and Tamara Rosenbaum Departamento de Neurociencia Cognitiva, División Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico, Mexico ABSTRACT The members of the superfamily of Transient Receptor Potential (TRP) ion channels are physiolo- gically important molecules that have been studied for many years and are still being intensively researched. Among the vanilloid TRP subfamily, the TRPV4 ion channel is an interesting protein due to its involvement in several essential physiological processes and in the development of various diseases. As in other proteins, changes in its function that lead to the development of pathological states, have been closely associated with modification of its regulation by different molecules, but also by the appearance of mutations which affect the structure and gating of the channel. In the last few years, some structures for the TRPV4 channel have been solved. Due to the importance of this protein in physiology, here we discuss the recent progress in determining the structure of the TRPV4 channel, which has been achieved in three species of animals (Xenopus tropicalis, Mus musculus, and Homo sapiens), highlighting conserved features as well as key differences among them and emphasizing the binding sites for some ligands that play crucial roles in its regulation. ARTICLE HISTORY Received 1 December 2023 Revised 17 January 2024 Accepted 18 January 2024 KEYWORDS Ion channel; TRPV4; structure; Cryo-EM; 4α-PDD; HC-067047 Introduction The members of the superfamily of Transient Receptor Potential (TRP) channels have been described in many organisms, from yeast to verte- brates [1], where they are expressed in different cell lineages and perform multiple functions in both excitable and non-excitable tissues [2,3]. Some TRP channels are expressed in the membranes of cell organelles, where they play functions in their biology and signal transduction in response to stimuli such as changes in pH, oxidants, and osmomechanical forces, regulating processes such as endosomal and lysosomal function and trafficking, mitochondrial function, regulation of endoplasmic reticulum stress, among others [4]. TRP channels participate in signal transduction by controlling membrane potential and regulating intracellular Ca2+ concentrations [5]. Most TRPs are nonselective cation channels that respond to different stimuli [6–8], and contribute to various physiological functions including phototrans- duction, Ca2+ homeostasis, cell cycle modulation, changes in temperature and pH and/or noxious sti- muli that may result in pain, among other roles [9–13]. The TRP superfamily of channels has been classi- fied into seven main subfamilies based on the homology of their sequences: TRPC (“Canonical”), TRPV (“Vanilloid”), TRPM (“Melastatin”), TRPA (“Ankyrin”), TRPML (“Mucolipin”), TRPP (“Polycystin”) and TRPN (“NOMPC-like” or no- mechanoreceptor potential C-like, only found in fish and invertebrates) [2,6,14]. Less well-described TRP subfamilies, which are found in invertebrates include TRPS (“Soromelastatin”), TRPVL (“TRPV- like”), TRPY/TRPF (“Yeast”), and even a subclade of TRPM [15–21]. In the case of the vanilloid subfamily, six mem- bers have been described: TRPV1-TRPV6. The first four have a lower selectivity for cations with a permeability ratio of PCa/PNa ~1–10; further- more, they are responsive to physical and chemical stimuli and play a significant role in thermosensa- tion, chemosensation, and nociception [7]. On the other hand, TRPV5 and TRPV6 channels are highly selective to Ca2+ (PCa2+/PNa+ >100) and participate in regulating Ca2+ homeostasis in the kidneys and intestines [22]. CONTACT Tamara Rosenbaum trosenba@ifc.unam.mx CHANNELS 2024, VOL. 18, NO. 1, 2313323 https://doi.org/10.1080/19336950.2024.2313323 © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. According to biochemical and structure–func- tion studies [17,23–32], the members of the vanil- loid subfamily are arranged as tetramers, where each subunit has six transmembranal regions, a pore-forming loop between the fifth and sixth transmembrane regions, and intracellular amino- and carboxyl-terminal regions with differences in their sequences and architecture, resulting in a distinctive biophysical feature [33–37]. In the early 2000’s, researchers started examin- ing TRP channel structures; still, the complexity of the hydrophobic environment where the ion chan- nels are located, along with the flexibility of certain protein regions [38], restricted their experimental methods. Eventually, X-ray crystallography suc- ceeded in solving crucial domains of these proteins [39–44] and then cryo-electron microscopy (Cryo- EM) techniques allowed scientists to determine the entire structure of several members of the TRPV subfamily of ion channels [35,36,45–53]. Here, we focus on pinpointing the structural details of one member of the TRPV subfamily of ion channels, which has been identified as an important regulator in normal physiology and dis- ease: the TRPV4 channel. Particularly, we focus on studies using Cryo-EM and comparing these stu- dies of TRPV4-channel structures solved in three different species (Xenopus tropicalis, Mus musculus, and Homo sapiens) to highlight the importance of different regions of the channel in its function. General properties of TRPV4 channel Biophysical properties TRPV4 is a nonselective cation channel with a permeability sequence as follows: Ca2+ > Mg2+> K+ ≈ Cs+ ≈ Rb+ > Na+ > Li+ [54]. Specifically, perme- ability with respect to Na+ is 6–10 and 2–3 for Mg2+ [55–57]. Since its early characterization more than 20 years ago as an osmosensitive Ca2+-permeable channel [55,56,58,59], it has been demonstrated that TRPV4 is crucial in maintaining cell function by regulating Ca2+ concentrations in different cell types and organisms [60,61]. TRPV4’s currents can be acti- vated by several stimuli, as mentioned below and, in the presence of Ca2+, the channel displays outward rectification in response to different voltages while when Ca2+ is absent, the current-voltage relation- ship, when only Na+ is present, becomes more linear and even slightly inwardly rectifying. The conduc- tance calculated from single-channel electrophysio- logical recordings for outward currents is 90–100 pS and 50–60 pS for inward currents [56,62]. Pharmacological properties At present, we know that the TRPV4 channel can be activated by physicochemical stimuli like tempera- tures near 27°C [63], changes in osmolarity [55], mechanical stress [64], and protons (pH 4.5–7.5) [65]. This channel is also activated through its inter- action with endogenously produced molecules, such as arachidonic acid (AA) and its derivatives, endo- cannabinoids [66], phosphatidylinositol 4,5-bispho- sphate (PIP2) [67], lysophosphatidylcholine (LPC) and its derivative, lysophosphatidic acid (LPA) [68,69], among others Figure 1(a). It has also been demonstrated that some ligands of TRPV4 are natural products found in plant extracts like apigenin from celery and chamomile flowers [70]; eugenol from cloves, basil, cinnamon and nutmeg [71]; and bisandrographolide A (BAA) from Andrographis paniculata, com- monly known as creat Figure 1(a) [72]. Additionally, synthetic molecules have been produced and reported to specifically regulate the activity of TRPV4 Figure 1(a) [73,74]. These include the following, which were used in experi- ments to solve the structure of TRPV4: Agonists ● 4-alpha-phorbol 12, 13-didecanoate (4αPDD) [75] ● (N-((1S)-1-([4-((2S)-2-([(2,4-Dichlorophenyl) sulfonyl]amino)-3hydroxypropanoyl)- 1-piperazinyl]carbonyl)-3-methylbutyl)- 1-benzothiophene-2-carboxamide (GSK1016790A) [76] Antagonists ● 2-Methyl-1-[3-(4-morpholinyl)propyl]- 5-phenyl-N-[3-(trifluoromethyl)phenyl]-1 H-pyrrole-3-carboxamide (HC-067047) [77] 2 R. SÁNCHEZ-HERNÁNDEZ ET AL. Figure 1. TRPV4 modulation and expression. a, the TRPV4 channel can respond to several stimuli, such as changes in temperature (27–35°C) or pH (4.5–7.5), shear stress, and endogenously produced molecules like LPC (lysophosphatidylcholine), AA (arachidonic acid), and PIP2 (phosphatidylinositol 4,5-bisphosphate). Some synthetic ligands of TRPV4 function as agonists (i.e. 4-αPDD (4-alpha- phorbol 12, 13-didecanoate) and GSK1016790A ((N-((1S)-1-([4-((2S)-2-([(2,4-Dichlorophenyl)sulfonyl]amino)-3hydroxypropanoyl)- 1-piperazinyl]carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide)) or antagonists (i.e. GSK2798745 (3-[[(5S,7S)-3-[5-(2-hydro- xypropan-2-yl)pyrazin-2-yl]-7-methyl-2-oxo-1-oxa-3-azaspiro[4.5]decan-7-yl]methyl]benzimidazole-5-carbonitrile) and HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1 H-pyrrole-3-carboxamide)) of the channel. Extracts from plants include BAA (bisandrographolide A), apigenin, and eugenol. b, examples of some types of cells that express TRPV4, along with their corresponding functions are depicted. PDB: 8T1B (3.0-Å resolution) [52]. Created with PyMOL and BioRender.com. CHANNELS 3 ● 3-[[(5S,7S)-3-[5-(2-hydroxypropan-2-yl)pyra- zin-2-yl]-7-methyl-2-oxo-1-oxa-3-azaspiro [4.5]decan-7-yl]methyl]benzimidazole-5-car- bonitrile (GSK2798745) [78] Functional roles and expression of TRPV4 When TRPV4 was discovered, it was initially named OSM-9 in the invertebrate C. elegans [58] and then VR-OAC [55], OTRPC4 [56], or VRL-2 [59] in vertebrates. Since then, our knowledge of its roles in physiology and advances in the details of its structure have come a long way. Activation of the TRPV4 channel translates into Ca2+ signals and, the responses vary depending on the cell type and tissue where it is expressed. TRPV4´s expression is widely distributed throughout the human body. It has been shown to be present in the cardiac, respiratory, urin- ary, muscle-skeletal, digestive, immune, endothelial, central, and peripheral nervous systems Figure 1(b). For instance, TRPV4 is expressed in several cells of the respiratory system and functions by maintain- ing homeostasis of osmotic pressure. In the lungs, TRPV4 transduces several stimuli into Ca2+ signals and regulates the relaxation of the main pulmonary artery and the vasoconstriction of pulmonary circu- lation. Importantly, this channel plays an important function in preserving the integrity of the alveolar epithelial barrier (and skin barrier), where its activity can impact the severity of chronic asthma and, due to its sensitivity to mechanical forces, it can influence pulmonary injury induced by ventilators used to treat respiratory failure [79–81]. Activation of TRPV4 by mechanical forces also regulates the func- tion of retinal cells such as ganglion cell soma- dendrite, microglia and Müller cells, suggesting that it plays roles in diseases like glaucoma and in the skeletal system where it is expressed in cells such as osteoblasts and chondrocytes, where it also partici- pates in mechanotransduction [82]. As for the kidneys, TRPV4 is expressed in the distal convoluted tubule and in regions where transcellular osmotic gradients can develop, regu- lating osmotic balance by modifying water secre- tion in the kidney. In nephrons expressed in regions of the kidneys where there is no water permeability, TRPV4 contributes to the detection of osmotic stimuli and regulation of blood pressure in the presence of increased salt intake [55]. Interestingly, TRPV4’s activation has been suggested to influence the severity and progression of polycystic kidney disease (PKD) [83]. Generalities of TRPV4’s interactions and structure Currently, we know that TRPV4 is assembled as a homotetramer [45,52,53,84]; although, it has been described that it can form heteromeric chan- nels with the TRPP2 in an alternating 2:2 stoichio- metry [39]; TRPC1 [85–88]; TRPC1 and TRPP2 [89], and most recently with TRPV3 [90]. In the case of the human TRPV4 homotetramer, each subunit consists of 871 residues and contains two layers: the bottom, also known as the cytoplas- mic layer, which encompasses the amino- and car- boxyl-terminal regions of the protein [67,91]; while the top layer, or transmembrane region, consists of six helices, where the first four α-helices (S1-S4) form a voltage sensor-like domain (VSLD), similar to other tetrameric voltage-gated ion channels (VGIC), while the S5 and S6 α-helices form the pore domain [45,52,53]. The N- and C-termini contain very characteristic domains such as a phosphoinositide binding domain (PDB), the ankyrin repeat domain (ARD), the proline-rich domain (PRD), the coupling domain (CD), the TRP box, the calmodulin-binding domain (CAM) and the PDZ-like domain Figure 2(a). Specific fea- tures of these regions are further detailed below. The bottom or cytoplasmatic layer of TRPV4 N-terminal region The N-terminal region of TRP channels has been shown to participate in the binding of molecules that regulate the activity of these proteins [23,29,96–100]. We will now detail some of these interactions in the N-terminus with different molecules and their effects on the structural con- formation of the TRPV4 channel. The N-terminus of TRPV4 contains a phosphoinositide binding site with the (++W++) characteristic sequence, where the positive charges correspond to lysine (K) or arginine (R) residues. This region allows the binding of PIP2, sensitizing 4 R. SÁNCHEZ-HERNÁNDEZ ET AL. Figure 2. Domain organization of the TRPV4 channel. a, representation of a subunit of the hTRPV4 channel. From left to right: N-terminal region, which consists of the PIP2 binding domain (PBD); proline-rich domain (PRD); Ankyrin repeat domain (ARD); and coupling domain (CD); the transmembrane region where the voltage sensor-like domain (VSLD, S1–S4 α-helices) and the pore domain (S5-S6 α-helices) are located; and the C-terminal region, which includes the TRP box and the calmodulin interaction site (CAM) and the PDZ-like domain. Capital and bold letters are amino acid residues that are part of the domains described or interaction sites with other proteins (numbers represent the positions of these amino acids). b, human TRPV4 in the apo state is CHANNELS 5 a 128 P Goretzki et al. 2018 142 P; 143 P Cuajungco et al., 2006 PRD Fan, Zhang, & McNaughton , 2009 Voltage Sensor-Like Oomain (VSLO) 51 52 53 54 Pore domain NHz COOH b C d PBD " , ' , ' , ' , , , ' , ' , , , , 121 KRWRK 125 Garcia-Elias et al. 2013 Out In hTRPV4ApO HLH Coupling domain (CO) TRPV4human TRPV1 human TRPV2 human TRPV3 mouse TRPV1 ral ' /""""'/""""""" 806 QVVGFSHTVGRLRRDRWSSWPRWE 831 5trotmann et al. 2003 SH3-PACSIN3 , l" , 2 · t ." , "/"" ' . YY the channel to thermal or osmotic stimuli Figure 2(b) [67]. Moreover, the N-terminal region of each TRPV4 subunit also contains a proline-rich domain (PRD) to which the PACSIN3 protein (pro- tein kinase C and casein kinase substrate in neu- rons 3) binds to the channel through proline residues 142 and 143 [101]; PACSIN3 acts as a negative modulator of the responses of TRPV4 to thermal and hypoosmotic stimuli [102] by reor- ienting and stabilizing the N-terminal region of TRPV4 away from PIP2, which positively regulates TRPV4 Figure 2(b) [92]. The N-termini of TRPV channels all contain six ankyrin repeats domains (ARD) composed of two antiparallel helical structures joined by a turn for each repeat (anti-parallel helix-turn-helix motif), and with each joining to the next one through a loop or finger [103]. In general, the ARD is conserved in the vanilloid TRP subfamily includ- ing the human TRPV1 [93]; rat TRPV1 [94]; human TRPV2 [95]; mouse TRPV3 [104]; or human TRPV4 channels [105] Figure 2(c). However, comparison of the structures resolved of the ARD of the TRPV4 channel by Inada et al. [43] with the structures of other vanilloid members show that the length and flex- ibility of the third finger of human TRPV4 differs when it is in the presence or absence of ATP. Moreover, this arrangement of finger 3 plays an important role in regulating channel activity; when ATP is bound to TRPV4, protein stability is favored. Studies have shown that several muta- tions reported along the ARD, including those in the third finger where the binding of ATP is impaired, cause dysregulation of the basal gating process or, in other words, an increase in the open probability or a “gain of function” pheno- type Figure 2(d). The ARD is involved in transport, anchoring, localization, and protein–protein interactions [106,107]. Currently, it is known that alterations in the sequence of TRPV4, especially in the ARD, can cause different diseases such as Brachyolmia, Charcot–Marie–Tooth type 2C disease, among other muscular atrophies and syndromes. Most of these have been associated with mutations in several amino acid residues within the ARD struc- ture, which can result in “gain of function” effects on the channel [61,108–111]. In this sense, previously reported interactions between the ARD of TRPV4 and a GTPase named RhoA [112–115], have recently regained attention in the field. RhoA is a small GTPase that plays a crucial role in transmitting signals from outside the cell to its cytoskeleton. The struc- ture of RhoA consists of a β sheet of six strands (β1–β6) surrounded by six short helices (α1–α6) joined by loops and a variable C-terminal region; additionally, regions corresponding to switches I and II regions or the ligand-binding pocket, adopt different conformations depending on whether RhoA is GDP or GTP bound Figure 3(a) [52,53,113,116]. Essentially, RhoA acts as a molecular switch by transitioning between an inactive GDP-bound state and an active GTP-bound state. In its active state, RhoA is bound to GTP and positively reg- ulates downstream cytoskeletal-modulating pro- teins and is involved in a variety of processes such as focal adhesions, actin assembly, transcrip- tional activation, exocytosis, and regulation of smooth muscle contraction [113,116]. Most recently, the details of the binding of RhoA to TRPV4 were shown in two different studies using the Cryo-EM technique while resol- ving the structure of the channel. Both research groups overexpressed human TRPV4 in HEK293 cells but reported obtaining another additional protein density, which after analysis and compar- ison with other previously solved structures [116], was identified as RhoA Figure 3(b). The stoichio- metry of RhoA molecules with respect to the shown and its interaction with PIP2 and PACSIN3 is represented; a zoom-in into the PRD shows that P142 and P143 are involved in these interactions with PACSIN3; other proline residues among the PRD are shown in purple sticks. c, comparison of the ARD between several members of the vanilloid subfamily, where the alignment shows mostly conserved structure among species. Only the human TRPV4 complete subunit is represented in a side view parallel to the membrane. d, zoomed-in view of the ARD of the human TRPV4 is shown, where finger 3 acquires a different conformation when it is unbound to ATP (yellow sticks). PDB 8T1B, 6L93, 2PNN, 2F37, 4N5Q, and 6F55 (resolutions were 3.0 Å, 4.47 Å, 2.70 Å, 1.70 Å, 1.9 Å and nuclear magnetic resonance structure, respectively) [43,52,92–95]. Created with PyMOL and BioRender.com. 6 R. SÁNCHEZ-HERNÁNDEZ ET AL. TRPV4 tetramer could be observed in a range from none to four molecules per channel, furthermore, this arrangement does not seem to result in major changes in the general structure of each subunit of TRPV4 [52,53]. However, in the hTRPV4-RhoA complex, no differences were observed in the presence of GDP or GTP [52,53]. On the other hand, the interface between hTRPV4-RhoA is generated through elec- trostatic interactions between the β1, β3, switch Figure 3. hTRPV4 interactions with RhoA GTPase. a, cartoon representation of the RhoA structure. The RhoA protein is bound to GDP (green sticks). b, the homotetramer of the human TRPV4 in complex with RhoA is shown; each subunit of TRPV4 is identified with a different color, RhoA (wheat) is shown interacting with the TRPV4 bottom layer; the stoichiometry is 1:1. c, lateral view of two subunits of TRPV4 in complex with RhoA and a close-up view of the interaction zone between the ARD of hTRPV4 (yellow sticks) and the β sheets of RhoA (blue sticks). PDB: 8FC9 (resolution 3.75 Å) [52,53]. Created with PyMOL and BioRender.com. CHANNELS 7 I and II of RhoA, and AR2-AR5 of TRPV4. The sites in TRPV4 where strong interactions with RhoA are found are residues: R232, R237, D263, R269, R315 and R316 of TRPV4, while in RhoA residues R5, E40, E54, and D76 are involved Figure 3(c) [52]. It has been proposed that RhoA anchors to the membrane through its prenylated C-terminal region, forming an interface through three β- strands and one α-helix with the loops that join the fingers of the ARD of TRPV4, limiting its movement until activation of the channel can occur in the presence of a given stimulus [52,53]. Hence, it was concluded that the interaction between hTRPV4-RhoA results in mutual inhibi- tion of the activity of these proteins, which was previously reported by structure–function studies of hTRPV4 [114,117–122]. Furthermore, it has been proposed that several mutations reported along the residues of the ARD of hTRPV4 [114,117–122], cause a loss of interac- tion with RhoA, leading to a bidirectional dysre- gulation since binding of RhoA to TRPV4 suppresses channel activity and, the binding of TRPV4 to RhoA inhibits activation of this GTPase [113]. These results agree with previous studies which suggested that mutations in the ARD of hTRPV4 can result in a gain-of-function, leading to the opening of the channel even when it is in a ligand-free state [61,108–110]. However, it is still unclear whether one single residue mutation is sufficient to cause the loss of interaction with RhoA, which leads to channelopathies due to the malfunction of TRPV4. Together, these results on the structure of TRPV4 provide valuable knowl- edge about its interaction with RhoA and its pos- sible consequences in cell physiology. It is important to also mention that RhoA plays pivotal functions in regulating cytoskeletal func- tions, modulating the transitions between the inac- tive GDP-bound state and an active GTP-bound state [123], which are important because the active state of RhoA leads to cell contraction and process extension by promoting actin polymerization and actomyosin contraction [114,124]. An interplay of regulation between RhoA and TRPV4 has been demonstrated by McCray et al. [114] to result in the modulation of the activities of both proteins and, in turn, this leads to cytoskeletal changes. The disruption in the interactions of TRPV4 and RhoA and to cellular outgrowth mediated by TRPV4, have been linked to mutations that lead to some neuropathies but not to mutations that produce skeletal dysplasias, which is conducive to a neuron-specific disease. The N-terminus of TRPV4 contains an intrinsi- cally disordered region (IDR) of about 150 amino acids, which remains unresolved. Interestingly, in a recent study, Goretzi et al. [125], have proposed that this region contains several constituents that can transiently couple and uncouple to regulate the activity of TRPV4. Such interactions were pro- posed to modulate the binding of lipids (i.e. PIP2), of proteins that regulate TRPV4’s activity and also participate in post-translational modifications in the IDR and ARD. By deleting stretches of several amino acids in the IDR and measuring the activity of the mutant TRPV4 channels, these authors pro- posed that there is an autoinhibitory region that acts on the PIP2-binding site and negatively reg- ulates TRPV4’s activity. It was also proposed that the binding of lipids to the IDR and the PIP2- binding region, wields a pull force on the ARD and helps transduce mechanical forces to the rest of the TRPV4 structure [125]. We will next discuss the features and relevance of another region in the bottom layer of the TRPV4 channel that also regulates its activity in homeostasis and pathological processes. C-terminal region All TRP channels contain a C-terminal region, which in turn encompasses important domains such as the TRP box, a calmodulin-binding domain (CAM), and a PDZ-like domain Figure 2(a). The TRP box (or TRP domain), is a sequence of six highly conserved amino acids found among the vanilloid subfamily, as well as in the TRPC, and TRPM subfamilies, that allow the functional coupling of the tetramers. In the case of the TRPA subfamily, although the sequence of amino acids differs in comparison to the other subfamilies mentioned, it has been demonstrated that the topology of the structure also acquires an alpha helix conformation [126]. In TRPV4 channels, this sequence interacts with the S4–S5 linker region of the same subunit 8 R. SÁNCHEZ-HERNÁNDEZ ET AL. through a hydrogen bridge that regulates gating processes, which are also influenced by the polar– nonpolar interface that is established between the protein and the membrane Figure 4(a) [128,129]. There are also interactions between the TRP box and the S4–S5 linker where the side-chain indole is the hydrogen donor of the W733 residue, one that is highly conserved throughout TRPV4 channels of different species Figure 4(b), and resi- due L596 of the S4–S5 linker, whose carbonyl oxygen is the bridge acceptor. The hydrogen bond formed between L596-W733 maintains the TRPV4 channel in a closed state by orienting the S4-S5 linker along with the TRP box, which Figure 4. The S4–S5 linker/TRP box interface. a, alignment of the TRPV4 interface of different species as described in the text is shown in a lateral view, only the S4–S6 and the TRP box are represented. b, zoomed-in view of the interaction zone between the S4–S5 linker and the TRP box, where the amino acid residues L596 (human and mouse) or L592/L594 (frog) form a hydrogen bond with the conserved residue W733. The frog TRPV4 linker has the most flexible interface. The amino acid residues are shown in orange sticks and the hydrogen bridges are shown in dotted lines. PDB: 8J1D, 8T1B, and 6BBJ (resolutions of 3.59 Å, 3.00 Å and 3.80 Å, respectively) [52,53,127]. Created with PyMOL and BioRender.com. CHANNELS 9 a b Mouse Human Frog impairs the movement of the S6 and the opening of the channel. Stimuli such as depolarization, membrane stretching, or heat, disrupt this hydro- gen bond leading to gate opening [128]. Furthermore, it has been described that mutations in these two amino acids (i.e. L596P and W733R) are associated with the generation of skeletal dys- plasias, associated with TRPV4 mutations with gain-of-function phenotypes due to increased open probability as a result of weak interaction between these amino acids [128,130,131]. When the human and mouse TRPV4 channels are compared, residue W733 in mouse TRPV4 is localized in a flexible loop, while in the human TRPV4, it is located at the end of the α-helix [52,127]. A closer look into the frog TRPV4 chan- nel structures reveals that it exhibits a flexible loop that could shorten the distance of the interaction between either L592 or L594 [45]. These variations in the position along the S4–S5 linker possibly influence the stability of the hydrogen bridge and therefore the switching between closed or open states; however, despite these differences between species, all three structures maintain the same function of the S4–S5 linker/TRP box interface, giving stability to the closed state when no stimuli are present Figure 4(b). Specific features of the S4- S5 linker region in TRPV4 channels from different species are further detailed in the transmembrane layer section below. In the C-terminal region, between residues 806 and 831, a CAM-binding site has been described and it has been shown that in the presence of [Ca2+]i, the spontaneous activity and the response of TRPV4 to hypotonic solutions has been sug- gested to be potentiated through a CAM- dependent mechanism [62]. This phenomenon is then followed by inhibition or inactivation, which is a stable non-conducting conformation of the open channel [62,132,133]. In the human TRPV4 channel, the CAM domain is also the binding site for inositol 1,4,5-trisphosphate (IP3), which sensi- tizes the channel´s response, not only to mechan- ical and hypotonic stimuli, but also to epoxyeicosatrienoic acids (EETs) [134]. In addi- tion, a self-inhibition domain has been proposed, where residues E797-P799 located immediately upstream of the CAM domain, participate in this type of down-regulation of the activity of the channel by establishing a salt bridge with an uni- dentified positively charged region that stabilizes the closed state of the channel [135]. Consistently, mutation of residue E797 leads the channel to a constitutively open state associated with the gen- eration of skeletal dysplasia [133,135]. Finally, the PDZ-like domain (also known as PSD95/Dlg/ZO-1-like) near the end of the C-terminal region consists of a short sequence of four amino acids (DAPL) and participates in membrane trafficking of the channel, oligomeriza- tion of the tetramer and in the interaction with different proteins that regulate downstream signal- ing processes such as Yes-associated protein/tran- scriptional co-activator with PDZ-binding motif (YAP/TAZ), which is a transcription factor involved in inflammatory process, which seems to be activated by the influx of Ca2+ through TRPV4 channel of immune cells [130,134,136–138]. Moreover, the rigidity of the extracellular matrix participates in pathophysiological processes such as fibrosis in which epithelial–mesenchymal transition (EMT) is involved. TRPV4 has been linked to EMT as a mediator of its response to matrix stiffness and transforming growth factor β1 (TGF-β1). Sharma et al. [139] have suggested that inhibition of the activity of TRPV4 blocks matrix stiffness and EMT in response to TGF-β1, further supporting the role of the PDZ-binding motif of this channel and its interaction with matrix com- ponents on the physiology of cells. The top or transmembrane layer of TRPV4 Voltage sensor-like domain The S1-S4 helices form the VSLD, which flanks the S5 and S6 helices (pore domain) of the adjacent subunit in a domain-swapped arrangement; how- ever, it has been demonstrated that the arrange- ment of the VSLD domain is different between TRPV4 channels from several species [45,52,53,127]. The structural arrangement of these regions in frog TRPV4 differs significantly from mouse and human [127], displaying closer proximity of the S3 and S4 from the VSLD to the pore domain Figure 4(b). The S3 establishes a key contact with 10 R. SÁNCHEZ-HERNÁNDEZ ET AL. S6 in its entire length generating a zipper [45]. Additionally, in the frog TRPV4 channel, the S4–S5 linker structure is an ordered loop with greater flex- ibility [45], as compared to the α-helix present in the structures of mouse [127] and human TRPV4 chan- nels Figure 4(b) [52,53]. Modifications in the S4–S5 linker, specifically shortening in length or changes in flexibility result in impairment of domain swapping, which affects the architecture and the gating of the channel [32,140,141]. For example, it has been shown that in the TRPV6 channel, the S4–S5 linker is a critical component in domain swapping, sup- porting the importance of this region among mem- bers of the TRPV subfamily of ion channels [32]. Pore domain In TRPV4, the pore domain is located in the center of the tetramer and is formed by the S5-S6 helices and by a reentrant loop and helix motif. In the upper part of the TRPV4 pore structures there is a first constriction corresponding to the selectivity filter region (TIGMGD/E) (Figure 5). In the apo state, the frog and mouse TRPV4 channels have a cross-pore dis- tance greater than 10 Å between the nearest amino acids in the narrowest region (residue G675, 10.6 Å; residue D682, 14.4 Å respectively) Figures (5a,b) [45,127]; both diameters are unable to prevent the passage of ions such Na2+, K+ or Ca2+, considering their Pauling radii [142]. Moreover, ion coordination in the mouse channel Figure 5(b) by this region is unlikely when its architecture is compared to other ion channels such as KcsA, considering that it requires greater closeness between the side chains of the residues [143]. In the human TRPV4 channel Figure 5(c), the distance between the carbonyls of G679 in the pore is reduced to 6.6 Å [52]; however, it is still wide compared to other channels of the same subfamily such as rat TRPV1 (4.6 Å, residue G643) [36]; rabbit TRPV2 (5.2 Å, residue G604) [144] or rat TRPV6 (4.6 Å, residue D541) [42], suggesting the entry of ions to the upper and middle pore cavity could occur in the closed state. On the other hand, at the bottom of the pore domain, there is a region corresponding to the intracellular gate that controls the permeation pathway, present in other members of the vanil- loid subfamily: M643 in rabbit TRPV2 [144]; M677 in mouse TRPV3 [47]; M578 in rabbit TRPV5 [48]; M577 in rat TRPV6 [42]) or I679 in rat TRPV1 [36] side chains are responsible for preventing the passage of cations in the closed state of the channel. Figure 5. Pore domain of TRPV4. the pore regions of the TRPV4 channel from a, frog, b, mouse, and c, human are shown in the apo state; all of them have a selectivity filter in the upper region which differs in their cross-pore distances. The lower region of the pore domain contains the intracellular gate, where the distance between the side chains of the nearest amino acids is less than 6 Å, preventing the passage of ions. Mammalian ion TRPV4 channels show similarity in structure in contrast to the frog TRPV4, which displays a “tighter” conformation. The amino acids in the selectivity filter and the intracellular gate are shown as sticks. PDB: 6BBJ, 8J1D, and 8T1B (resolutions of 3.80Å, 3.59 Å and 3.00 Å, respectively) [45,52,127]. Created with PyMOL and BioRender.com. CHANNELS 11 In the frog and mouse TRPV4 channel, residues M714 and M718 are positioned homologously, and constrict the pore to a diameter of 5.3 Å and 5.4 Å Figures 5(a,c) [45,127]; however, in the human channel, the identity and position of the amino acid that restricts ionic conduction is I715, which has a diagonal distance of 5.9 Å between side chains or 10.2 Å between Cα Figure 5(c) [52]. The side chains of all three structures are directed to the central cavity and form an ion- and water- impermeable barrier (Figure 5) [52]. Interactions between the cytoplasmic and transmembrane layers The TRPV4 channel tertiary structure has an arrangement where the N- and C-terminal regions interact through the coupling domain (CD) Figure 6(a). At the N-terminal region, the CD is located between the ARD and the S1 Figure 6(b), involving two beta strands (β1 and β2) which estab- lish hydrogen bonds with a third beta strand (β3, black arrows in Figure 6(b) of the C-terminal region, and form an antiparallel beta sheet [52,53,127]. The interaction of the cytoplasmic domains pro- motes the nearing of distant regions in the primary structure that are important in the assembly and gating process of the channel. These regions include the TRP box, that runs along the lower part of the VSLD and comes into contact with key sites like the S4–S5 linker, the vanilloid pocket, and the helix-loop -helix motif (HLH) Figure 6(b). Any alteration in these sites of the structure influences the gating processes of the channel [52,53,127]. Modulation of TRPV4 channel by ligands As previously mentioned, the TRPV4 channel has an exceptionally polymodal ability to respond to many physicochemical stimuli; additionally, its extensive distribution in different organs and its participation in physiology as well in pathologies caused by its malfunction or dysregulation have attracted the atten- tion of different research groups, identifying TRPV4 as an important therapeutic target from which we yet need to learn more details of its function. Several biophysical and biochemical studies have been performed and improved our under- standing of the gating mechanisms and the role of modulators (endogenous or synthetic) of TRPV4 on its activity [61,73,145]. However, it is still necessary to complement this knowledge with structural studies that provide us with information about the changes, binding sites of modulators, and mutations that occur in TRPV4 [146]. Solved human and mouse TRPV4 structures have revealed details of agonist- and antagonist-bound open and closed states, giving us new insights into channels’ gating processes. We will further discuss the binding site of synthetic modulators of TRPV4 and the structural mechanisms that determine both open and closed states. The vanilloid pocket in the TRPV4 channel According to the recently solved human and mouse TRPV4 structures, it seems more clear that certain compounds bind to the channels in a similar region, the vanilloid pocket, which lies within a cytosol- facing cavity between S1–S4 and the TRP box Figure 7(a) [52,53,127]. The apo structure of hTRPV4 shows that this cavity is composed mainly of aromatic and polar residues, where the polar side chains are distributed around the transmembrane helix top, while the aromatic rings are positioned in a central fashion Figure 7(b) [52]. This particular arrangement of the amino acid residues allows dif- ferent ligands (agonists and antagonists) to enter the cavity and stably position themselves inside it. The first synthetic TRPV4 selective agonist described was 4α-PDD, a non-PKC-activating phorbol ester [EC50 200 nM in human, 370 nM in mouse] that has been widely used in many func- tional experiments [75,145,147–150]. Pioneering site-directed mutagenesis experiments had determined a tentative binding site for 4α-PDD within a pocket between S3 and S4, proposing resi- dues Y556, L584, W586, Y591, and R594 as essential structural elements in the mechanisms of 4α-PDD- dependent TRPV4 gating [149,151]. In the currently solved open-state conformation structure of the human TRPV4 channel in complex with 4α-PDD, the binding site for this molecule has been identified within the cavity between the S1-S4 and the TRP helix bundle [52,53]. There, 4α-PDD is surrounded by residues S470, N474, S477, F524, Y553, Y591, S747, and F748, maintaining hydrogen bonds with N474 and Y591 Figure 7(c) [52]. 12 R. SÁNCHEZ-HERNÁNDEZ ET AL. Furthermore, docking calculations and molecular dynamics simulation determined high flexibility within the 4α-PDD molecule with different conforma- tions within the binding pocket [53]. However, these different modeled conformations showed that the ali- phatic chain of 4α-PDD is stably localized outside the pocket toward the hydrophobic S1-S4 region, empha- sizing the importance of the acyl-chain length as an essential element for the correct positioning of the terpenoid within the binding site Figure 7(c) [147]. It is also important to highlight that this binding site at the base of the S1-S4 bundle is an allosteric modulation site, which has been identified in all TRP members of the vanilloid subfamily [152], explaining why mutations that antagonize 4α-PDD effects also affect activation of the channel by other agonists such as 5,6-ETT and BAA, which could be sharing common binding sites [149,151]. Additionally, another human TRPV4 structure in the open-state conformation was solved, in Figure 6. Contact domain of TRPV4. a, the cytoplasmic domains and helices S1 and S6 of the mouse and human TRPV4 channel are represented. b, zoomed-in views of the coupling domain (CD), where the β1 and β2 strands of the N-terminal regions interact with the β3 strand of the C-terminal region, which brings these two regions closer to each other. The arrangement of the tertiary structure allows certain domains such as the TRP box to come into contact with key areas of the protein for its regulation. The black arrows represent the movement of the TRP box toward the plasma membrane facilitated by the HLH motif. PDB: 8J1D and 8T1B (resolutions of 3.59 Å and 3.00 Å, respectively) [52,127]. Created with PyMOL and BioRender.com. CHANNELS 13 Figure 7. Human TRPV4 ligand binding site. a, schematic representation of the apo structure of TRPV4 channel (white ribbon) with the identified ligand binding site highlighted in light purple. b, zoomed-in view of the ligand binding pocket shown in a, (purple ribbon). c, zoomed-in view of the structure of TRPV4 in the open-state in complex with agonists 4α-PDD (orange ribbon) and d, GSK1016790A (pink ribbon). Closed-state structures in complex with antagonists are shown in e, HC-067047 (blue ribbon) and f, GSK2798745 (cyan ribbon). The side chains of the polar and aromatic residues essential for binding agonists and antagonists (shown in gray) are represented as sticks. Hydrogen bonds are represented as dashed lines. Both agonists and antagonists are stably positioned within the ligand binding pocket and share some residues with which they interact, such as S470, N474, F524, N528, Y553, Y591, D743, and S747. However, they are also closely surrounded by particular residues: 4α-PDD (F478); GSK1016790A (Q550, D531, F549, L523); HC-067047 (Y478, F592) and GSK2798745 (Y478, F524). PDB: 8T1B, 8FCA, 8FC7, and 8FC8 (resolutions of 3.00 Å, 3.41 Å, 3.30 Å and 3.47 Å, respectively) [52,53]. Created with PyMOL and BioRender.com. 14 R. SÁNCHEZ-HERNÁNDEZ ET AL. complex with the synthetic compound GSK1016790A [EC50 = 2 nM in human, 18 nM in mouse] [76,153]. This structure shows that this agonist shares the same ligand-binding site as 4α- PDD Figure 7(d) [53]. GSK1016790A is a highly potent and selective agonist for the TRPV4 chan- nel, used extensively in pharmacology studies of TRPV4 activation [154–161]. Also, molecular dynamics simulations showed that, unlike 4α-PDD, the conformational arrange- ment of GSK1016790A remains stable within the binding site and that among the closest residues surrounding it, both GSK1016790A and 4α-PDD share residues S470, S477, N474, F524, N528, Y591, T553, D743, and D747 for their binding to the chan- nel. In contrast, residues L523, T527, D531, F549, Q550 and T478 seems to interact specifically with GSK1016790A only Figure 7(d) [53]. On the other hand, according to recent cryo- EM structures, these agonists are proposed to share the same ligand-binding site with synthetic antagonists. Nadezhdin et al. [52] have solved the human TRPV4 structure in closed-state conforma- tion in complex with the molecule HC-067047 [IC50 48 nM in human, 133 nM in rat, 17 nM in mouse] [77], which is a potent and selective antagonist for TRPV4 that has been used to study the pathophysiology of TRPV4 channel in both in vitro and in vivo models [61,162]. The structure shows that the HC-067047 mole- cule is positioned within the vanilloid pocket, sur- rounded by some residues shared with the agonists 4α-PDD and GSK1016790A (i.e. N474, S477, Y553, Y591, and D743), and, particularly with the residues F471, D546 and F592 Figure 7(e). Moreover, these residues identified for the binding of HC-067047 in the Cryo-EM structure, were also confirmed by functional assays in which substitutions for alanines were introduced in these sites, entailing a decrease in the inhibitory effect of the compound [52]. The compound GSK2798745 [IC50 1.8 nM in human] [78,163] is the first TRPV4 antagonist to advance to clinical trials [163]. Kwon et al. [53] have solved the human TRPV4 structure in the closed-state conformation in presence of this syn- thetic molecule. They found that GSK2798745 is also surrounded by the residues shared with the agonists 4α-PDD and GSK1016790A (N474, S477, N528, Y553, Y591, D743 and S747), and like HC- 067047, it interacts closely with F471 and D546, and with Y478 and F524 particularly Figure 7(f). The solved mouse TRPV4 channel structure [127] has also provided detailed information about the mTRPV4 channel in a ligand-free state (apo), in the open-state in complex with agonist GSK1016790A, in complex with both GSK1016790A and ruthenium red (RR, a nonselective pore-blocking molecule) and, in complex with both GSK1016790A and Agonist-1 (or quinazoline-4(3 H)-one derivative 36) [164]. The apo structure of the mTRPV4 channel resembles a similar arrangement as the one described for the hTRPV4 channel Figure 8(a). In both structures, the intracellular gate shows the amino acid residue M718 (mouse) or I715 (human) of each subunit at a distance of less than 6 Å, which impairs ions influx Figure 8(a), right panel). On the other hand, the open-state structure of mTRPV4 in complex with GSK1016790A reveals that this agonist binds in the same pocket between the VSLD and the TRP box region as in the human TRPV4. The key residues for the binding of GSK1016790A in the mTRPV4 channel are S470, F524, and F549, while in the hTRPV4 the residues N474, Y478, Y591, Q550, T527, and D743 are involved Figure 8(b) [53,127]. Although in this review we only discuss the mTRPV4 apo and open structure in complex with GSK1016790A in order to compare it with the hTRPV4 structures, we consider that is also impor- tant to briefly highlight that Agonist-1 shares the binding site within the vanilloid pocket as well [127]. As for the structure solved with both GSK1016790A and RR, the structure solved by Zhen et al. [127] is the second TRPV channel struc- ture resolved in the presence of RR, since the last one was in complex with the human TRPV6 channel [165]. The mTRPV4 structure with RR shows a classic pore-blocking mechanism in which RR binds to the extracellular region of the selectivity filter near residues D682 and M680 [127,166–174]. Structural mechanisms in the hTRPV4 gating The structural changes of the human TRPV4 asso- ciated with channel activation by the binding of an agonist, whether 4α-PDD or GSK1016790A; or channel inhibition by the binding of an antagonist, CHANNELS 15 whether HC-067047 or GSK2798745, involve transi- tions in different key domains, beginning in the amino acid residues within the binding site in the VSLD and ending until they reach the pore domain. It has been also proposed that residues D531, D546, Q550, and R594 within the S1–S4 region interact through polar contacts in the closed state Figure 9(a). When the interactions between residues within the S1–S4 are broken by the binding of an agonist, new hydrogen bonds are formed with both the agonist and residues of the TRP helix. In this fashion, residue Q550 interacts with the agonist, T594 forms a salt bridge with residue D743, and D531 forms a hydrogen bond with R746, which in turn, allows residue R746 to maintain an interaction with Y439, located in the CD that links the N- and C-terminal regions Figures (9a,b) [52,53]. Regarding the architecture of the intracellular gate in the open state, the solved structures [52,53] sug- gest that this region exhibits an increased distance between the gating residues (I715) of each subunit, allowing the movement of a hydrated ion through the channel Figure 9(c). As for the selectivity filter in the structures activated by 4α-PDD and Figure 8. Comparison between hTRPV4 and mTRPV4 channels. a, schematic representation of the apo structure of human (pink ribbons) and mouse (cyan ribbons) TRPV4 channels along with a close-up of the pore domain with the identified intracellular gate residues at I715 (human, green sticks) or M718 (mouse, orange sticks) and its cross-pore distances. b, TRPV4 channel structure in the open state in complex with the agonist GSK1016790A. A zoom-in of the ligand binding pocket between the S1–S4 and the TRP box (human, pink ribbons; mouse, cyan ribbons) is shown. The key amino acid residues and the agonist structure are shown in purple sticks. PDB: 8J1D, 8FC9, 8J1F, and 8FC8 (resolutions are 3.59 Å, 3.75 Å, 3.62 Å and 3.47 Å, respectively) [53,127]. Created with PyMOL and BioRender.com. 16 R. SÁNCHEZ-HERNÁNDEZ ET AL. Figure 9. Structural changes in the closed and open states of hTRPV4. Close-up view of the ligand binding pocket showing the key residues that form the coupling interface between the S1–S4, CD, and TRP domains in the a, closed and b, open states. Dashed lines indicate hydrogen-bonds and salt bridges. Representation of the structural changes in the selectivity filter and the intracellular gate of the pore region caused by the binding of the agonists c, 4α-PDD (yellow structure) and GSK1016790A (pink structure), or antagonists d, HC-067047 (blue structure) and GSK2798745 (cyan structure). Dashed lines indicate the distances between gating (I715 in the open state and M718 in the closed state) and selectivity filter (G679, M680) residues in opposite subunits. Upon activation by the agonist, a transition from α to π secondary structure occurs in the S6 helix, inducing a helical bend (π-hinge). The binding of the antagonist promotes a transition from π to α secondary structure, inducing the formation of an α-helix. The position of residue F707 is highlighted since it putatively stabilizes the π-helices structure through H-C···π interactions. PDB: 8FCA, 8FC8, 8T1F, and 8FC7 (resolutions are 3.41 Å, 3.47 Å, 3.49 Å and 3.30 Å, respectively) [52]. Created with PyMOL and BioRender.com. CHANNELS 17 GSK1016790A [52,53], the distance between resi- dues in the selectivity filter (G679 and M680) seems to be decreased Figure 9(c), as compared to TRPV4 structures in the closed state Figure 9(d), possibly allowing the coordination of cations [52,53]. Structure changes promoted by the binding of the antagonists HC-067047 and/or GSK2798745 [52,53] include the rotation in the middle of the S6, which allows for the transition from a π to α structure and a conformational change at the C-terminal end of the S6 that positions residues M718 of each subunit to form the intracellular gate and create a hydrophobic seal in the pore that blocks the passage of cations. Furthermore, the closed state bound to antagonists acquires a wide selectivity filter (G679 and M680) that inhibits the direct coordination of cations Figure 9(d) [52,53]. These studies also showed that the radius of the pore is wider when HC-067047 is bound to the channel (11.6 Å cross-pore distance) Figure 9(d), Figure 10. The “vanilloid pocket” in TRPV4. a, schematic representation of the apo structure of human TRPV4 channel in a parallel view with the membrane. Each subunit of the homotetramer is shown by a different color. A zoom-in of the ligand binding pocket between the S1–S4 and the TRP box where key amino acid residues for the binding of b, endogenous (pink ribbons, orange sticks) or c, synthetic (cyan ribbons, orange sticks) ligands are represented. The chemical structures of the modulators of the TRPV4 channel discussed in this review are shown as well. PDB: 8T1B (resolution 3.00 Å) [52]. Created with PyMOL and BioRender.com. 18 R. SÁNCHEZ-HERNÁNDEZ ET AL. left panel), compared to the apo state (6.6 Å cross- pore distance) Figure 5(c) [52]. When GSK2798745 is bound to hTRPV4, the selectivity filter acquires a wide structure that impairs the coordination of cations (a distance of 14.5 Å between residues M680 from each subunit is observed), while the intracellular gate exhibits a distance which is too narrow for the flux of ions to occur (the distance between residues M718 among subunits is 4.9 Å) (Figure 9(d), right panel) [53]. In summary, based on the above-mentioned findings, it can be concluded that both agonists and antagonists can interact with the same ligand- binding sites of the TRPV4 channel, even sharing some of the residues in these sites. However, the molecular details of types of interactions or bonds vary with each ligand, possibly explaining the dif- ferences in the effects they exert and the potency of their modulation on the TRPV4 channel. It is worth mentioning that very few endogenous ligands have been described for TRPV4. Our work group has recently shown that there is an interac- tion between LPA 18:1, an endogenous lipid asso- ciated with the generation of pain and many other functions [175], with the TRPV4 channel. Interaction of LPA18:1 with TRPV4 involves resi- dues R746 of the TRP box, K442, R446 of the N-terminal region and K535 of the S2–S3 linker (Figure 10). When compared to activation of TRPV4 by GSK1016790A, single-channel current amplitude values in the presence of LPA18:1 are similar to those with GSK1016790A [69]. TRPV4 is also activated by the precursor of LPA18:1, LPC18:1 [68], by a mechanism that involves interaction with a positively charged residue (R746) localized in the TRP box of the channel. However, activation of TRPV4 by LPA18:1 and LPC18:1 occurs with dif- ferent open probabilities (higher Po for LPC than for LPA) but with different single-channel conduc- tances (lower for LPC), suggesting that this happens through a mechanism in which these agonists pro- mote different open states. A similar phenomenon was observed by our group for LPA18:1 and TRPV1, where LPA18:1 activates the channel, par- tially through interactions with residue K710 in the TRP box of the channel [176], but also leads to a different conformational open state with higher conductance, as compared to the one produced by capsaicin [177]. Although no structure has been obtained for the endogenous ligands of the TRPV4 channel, Figure 10 highlights the binding sites we have found to be important for the binding of LPA18:1 and LPC18:1 and emphasizes the impor- tance of the vanilloid pocket in the modulation of the gating process of the TRPV4 channel. Conclusions The TRPV4 channel is a tetrameric protein loca- lized in the plasma membranes of different cells, where it regulates ion fluxes, particularly Ca2+, in response to several stimuli. TRPV4 plays a crucial role not only in normal physiology but also in diseases or syndromes where its regulation is com- promised. Although the different structures cur- rently solved exhibit differences between them, they all agree that each subunit consists of an N-terminal region, where the PBD, PRR, and ARD are located, while the C-terminal region con- tains the TRP box, CAM binding site, and PDZ- like domain. The transmembrane region contains helices S1– S6 and the VSLD, but among different species of TRPV4, especially the pore region and the S4–S5 linker, exhibit the most significant changes. Solved structures seem to share common binding sites for the synthetic modulators 4αPDD, GSK1016790A, GSK2798745, and HC-067047 in the vanilloid pocket, which also seems to play a crucial role in the modulation of the channel by endogenously produced molecules as well. Recent advances in imaging techniques, such as Cryo-EM, have provided us with greater knowledge about the structure, function, and gat- ing processes of TRPV4. It is crucial to emphasize the significance of not only obtaining the details of the structure of the human TRPV4 channel but also of its homologs in other species of animals (Xenopus tropicalis, Mus musculus), which serve as models for comparison and better understand- ing of how this biologically relevant protein func- tions. It is also important to consider that the resolutions of the TRPV4 structures are mostly around 3–3.8 Å, which are values like some of the other TRPV channels, although for a few of the latter, better quality structures have been obtained (~1.9–3.5 Å). Nonetheless, information CHANNELS 19 on specific molecular interactions of ligands with TRPV4 can be attained from the available struc- tures for this channel. We still have much to learn about how TRPV4 is regulated and surely many endogenous regulators of the activity of this channel have yet to be discovered. Acknowledgments We thank Itzel Llorente from Instituto de Fisiología Celular in Universidad Nacional Autónoma de México for proof- reading of the manuscript and administrative support. Raúl Sánchez-Hernández wrote this review to fulfill the require- ments of Programa de Doctorado en Ciencias Bioquímicas of Universidad Nacional Autónoma de México, and received a doctoral scholarship from Consejo Nacional de Humanidades, Ciencias y Tecnologías (CVU#894531). Disclosure statement No potential conflict of interest was reported by the author(s). Funding T.R. received funding from Consejo Nacional de Humanidades, Ciencias y Tecnologías (#A1-S-8760) and from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México, (PAPIIT #IN200423). Author contributions R.S.H. and T.R. conceived the manuscript. R.S.H., M.B.A., and A.M.H.V. performed the literature search and produced the figures. R.S.H., M.B.A., A.M.H.V. and T.R. wrote the paper. T.R. supervised the work, reviewed and edited the manuscript. 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International Journal of Molecular Sciences Review TRPV4: A Physio and Pathophysiologically Significant Ion Channel Tamara Rosenbaum 1,* , Miguel Benítez-Angeles 1, Raúl Sánchez-Hernández 1, Sara Luz Morales-Lázaro 1, Marcia Hiriart 1 , Luis Eduardo Morales-Buenrostro 2 and Francisco Torres-Quiroz 3 1 Departamento de Neurociencia Cognitiva, División Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico; mbenitez@ifc.unam.mx (M.B.-A.); rsanchez@ifc.unam.mx (R.S.-H.); saraluzm@ifc.unam.mx (S.L.M.-L.); mhiriart@ifc.unam.mx (M.H.) 2 Departamento de Nefrología y Metabolismo Mineral, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City 14080, Mexico; luis.moralesb@incmnsz.mx 3 Departamento de Bioquímica y Biología Estructural, División Investigación Básica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico; ftq@ifc.unam.mx * Correspondence: trosenba@ifc.unam.mx; Tel.: +52-555-622-56-24; Fax: +52-555-622-56-07 Received: 3 May 2020; Accepted: 24 May 2020; Published: 28 May 2020   Abstract: Transient Receptor Potential (TRP) channels are a family of ion channels whose members are distributed among all kinds of animals, from invertebrates to vertebrates. The importance of these molecules is exemplified by the variety of physiological roles they play. Perhaps, the most extensively studied member of this family is the TRPV1 ion channel; nonetheless, the activity of TRPV4 has been associated to several physio and pathophysiological processes, and its dysfunction can lead to severe consequences. Several lines of evidence derived from animal models and even clinical trials in humans highlight TRPV4 as a therapeutic target and as a protein that will receive even more attention in the near future, as will be reviewed here. Keywords: TRP channels; TRPV4; structure; disease 1. Introduction Ion channels are proteins that participate in multiple cellular functions including the generation of electrical signals in the nervous and muscular systems, in the transport of electrolytes, the secretion of hormones, etc. Some of these ion channels work as receptors to changes in temperature, mechanical stimuli, osmolarity, and acidity [1–3]. Among these are members of the Transient Receptor Potential (TRP) superfamily that are characterized by being weakly voltage-dependent nonselective cation channels. The importance of the more of 30 TRP channels described to date is well exemplified by their roles in physiology including phototransduction in invertebrates [4–6], responses to painful stimuli and to temperature [7–9] and to intracellular Ca2+ store depletion [10,11], modulation of the cell cycle [12,13] and regulation of the function of several organs such as the pancreas, lung, kidney, etc. [14]. The general structure of TRP channels, resolved by single-particle cryo-electron microscopy, for several members of this family of proteins shows the presence of intracellular amino (N)- and carboxyl (C)-terminal regions and six (S1–S6) transmembrane domains, where S5–S6 give rise to the pore or ion conduction pathway [15]. Each of these regions in the channels plays a role in the changes associated to their activation and their ability to alternate among conformations, allowing these ion channels to regulate several cellular functions. Thus, it is important to understand the molecular details of the modulation of these proteins by several types of stimuli that are relevant during physiological and pathophysiological contexts. Int. J. Mol. Sci. 2020, 21, 3837; doi:10.3390/ijms21113837 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3837 2 of 36 In this sense, important roles for several TRP channels have been associated to various diseases [16–20]; however, there is still quite a bit of work to be done since the roles of some of these proteins in physiology are still being uncovered. It is important to note that, while some TRP channels have been extensively studied (i.e., TRPV1, the first mammalian TRP channel to be cloned) [7], other members of this family of ion channels have proven to be somewhat more elusive. This review will focus on the TRPV4 channel for which only few endogenously produced agonists have been described, although it is clearly a physiologically important protein. This channel has been shown to regulate the homeostasis of intracellular calcium concentrations [Ca2+]i [21] participating in the integrity of osmoregulation [22,23], endothelial barriers [24–26], control of vascular tone [27], nociception [28], bone homeostasis [29], pulmonary [30,31], and renal function [23], as well as in itch [32]. In fact, several mutations in TRPV4 have been associated to congenital diseases, some of which will be reviewed and discussed here and we will mention recent discoveries in this rapidly changing field of study. 2. General Properties and Structure of TRPV4 Described for the first time in the year of 2000, TRPV4 has acquired the following names according to the functional features observed when it was studied: OSM-9 [33], VRAC or VR-OAC [22], VRL-2 [34], and TRP12 [35]. TRPV4 is expressed in smooth muscle cells in the pulmonary aorta and artery, brain arteries [36,37], vascular endothelium [38–40], epidermal keratinocyte cells [41], epithelia of the trachea and lungs (specially in cilia of the bronchial epithelium) [42], Fallopian tubes [43], ciliated epithelia of bile ducts [44], epithelial cells of the human cornea [45], insulin secreting β cells of the pancreas [46], epithelial cells of the urinary system [47], urothelial cells in the renal pelvis, ureters, urethra, urinary bladder [48,49], primary afferent sensory neurons that innervate the gastrointestinal tract [50], as well as in enterocytes and enteroendocrine cells [51,52], etc. TRPV4 is a nonselective cation channel with higher permeability for Ca2+ (due to interaction of the ion with residues D672, D682, and M680 in the pore region) than for Mg2+ as compared to Na+ [53,54] and that has been described to be both an inward and outward rectifier depending on whether Ca2+ is absent or present [22,53]. In fact, this channel has been shown to be inhibited or potentiated in a Ca2+ concentration-dependent fashion [55], a process that has been described to rely on the presence of residue F707 [56]. Like many other TRP channels, TRPV4 is a polymodal protein activated by temperatures around 27 ◦C, hypoosmotic conditions, and mechanical stress [22,23], and blocked or antagonized by GSK3527497 [57], GSK205 [58], derivates of GSK205 [28], ruthenium red [59], and Gd3+ (which has also been shown to block stretch-activated channels in bacteria) [58,60], RN-1734 [61], and RN-9893 [62]. Ligands for this channel include plant chemicals such as bis-andrographolide from Andrographis paniculate [63]; phorbol derivatives (i.e., 4α-phorbol 12,13-didecanoate or 4αPDD [53]), the flavonoid apigenin [64], and the widely used synthetic agonist GSK1016790A [65]. Endogenous agonists or activity modulators of TRPV4 are phosphatidylinositol-4,5-biphosphate (PIP2), which binds to the 121KRWRK125 region and allows for activation by temperature and osmotic changes [66]; 5,6-epoxyeicosatrienoic acid (5,6-EET), a product of the metabolism of arachidonic acid through cytochrome P450, which binds to residue K535 [67,68] of the channel activating it, and polyunsaturated fatty acids that affect the activity of the channel through changes in membrane fluidity [69]. A structure for the Xenopus tropicalis TRPV4 channel was recently resolved from a nearly complete protein (amino acids 133–797) in which only a few residues from the N- and C- termini were deleted and a glycosylation site was modified (N647Q) [70]. Deng and collaborators found that the TRPV4 channel is a symmetric tetramer formed by subunits with six transmembrane domains with linking loops that resemble those of voltage-gated ion channels (VGICs), where S1–S4 domains encircle the pore formed by S5–S6 [70]. Int. J. Mol. Sci. 2020, 21, 3837 3 of 36 The intracellular N-terminal regions contain ankyrin repeat domains (ARD) followed by linkers with two β-strands, β1 and β2, which precede the S1–S4 domains of each subunit and that, together with a β-strand β3 in the C-terminus, give rise to a three-stranded β-sheet that tethers the ARD from the N-terminus to the C-terminus of the same subunit, allowing for interaction with the ARD of another subunit. Following the β-strands, a helix-turn-helix motif and a pre-S1 helix lodge an amphipathic TRP helix in the C-terminus [70]. This TRP helix, a conserved sequence among TRP channels, has been proposed to regulate the gating of these channels and it follows the S6 transmembrane domain in such a fashion that it runs parallel to the membrane and comes nearby to the S4–S5 linker. Thus, it ends up being localized between the cytosolic regions and the transmembrane domains [71–73] (Figure 1A). β strand β3 in the C stranded β β β β β Figure 1. Structure of the TRPV4 channel. (A) Frontal view (top) and extracellular view (bottom) where one single subunit of the tetramer and each of its domains are shown in a different color (ARD in orange, β1 and β2 in blue, Helix Turn Helix in green, PreS1 in pink, S1–S4 in purple, S5–S6 in red, TRP box in magenta, and β3 in cyan). The membrane boundary is delimited by the black lines. (B) TRPV4 pore diameter. The structures of S6 and the pore helix of two subunits are shown. The distance between the two M714 residues is represented by the black line. (C) Top view of S1–S4 and S6 domains. Subunit domains follow the same color scheme as in A. The 6BBJ PDB file was used to produce this figure [70]. It must be noted that Arniges and collaborators have shown that the ARDs are important for oligomerization and trafficking of the channel since the absence of these domains results in accumulation of TRPV4 protein in the endoplasmic reticulum [74]. This led these authors to propose a reevaluation of the role of the ARDs in TRPV4 function with respect to previous findings where it was suggested that deletion of these regions still results in functional proteins, albeit less responsive to activation by hypoosmotic stimuli [1]. The ARDs have also been shown to be important for activation of mouse TRPV4 by temperature. In this sense, Watanabe and collaborators have shown that deletion of the first three proximal ARDs results in mutant channels that do not respond to heat [75]; hence, these structures play an important role in temperature detection in this channel. On the other hand, Liedtke et al., showed that in channels where the ARDs have been removed, cell swelling can still activate TRPV4, leading these authors to conclude that the link that the ARDs provide for TRPV4 to the cytoskeleton is Int. J. Mol. Sci. 2020, 21, 3837 4 of 36 not necessary for its response to osmotic stress [22]. Moreover, as will be discussed here, mutations in the ARDs of TRPV4 are associated to genetic diseases such as Charcot-Marie-Tooth disease type 2C [76]. As for the pore of TRPV4, unlike TRPV1 [77], only one constriction was found with the narrowest region (5.3 Å in diameter) being located at residue M714 and it was designated as the lower gate (Figure 1B). Its selectivity filter was shown to be remarkably wide, as compared to other TRP channels, and to coordinate a single hydrated cation, irrespective of the ion valency, at basically the same location [70]. An interesting difference between the TRPV1 and TRPV4 channels is that, in TRPV4 the S1–S4 domain is rotated counterclockwise by approximately 90 ◦C around the S4 helix, leading to a very characteristic packing interface between S1–S4 and the pore domains. Furthermore, also the way S3 points to the pore and contacts with S6 led the authors to speculate on the possibility of S3 directly interacting with S6 to open the gate [70] (Figure 1C). We will next explain the function of TRPV4 in the normal physiology of some systems and/or organs that highlight its role as a mechanosensor, osmosensor, and as a chemosensor. It is worth mentioning, that the role of a protein is many times more evidently illustrated when it exhibits a dysfunction that, in turn, often leads to a pathology. Hence, here we have also described some of the diseases that are generated as a consequence of a change in the function of TRPV4. 3. TRPV4 and the Vascular Endothelium Adequate Ca2+ signals play a fundamental role in the function of endothelium and in the systems and organs that express endothelial cells, contributing to their homeostasis. Aside from intracellular Ca2+ stores that contribute to homeostasis [78], the influx of this ion from the extracellular milieu is extremely relevant for the function of the endothelium and TRPV4 is one of the molecules that participate in this process [27]. Watanabe and collaborators described the activation of TRPV4 in mouse aortic endothelial cells (MAEC), and they corroborated the activation of this channel using a heterologous expression system in HEK293 cells. These authors showed that, in both types of cells, temperatures above 25 ◦C activated TRPV4 allowing for the influx of Ca2+ into the cells and this was reproduced when 4α-phorbol 12,13-didecanoate (4αPDD) was used to activate the channel [53]. Then, the same research group showed that arachidonic acid and anandamide activated TRPV4 in endothelial cells indirectly via cytochrome P450 [68]. Further work carried out by Vriens and colleagues confirmed these results since the block of phospholipase A2 and cytochrome P450 resulted in the inhibition of TRPV4 when activated by osmotic swelling, while activation by temperatures above 25 ◦C or by 4αPDD remained unchanged [79]. These studies suggested that activation of TRPV4 in vascular endothelial cells contributes to relaxant effects of endocannabinoids on the vascular tone. Later, by using a combination of techniques including patch-clamp and [Ca2+]i measurements, it was shown that TRPV4 was activated in MAEC in response to heat, swelling, 4αPDD, and arachidonic acid, but this activation did not occur in MAEC harvested from trpv4−/− mice [80]. This prompted several other research groups to broaden the study of the functions of TRPV4 in endothelial cells since it could be of importance for diseases such as hypertension, atherosclerosis, diabetic vasculopathy, vascular tumors, stroke, etc. It was then shown that the loss-of-function of TRPV4 in endothelial cells of the carotid artery affected the process of vasodilation due to the lack of activation of the channel in the presence of mechanical stimuli (or shear stress), leading to problems in the regulation of the vascular tone, blood pressure, and ultimately affecting the supply of glucose and oxygen to the organs [81]. More recently, the technique of total internal reflection fluorescence microscopy (TIRFM) was used to study microvascular human endothelial cells and it was shown that TRPV4 is present at the plasma membranes of these cells. Moreover, the same authors proposed that GSK1016790A acted by recruiting and activating TRPV4 channels that had previously remained inactive and not by increasing their basal activity [82]. Int. J. Mol. Sci. 2020, 21, 3837 5 of 36 It has been demonstrated that TRPV4 channels give rise to Ca2+ transients in the plasma membranes of endothelial cells that lead to the recruitment of other downstream effectors that result in a magnified Ca2+ signaling [83]. McFarland and collaborators used an endothelium-specific trpv4−/− mouse model (ecTRPV4−/−) where the channel was specifically knocked down in keratinocytes, to newly show that the artery endothelium from the intact arterial intima of these animals produces larger Ca2+ events (via release from internal stores), as compared to the wild-type counterparts but these mutant mice also display deficiencies in “small events”, meaning that TRPV4 channels generate focal Ca2+ transients and are essential for vascular homeostasis [84]. Another important aspect of the roles of TRPV4 in the organism are the effects of the influx of Ca2+ through this channel at the tight junctions among vascular endothelial cells. There are two important mediators of endothelium-derived hyperpolarization (EDH(F)-mediated) relaxation: TRPV4 and connexins or gap junctions. Both these proteins control vascular tone in concert with nitric oxide (NO) that can mediate relaxation. It has been shown that when ischemia and reperfusion are emulated in arteries from in vivo preconditioned mice that were exposed to repeated cycles of hypoxia-reoxygenation, NO-mediated relaxation is decreased but EDH(F)-mediated relaxation is increased in superior mesenteric arteries [85]; hence, TRPV4 channels and connexins might be important for maintaining vasorelaxation under hypoxia. Rath et al., also found that preconditioning can preserve vascular function through a mechanism that restores relaxation induced by NO and improves the EDH(F)-mediated response. Furthermore, by using cultured endothelial cells and mice aortas, they found that TRPV4 channels (whose activity and expression levels are increased during hypoxia) and connexins Cx40 and Cx43, help protect the vasculature under an experimental scenario where protection to the vessels is achieved by hypoxic preconditioning and they further strengthen the signaling pathway that depends on NO for these effects to be achieved [85]. Mendoza and collaborators investigated the role of TRPV4 in mouse mesenteric arteries, which maintain a low resistance to changes in blood flow or mechanical stimuli. First they showed that TRPV4 was present in this type of endothelial cells and then they activated the channel with GSK1016790A that allowed for an increase in [Ca2+]i and demonstrated that it acted as a second messenger that induced relaxation of the arteries of wild-type mice but not trpv4−/− mice, concluding that TRPV4 may act as one of the mechanosensitive channels responsible of transducing shear stimuli into Ca2+ signaling in vascular endothelial cells [86]. The fundamental roles of TRPV4 in the vascular endothelium are consistent with the role of this channel in cardiovascular diseases. For example, it is known that vascular hardening (i.e., atherosclerosis) is associated to the outcome of a cardiovascular (CVD) disease. It has been proposed that activation of TRPV4 can limit vascular inflammation and atherosclerosis [87]. Thus, it follows that endothelial dysfunction in which reduced vasodilation and increased vasoconstriction occur, not only leads to changes in vascular tone but it accelerates the progression of CVDs. Until most recently, the mechanisms by which stiffening of the vascular tone happens had not been clarified. However, Song and collaborators performed experiments using human umbilical vein endothelial cells, and by increasing substrate stiffness, they showed that expression and activity of TRPV4 was reduced, an effect that was followed by an increase in expression of the potent vasoconstrictor endothelin 1 (ET-1) and a decrease in endothelial nitric oxide synthase (eNOS) expression [88] (Figure 2). These authors also identified a stiffness-sensitive microRNA (miR-6740-5p), whose levels were also decreased in stiff substrates. In summary, Song et al., proposed that decreasing the activity of TRPV4 leads to an increase in ET-1 by inhibiting miR-6740-5p and this, in turn, is associated to vascular stiffening that worsens the prognosis of CVD patients [88]. Int. J. Mol. Sci. 2020, 21, 3837 6 of 36 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 37 was shown that GSK1016790A augmented permeability of retinal blood vessels in wild-type mice but not in trpv4−/− mice. Therefore, TRPV4 seems to be pivotal for Ca2+ homeostasis and barrier function i retinal capillaries and probably contributes to inner versus outer blood-retinal barrier function (BRB) [24]. Figure 2. Function of TRPV4 in the vasculature. (A) Representation of a region of human vasculature and a cross section of an arteriole. (B) Activation of TRPV4 by different stimuli (i.e., 4α-phorbol 12,13- didecanoate or 4αPDD) leads to changes in intracellular Ca2+ levels, promoting the activity of the enzyme nitric oxide synthase (eNOS) and then increasing the levels of nitric oxide (NO). NO can cross endothelial cell membranes and activate other signaling pathways in smooth muscle cells, inducing vasodilation. Created with Biorender.com. Retinal diseases such as diabetic retinopathy exhibit a phenomenon in which there is a breakdown of the blood-retinal barrier that, in turn, leads to neuronal tissue damage and vasogenic edema and then loss of vision [90]. In 2017, Arredondo et al., showed that TRPV4 is expressed in the endothelium and retinal pigment epithelium (RPE) of the BRB and that the use of TRPV4 antagonists could resolve breakdown of the BRB in diabetic rats. They also studied human RPE cell monolayers and endothelial cell systems and found that vasoinhibins (that are endogenous regulators of angiogenesis and vascular function and which have been shown to exhibit lower circulating concentrations in diabetic patients) can block TRPV4, concluding that inhibition of the channel can contribute to conserve the integrity of the BRB and endothelial permeability [91]. Later it was also shown that TRPV4 regulates the migration and tube formation of human retinal capillary endothelial cells, again highlighting TRPV4 as a likely therapeutic target in retinal vascular diseases [92]. In fact, TRPV4 has also been pinpointed as an important molecular effector in glaucoma since new evidences show that its mechanotransducing roles are important within cells in the retina such as: Müller cells, ganglion cell soma-dendrite, and microglia. A dysfunction of the mechanotransduction mechanisms in these cells precedes damage to the retina due to changes in pressure and the antagonism of these mechanosensing protein (among others) could lead to beneficial effects [93]. Figure 2. Function of TRPV4 in the vasculature. (A) Representation of a region of human vasculature and a cross section of an arteriole. (B) Activation of TRPV4 by different stimuli (i.e., 4α-phorbol 12,13-didecanoate or 4αPDD) leads to changes in intracellular Ca2+ levels, promoting the activity of the enzyme nitric oxide synthase (eNOS) and then increasing the levels of nitric oxide (NO). NO can cross endothelial cell membranes and activate other signaling pathways in smooth muscle cells, inducing vasodilation. Created with Biorender.com. TRPV4 is also expressed in the endothelial microvasculature of the retina and has been associated to diabetic retinopathy. In bovine tissue it was shown that TRPV4 is expressed in this system and when cells were cultured under hyperglycemic conditions, a decrease in the expression of TRPV4 was observed, as compared to control cultures [89]. It was later demonstrated that activation of TRPV4 with GSK1016790A in vitro reversibly increases the permeability of human retinal microvascular endothelial cell monolayers and in vivo it was shown that GSK1016790A augmented permeability of retinal blood vessels in wild-type mice but not in trpv4−/−mice. Therefore, TRPV4 seems to be pivotal for Ca2+ homeostasis and barrier function in retinal capillaries and probably contributes to inner versus outer blood-retinal barrier function (BRB) [24]. Retinal diseases such as diabetic retinopathy exhibit a phenomenon in which there is a breakdown of the blood-retinal barrier that, in turn, leads to neuronal tissue damage and vasogenic edema and then loss of vision [90]. In 2017, Arredondo et al., showed that TRPV4 is expressed in the endothelium and retinal pigment epithelium (RPE) of the BRB and that the use of TRPV4 antagonists could resolve breakdown of the BRB in diabetic rats. They also studied human RPE cell monolayers and endothelial cell systems and found that vasoinhibins (that are endogenous regulators of angiogenesis and vascular function and which have been shown to exhibit lower circulating concentrations in diabetic patients) can block TRPV4, concluding that inhibition of the channel can contribute to conserve the integrity of the BRB and endothelial permeability [91]. Later it was also shown that TRPV4 regulates the migration and tube formation of human retinal capillary endothelial cells, again highlighting TRPV4 as a likely therapeutic target in retinal A B Endothelial eells Human vasculature (thígh) Smooth muse le eells Arteriole (cross-section) \ 'fH ir-- Endothelial eells ~~;:,; ~ ",, "'----Smoo th musele eells 4aPDD GSK1016790A Mechanical stimuli Vasodilation TRPV4 Int. J. Mol. Sci. 2020, 21, 3837 7 of 36 vascular diseases [92]. In fact, TRPV4 has also been pinpointed as an important molecular effector in glaucoma since new evidences show that its mechanotransducing roles are important within cells in the retina such as: Müller cells, ganglion cell soma-dendrite, and microglia. A dysfunction of the mechanotransduction mechanisms in these cells precedes damage to the retina due to changes in pressure and the antagonism of these mechanosensing protein (among others) could lead to beneficial effects [93]. In the endothelial microvasculature of the human brain (HBMEC) the expression of TRPV4 was also confirmed and shown to regulate [Ca2+]i, and thus proposed to act as a protective molecule during damage to brain vasculature [94]. Nevertheless, the angiogenesis that TRPV4 drives after ischemic neuronal death has not been finely examined and it has not been clearly determined if activation of this channel can serve as an effector of neurorestorative events. Recently, it was evaluated if the activation of TRPV4 could aid functional recovery in rats subjected to transient brain ischemia. By using 4αPDD that was intravenously injected via the tail veins of these animals, it was shown that the infarct volume was reduced by nearly 50% and this was accompanied by better functional outcomes [95]. In this same study, it was also shown that eNOS expression was increased and the expression of vascular endothelial growth factor A (VEGFA) and its receptor VEGFR2 were also augmented, resulting in amplified microvessel density and enhanced neural stem/progenitor cell proliferation and migration. This suggests that activation of TRPV4 may improve poststroke outcomes. 4. TRPV4 in the Respiratory Airways and Lung Function The respiratory system presents sensory innervation of the trachea, glands of the larynx, smooth muscle, bronchial tree and lungs [96,97]. Changes in breathing patterns, dyspnea, and cough are all due to the activation of sensory afferent nerve impulses that travel through the vagal nerve to the central nervous system [98]. There are afferent nerve endings in the upper airway that shield the lower airway from foreign noxious and/or irritant substances [98]. TRP channels are well known to respond to a wide range of molecules that include those that are potentially harmful to an organism [1,99]. In the respiratory airway, TRP channels induce inflammation, airway constriction, mucus secretion, and sneezing and coughing, all of which constitute a mechanism of defense for the respiratory airways [20,98,100]. The TRPV4 channel is expressed in the smooth muscle, fibroblasts, macrophages, submucosal glands, vascular endothelial cells, and in tracheal, bronchial, and alveolar epithelia [20,22,34,101–103]. Aside from maintaining the homeostasis of osmotic pressure in these tissues, TRPV4 also integrates stimuli of a diverse nature that translate into Ca2+ signals, promoting different responses in the tissues of the respiratory system [104]. Genetical, molecular, and physical regulators come together to ensure adequate pulmonary development. The TRPV4 channel is expressed in apical and basal surfaces of the respiratory airway epithelium, in the subepithelial mesenchyme and in the main pulmonary vessels, all of which are essential for the generation of the dynamic mechanical forces during pulmonary development [30]. Although there is no detailed pathway yet described, TRPV4 has been portrayed as a positive regulator of pulmonary development in the embryonic phase and seems to be important for pulmonary growth based on the mechanism of smooth muscle-contraction [105]. In a murine model of ex vivo embryonic lung explants from mice it was shown that TRPV4 is a modulator of the morphogenesis of the respiratory airway. The activation of the channel promotes the ramification of the respiratory airways, regulates contractility of the smooth muscle favoring its differentiation and augmenting the density of vascularization that supports the growth of the lung in general [30]. In fully-developed animals, Pankey and collaborators studied the response of TRPV4 to the synthetic agonist GSK1016790A in the vascular system of the lungs of rats. They found that when the agonist was injected at low doses it produced a decrease in the pulmonary arterial pressure. Their Int. J. Mol. Sci. 2020, 21, 3837 8 of 36 results showed that GSK1016790A had vasodilation activity in the vascular and pulmonary systems of rats, implicating TRPV4 as a molecular effector of this response [106]. Another research group showed the presence of TRPV4 in the cells of the pulmonary endothelial microvasculature of the mouse (MLMVEC) and of the human (HLMVEC) by studying the effects of oxygen reactive species on [Ca2+]i due to TRPV4 activity. In this particular case, it was revealed that H2O2 activates TRPV4 through a mechanism that requires the presence of a Fyn kinase and, it was concluded that the H2O2 induces an increase in [Ca2+]i as a result of a decrease in transmembrane electrical resistance in microvascular endothelial cells and that agonism of TRPV4 and reactive oxygen species worsen barrier function [107,108]. Ke and collaborators also performed experiments that showed the participation of TRPV4 in pulmonary vasculature. These authors used the myographic technique and GSK1016790A to activate the TRPV4 channel and studied regulation of the vascular tone in arterial rings from the main pulmonary arteries. Their results showed that GSK1016790A relaxed the main pulmonary artery and augmented vascular resistance (vasoconstriction) of pulmonary circulation in isolated perfused lungs, effects that were inhibited by the TRPV4 antagonist AB159908 [109]. These experiments led Ke et al., to propose a mechanism of action of TRPV4 in lung vasculature where the entry of Ca2+ through the channel into endothelial cells results in the activation of the intermediate conductance potassium channel (IKCa) and the small conductance potassium channel (SKCa) that leads to the contraction of the pulmonary vascular bed. Conversely, the activation of NO-dependent signaling pathways primes the relaxation (vasodilation) of the main pulmonary arteries [109], suggesting opposing effects for TRPV4 in the lung vasculature. Interestingly, it was found that there is a close interplay between three molecules that are thought to be pivotal for airway sensory nerve reflexes: TRPV4, ATP (adenosine triphosphate), and P2X3R (P2X purinergic receptor 3, which is activated by ATP) [110]. The use of agonists for TRPV4 and of hypoosmotic solutions led to depolarization of the vagal nerves in humans, mice, and guinea pigs, while antagonists of TRPV4 resulted in a decrease in cough [110]. Hence, it was proposed that TRPV4 is a molecular effector of airway protection and it comes as no surprise that changes in the function of this protein result in respiratory diseases. Furthermore, Gu and collaborators performed experiments directed at determining the role of the TRPV4 channel in the regulation of breathing in rats. The activation of TRPV4 with GSK1016790A in anesthetized rats induced rapid superficial breathing and potentiated the effects of apnea induced by capsaicin (i.e., chemoreflex) through the activation of TRPV1 in sensory neurons. By using a selective antagonist of TRPV4 (GSK2193874) and by dissecting vagal nerves, these effects were prevented and an indirect role for TRPV4 was suggested in breathing [111]. However, immunocytochemistry and electrophysiological experiments showed that the channel is not functionally-expressed in fibers from sensory neurons, although it is present in macrophages, epithelial and endothelial cells and these authors concluded that regulation of ventilation is due to indirect activation of bronchopulmonary sensory neurons through stimulation of other cells that express TRPV4 in the respiratory system [111]. The endothelial tissue functions as a semipermeable barrier between blood and subjacent tissue that plays a fundamental role in the movement of gases, fluids and molecules of different natures among compartments and maintains cellular homeostasis. It has been proposed by Parker and collaborators that ion channels activated by cell-stretching were responsible of the loss of vascular permeability in the face of increases in pressure [100] and later TRPV4 was identified as the architect of the loss of endothelial permeability [112] and as promoter of the entrance of Ca2+ into cells that leads to the reorganization of the cytoskeleton and to the loss of interendothelial junctions [113]. The activation of TRPV4 with 4αPDD and 14,15-EET induces tearing of the septal endothelium although it maintains the integrity of the interendothelial junctions in the lung of rats and mice, while thapsigargin (an ATPase-mediated inhibitor of the transport of Ca2+ into the endoplasmic reticulum) promotes the generation of holes in the junctions of endothelial cells in the extra-alveolar vessels. These Int. J. Mol. Sci. 2020, 21, 3837 9 of 36 observations have prompted the conclusion that damage to extra-alveolar vessels entails different functional consequences to the interruption of the alveolar basal barrier. This, in turn, results in the accumulation of liquid in the alveoli, disallowing for the exchange of gases in isolated lungs from mice and rats, a characteristic symptom of acute lung injury (ALI) [101]. Nonetheless, it was later reported that activation of TRPV4 in wild-type mice increased the permeability in lung endothelial cells without affecting adhesion proteins (i.e., selectins) and without affecting the lung’s wall integrity, a phenomenon that was not observed in trpv4–/– mice [114]. ALI and the acute respiratory distress syndrome (ARDS) are characterized by an acute increase in the permeability of pulmonary vascular and endothelial barriers [115]. In animal models of ventilator induced pulmonary injury [116], by liquids [31] and by exposure to chemical compounds (hydrochloric acid or chloride vapors) [117], it was demonstrated that TRPV4 plays a crucial role in the injury to the lungs (Figure 3). The use of inhibitors of TRPV4 after the induction of ALI was achieved, results in reduced hyperreactivity of the airway pathways, prevents edema, and produces a decrease in arterial pressure. Moreover, it has also been shown that there is a reduction in the pulmonary elastance (elastic deformation by a stressful event that is implicated in alveolar damage) and in the tensoactive-related leakage of proteins, in the infiltration of neutrophils and macrophages to the lung and in the production of inflammatory molecules such as cytokines and promoters of phospholipase A2 [31,117]. On the other hand, when pulmonary injury was promoted through the use of a ventilator, the generated heat induced an increase in pulmonary endothelial and epithelial permeability of wild type mice, which was absent in trpv4–/– mice or when inhibitors of the channel or of cytochrome P450 were used [116]. A later study by this same research group showed the importance of the expression of TRPV4 in alveolar macrophage cells. By activating TRPV4 with 4αPDD increments in [Ca2+]i, superoxide and NO in cells from wild type animals were observed and the role of TRPV4 in signaling pathways associated to pulmonary function was further supported [112] (Figure 3). Moreover, it was reported that the activation of TRPV4 and subsequent Ca2+ entry promote the expression of matrix metalloproteinases (MMP2 and MMP9) and a reduction in the TIMP1 (principal metallopeptidase inhibitor 1) in the broncho-alveolar lavage fluid (BALF) of the mouse lung, which is not observed in trpv4–/– mice. Hence, it was suggested that TRPV4 participates in the recruitment of macrophages and neutrophils during lung injury. Pulmonary epithelium is formed by different cell lineages: while the larynx and the trachea are coated by squamous epithelia, other tissues of the superior region are coated by cylindrical epithelium that secretes mucus or by caliciform cells that play a similar role in both, the superior and inferior respiratory airways. Clara non-ciliated and non-mucous secretory cells line the most distal region of the pulmonary tree, while epithelial cells type 1 and 2 line the alveoli [118]. All of these different cell types are in charge of preserving the structural integrity of the lungs by allowing for gas exchange, ion transport, growth factor secretion, and by protecting the organism against pathogens and contaminating particles [118]. In primary human bronchial epithelial cells (NHBE) and in the intact trachea of mice, activation of TRPV4 and low-voltage-activated (LVA) calcium channels was reported in response to gentle shearing, a process that allows for a better function of the epithelial barrier since the entrance of Ca2+ results in the reorganization of actin and in the formation of stress fibers. Notably, these effects are hindered when there is no Ca2+ in the media or in the presence of blockers and/or antagonists of LVA calcium channels or of TRP channels. Hence, it was concluded that shear forces regulate barrier function and the role of these proteins, as well as that of aquaporin 5, was highlighted. Specifically, it was proposed that the entry of Ca2+ followed activation of TRPV4 and voltage-gated calcium channels, resulting in the block of solute permeability [119]. Int. J. Mol. Sci. 2020, 21, 3837 10 of 36 As mentioned above, a strong mechanical force such as the one applied by a ventilator to induce pulmonary injury (VILI) will hamper lung function. Excessive mechanical stretch applied in vitro (i.e., using NCI-H292 human pulmonary epithelial cells) or in vivo (murine ventilation model), promotes distension and the consequent opening of the TRPV4 channel and the entrance of Ca2+ into the epithelial cells of the lung (Figure 3). This, in turn, leads to the release of cytokines such as interleukins -6, -8 and -1α (IL-6, -8 y -1α). Under this scenario, it was shown that the exposure to the selective TRPV4 antagonist, GSK2193874, reduced Ca2+ concentration, and levels of proinflammatory molecules only by about 30%, so this suggested that other mechanically-gated ion channels were involved. Nonetheless, the data demonstrated that TRPV4 also participates in this type of stress induced by stretch and plays a role in the permeability of the pulmonary barrier [25]. Nonetheless, it has also been shown that lipopolysaccharides (LPS) from gram-negative bacteria [120] activate TRPV4 from airway epithelial cells occur independently of Toll-like receptor 4 (TLR4, a protein whose activation leads to the production of inflammatory cytokines) [121], unlike what had been suggested by other groups [122]. Alpizar et al., showed that LPS produce increases in [Ca2+]i in mouse and human airway epithelial cells after a few seconds of their application and lead to an increase in CBF and of NO (that has direct antimicrobial and bronchodilation actions). Accordingly, when TRPV4 was pharmacologically inhibited or genetically deleted, augmented ventilatory and inflammatory responses were observed when mice were defied with LPS. In conclusion. TRPV4 is a central molecule in the defenses against bacterial endotoxins [121]. Ciliated cells are in charge of allowing for the propulsion of the mucus gel layer in the respiratory airways and providing for a first line of defense against inhaled particles and pathogens [123]. It has also been demonstrated that TRPV4 is expressed in the ciliated cells of mice trachea and that Ca2+ entry through this channel influences the frequency of the ciliary beat (CBF). By using 4αPDD and activating TRPV4, the CBF was increased and this effect was not observed in trpv4–/– mice. Similarly, it was also described that at 30 or 40 ◦C and under conditions of high viscosity where TRPV4 was activated, these Ca2+ signals also influenced CBF [42]. Interestingly, TRPV4 has also been linked in the response to contaminating particles, such as the diesel exhaust particles (DEP). Although these particles do not seem to directly activate the channel, they promote the activity of proteins, as briefly summarized next: the organic fraction of DEP activates proteinase 2 (PAR-2) and this, in turn, activates TRPV4 (in the moving cilia of the bronchial epithelia) through phospholipase Cβ3 (PLCβ3) and phosphatidylinositol 3-kinase (PI3K) allowing for the entrance of Ca2+, which is pivotal for the expression of matrix metalloproteinase 1 (MMP-1), an important molecule for tissue remodeling during development, migration of inflammatory and malignant cells, and diseases of the respiratory airways [124]. Silica nanoparticles (SiNP) are extensively used in cosmetics, food, biotechnology, medical, pharmaceuticals, and chemical industries. These SiNP have been shown to affect the function of the respiratory airways [125]. In this sense, Sanchez and collaborators evaluated the effects of SiNP on the activation of TRPV4 channels from cultured human airway epithelial cells 16HBE and primary cultured mouse tracheobronchial epithelial cells [125]. The authors found that SiNP inhibit the entrance of Ca2+ through GSK1016790A-activated TRPV4 channels in both cell types. Moreover, these authors also showed that TRPV4-current inhibition by SiNP (which was dose-dependent) occurred regardless of whether the channels were activated with GSK1016790A or 4αPDD and estimated that nanoparticles rapidly exerted their effects on the channel. In contrast, SiNP produced potentiation of TRPV1 currents in response to capsaicin, leading the authors to conclude that the antagonistic effects of these molecules on TRPV4 were specific [125]. Finally, it was also shown that SiNP decreased the effects of GSK1016790A on ciliary beat frequency. Since SiNP are approximately the same size as the whole TRPV4 channel protein (and double the size of the length of the transmembrane segments of TRPV4), the authors concluded that it is unlikely that these molecules bind to the channel but they might rather produce mechanical disturbances in the plasma membrane that lead to inhibition of TRPV4 [125]. Int. J. Mol. Sci. 2020, 21, 3837 11 of 36 Li et al., went further into determining the steps by which the above described phenomenon takes place. By using human bronchial epithelial cells (BEAS-2B) and human bronchial epithelium (HBE), they activated TRPV4 with 4αPDD and hypotonicity and observed that MMP-1 secretion was increased, while secretion was inhibited with an antagonist of TRPV4 [124]. Moreover, it was shown that the pathological activation of MMP-1 by the enhanced entrance of Ca2+ through a mutant TRPV4 with a gain-of-function mutation (TRPV4-P19S) [124] present in a population of humans, could play an important role in chronic obstructive pulmonary disease exhibited by individuals with this genetic polymorphism [126]. A year after this, an artificial lung model was recreated using coats of endothelial and epithelial cells with an alveolo-capillary interphase (a compartment with air and liquid, respectively) and was shown to be capable of imitating the movement of breathing in a normal sate [127]. It was demonstrated that interleukin-2 (IL-2) induces the infiltration of the alveolar canal, that is, an edema due to mechanical tension induced by this molecule. Nonetheless, activation of TRPV4 impedes the leakage, again providing evidence in support of the role of this channel in the face of mechanical forces [127]. In in vitro experiments (using NCI-H292 cells), activation of TRPV4 with various agonists (4αPDD, GSK1016790A, and 5,6-EET) promoted a dose- and time-dependent release of proinflammatory molecules (cytokines, chemokines, etc.) such as interleukin-8 (IL-8) and prostaglandin E22 (PGE2). In vivo experiments, after 24 h of intranasal delivery of 4αPDD, showed that it was possible to detect an increase in keratinocyte chemoattract (KC) and in PGE2, as well as an infiltration of neutrophils [128]. These results suggest that TRPV4 may be a target for treatment of diseases such as cystic fibrosis, as will be detailed below. Preservation of the fluid compartment localized at the apical surface of epithelia is pivotal for the function of several organs. In the respiratory airways, the surface liquid layer is important for mucociliary clearance. An adequate balance in the secretion of Cl−, HCO3 and other anions is crucial for normal physiology of epithelial apical membranes since the secretion of Cl− determines an electrical driving force for the secretion of sodium in the trans-epithelia and, as a result, correct osmotic driving forces for water and secretion products are established [129]. Both production of luminal fluid and its secreted volume are hindered in cystic fibrosis, resulting in the pathophysiological phenomena that typify this disease. This disorder was shown to be mainly caused by mutations in the cystic fibrosis transmembrane regulator protein (CFTR, that is regulated by cyclic adenosine monophosphate or cAMP), which is characterized by severe chronic pulmonary inflammation [130]. Nonetheless, other molecular effectors have been assayed for their importance in the generation or progression of this disease, as are the examples of the transmembrane member 16A (TMEM16A, an anion channel that is activated by Ca2+) [131] and of the TRPV4 channel. It has also been demonstrated that, in vitro and in vivo, TRPV4 is constitutively a part of the signaling pathways of cytosolic phospholipase A2 (cPLA2α), MAP-kinases, and NF-κB, supporting its involvement in the inflammatory response and potentially in cystic fibrosis pathogenesis [128]. Experiments showed that, in the absence or presence of CFTR, 4αPDD, or GSK1016790A caused a higher degree of mobilization of Ca2+, secretion of inflammatory components, KC release, and recruiting of neutrophils [128]. Hence, TRPV4 plays extremely important roles in the airways and is involved directly or indirectly in its pathological states. Since Cl− channels are fundamental for mucociliary clearance [132], Genovese and collaborators recently tested whether TMEM16A’s activity could be targeted to regulate Cl− transport in the airway epithelia. In their experiments, these authors used the N-(2-methoxyethyl)-N- (4-phenyl-2-thiazolyl)-2,3,4-trimethoxybenzeneacetamide (Eact) small molecule to directly activate TMEM16A and found that it displayed a mild effect on Cl− transport in airway epithelial cell; however, they discovered that Eact activates TRPV4. Moreover, these observations led this research group to highlight a coupling mechanism between TRPV4 and CFTR-dependent Cl− secretion through the activation of CFTR by the increase in [Ca2+]i that results from the activation of TRPV4. The authors also concluded that in non-ciliated cells, Ca2+-dependent signaling pathways are elicited by purinergic Int. J. Mol. Sci. 2020, 21, 3837 12 of 36 receptor stimulation leading to TMEM16A (and mucin exocytosis) activation, while in ciliated cells, it is the flux of this ion through TRPV4 the phenomenon that seems to regulate ciliary beat frequency in response to mechanical stress or chemical stimulation. These data imply that the Ca2+-dependent signaling events are different depending on the cell type [133]. The smooth muscle is part of the respiratory airways and is present in the trachea and the bronchial tree where it functions as an effector and regulator of the bronchomotor tone, the luminal diameter of the airway and modulates the resistance of the latter. The smooth muscle also plays important roles in embryonic development and in the secretion of cytokines, chemokines, and extracellular matrix proteins [134]. The TRPV4 channel was identified in primary cultures from human smooth muscle cells in the respiratory airway and in the intact tract of guinea pigs, where 4αPDD induced the entrance of Ca2+ to the cells and muscle contraction. Interestingly, the activity of other ion channels and signaling pathways was discarded and TRPV4 was pinpointed as essential for the translation of osmotic stimuli in the smooth muscle cells of the respiratory airway [135]. Accordingly, it was also demonstrated that activation of TRPV4 with GSK1016790A promoted the entrance of Ca2+ and a strong constriction in samples of ex vivo human bronchia and guinea pig trachea. These effects were abolished using antagonists of TRPV4 and inhibitors of 5-lipoxygenase (an enzyme that synthesizes cysteinyl leukotrienes that are reversible lipophilic constrictors of the respiratory pathway) and of the cysteinyl-leukotriene 1 receptor (cysLT1), suggesting that there is an indirect mechanism by which TRPV4 promotes the production of cysteinyl leukotrienes and muscle contraction [136]. A feature of chronic asthma is the proliferation of airways that contributes to the remodeling and obstruction of these structures and a role for TRPV4 in this process has been suggested [137]. In rats, activation of the channel with GSK1016790A and 11,12-EET produced the entrance of Ca2+ to the cells in microdomains termed “Ca2+ sparkles”, promoting proliferation of the cells through a signaling cascade that begins with calcineurin that dephosphorylates and promotes the nuclear translocation of NFATc3, which is important for cell proliferation of the smooth muscle under pathological conditions [137]. Finally, it was also reported that KCa3.1 and TRPV4 channels colocalize to human bronchial smooth muscle cells (HBSM) and it was proposed that hyperpolarization of the membrane induced by KCa3.1 leads to an increase in [Ca2+]i through TRPV4 and to the subsequent proliferation of asthmatic HBSMs. Furthermore, silencing of any of the genes coding for these ion channels attenuated the entrance of Ca2+ and cell proliferation, a process associated to chronic asthma [138]. Another type of cell present in the respiratory airways and the lungs are macrophages that play essential roles in inflammatory processes [139]. Pairet and collaborators had shown, as mentioned above, that equibiaxial stretch in macrophages M1 promotes the release of IL-1α, IL-1β, IL-6, and IL-8, an effect that is prevented when TRPV4’s activity is antagonized with GSK2193874. The physiological implication of this process, according to the authors, is that an increase in the permeability of the endothelia and epithelia of the respiratory airways results in edema and alveolar flooding [25]. Additionally, macrophages have been shown to contribute to the increase in vascular permeability after VILI. Filtration coefficients (Kf) were measured in the lungs of wild-type mice, of trpv4−/− and of trpv4−/− mice inoculated with macrophages from wild-type mice, after which they were exposed to periods of 30 min of ventilation at 9, 25, and 35 cm H2O. These experiments showed that wild-type macrophages restored the Kf of trpv4−/− mice to values similar to those of wild type animal lungs [112]. In the same study, it was also demonstrated that activation of TRPV4 with 4αPDD in macrophages promotes an increase in [Ca2+]i and of superoxide and NO, which does not occur in trpv4−/− cells. The authors then suggested that the channel participates in the pathways associated to reactive nitrogen and oxygen signaling, producing a fast increase in the permeability of the lungs after high pressure and volume ventilation. Hence, TRPV4 seems to be a driving force at initiating this type of injury [112]. In infectious processes, roles for TRPV4 have also been demonstrated. For example, it has been shown that this channel constitutes a part of the response mechanism associated to macrophages in the Int. J. Mol. Sci. 2020, 21, 3837 13 of 36 face of pathogens or, as discussed above, of their lipopolysaccharides in murine in vivo and in vitro models [121,140,141]. It has also been recently suggested that TRPV4 can participate in reducing viral infectivity in diseases such as dengue, Hepatitis C, and Zyka [142]. Doñate-Macián and collaborators have shown that TRPV4 can regulate RNA metabolism dependent on DDX3X [142] (a commonly expressed DEAD-box RNA-binding helicase that is sequestered by many RNA viruses [143,144]). Another viral disease of great importance is COVID-19, which was recently discovered at the end of the year 2019 in China and has been shown to be caused by a beta-coronavirus in assays where human airway epithelial cells were isolated from patients suffering from this disease [145]. The virus was named 2019-nCoV and is now known as the SARS-CoV-2 (Severe Acute Respiratory Syndrome) [145]. Because of its rapid and effective transmission among humans, this disease has been recently (March of 2020) declared a pandemic by the World Health Organization. People infected with this virus may or may not present clinical symptoms, but when they do these are characterized by high temperature, chest discomfort, and cough, which underlie a pneumonia condition [145]. We know that TRPV4 is activated by cytokines [146]. For example, in an asthma mice model, TRPV4 activation in the membrane promotes an increase in transforming growth factor- beta 1 (TGF-β1) through a signaling pathway involving PI3K and leads to stimulation of the myocardin-related transcription factor A (MRTF-A), which depends on Rho/myocardin. Then, the TRPV4/Rho/MRTF-A pathway activates the expression of fibrosis-related genes that depend on the mitogen-activated protein kinase (MAPK), p38. This, in turn, leads to a higher production of collagen and endothelial fibronectin and to the activation of the inhibitor of the plasminogen activator inhibitor 1 (PAI-1), producing a reduction in the degradation of the matrix [147]. As mentioned in Section 4, both CFTR and TRPV4 play important roles in cystic fibrosis. Specifically, the activation of TRPV4 had been proposed to induce the secretion of pro-inflammatory cytokines/chemokynes from epithelial cells (i.e., PLA2, IL-8, prostaglandin E2, and NF-κB), leading to neutrophil infiltration in response to lipopolysaccharide (LPS) from gram-negative bacteria. Secretion of IL-8 from bronchial epithelial cells in culture, as well as from lungs from intact mice, in response to the activation of TRPV4 was increased when CFTR was inhibited [128]. Finally, in pulmonary injury produced by hydrochloric acid, inflammation of the airways is accompanied by a dramatic increase in pro-inflammatory factors such as keratinocyte-derived chemokine (CXCL1), granulocyte colony-stimulating factor (GCSF), VEGF, IL-1β, IL-6, monocyte chemotactic protein-1, etc. [117,148]. A prominent feature of infection with SARS-CoV-2 it that there is an intense activation of cytokine-driven inflammation cascades [149,150]. Notably, it was recently proposed that TRPV4 may constitute a pharmacological target for patients with COVID-19. It has been proposed that patients in which an inhibitor of TRPV4 is administered (that has already been proven to be safe in a clinical trial) [151], preserve the alveolo-capillary barrier that, in turn, may result in reduced lethality in them (Figure 3). In summary, the TRPV4 channel plays highly important roles in lung physiology with its activation directly or indirectly influencing the endothelial and epithelial barriers, the smooth muscle and innate immune cell activity. Int. J. Mol. Sci. 2020, 21, 3837 14 of 36 t. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 14 o igure 3. TRPV4 and lung damage. Stimuli such as the exposure to chemicals or mechanical stress (induced by liquids or a ventilator), lead to failure in the function of e alveolo-capillary (endothelial and epithelial cells) barrier with a consequent increase in their permeability, allowing for build-up of liquids in the alveoli. In addition this, activation of TRPV4 in the alveolar macrophages results in an increase in intracellular Ca2+ and in the production of superoxide and nitric oxide (NO). Recently, e SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was identified and shown to cause the COVID-19 (coronavirus disease 19) that affects epithelial cells type I and II) of the alveoli, promoting edema. TRPV4 could be involved in the inflammatory response caused by the SARS-CoV-2 virus. Since TRPV4 overactivation or overexpression can lead to damage in the alveolo-capillary barrier, it has been proposed that inhibitors of TRPV4 could result in a better outcome for COVID-19 patients [147], alveolar epithelial type I and II (ATI and ATII), 4α-phorbol 12,13-didecanoate (4αPDD) and 5, 6-epoxyeicosatrienoic acid (5, 6-EET). ROS, reactive oxygen species. reated with Biorender.com. Figure 3. TRPV4 and lung damage. Stimuli such s the exposure to chemicals mechanical stress (induced by liquids or a ventilator), lead to failure in the function of the alveolo-capillary (endothelial and epithelial cells) barrier with a consequent increase in their permeability, allowing for build-up of liquids in the alveoli. In addition to this, activation of TRPV4 in the alveolar macrophages results in an increase in intracellular Ca2+ and in the production of superoxide and nitric oxide (NO). Recently, the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was identified and shown to cause the COVID-19 (coronavirus disease 19) that affects epithelial cells (type I and II) of the alveoli, promoting edema. TRPV4 could be involved in the inflammatory response caused by the SARS-CoV-2 virus. Since TRPV4 overactivation or overexpression can lead to damage in the alveolo-capillary barrier, it has been proposed that inhibitors of TRPV4 could result in a better outcome for COVID-19 patients [147], alveolar epithelial type I and II (ATI and ATII), 4α-phorbol 12,13-didecanoate (4αPDD) and 5, 6-epoxyeicosatrienoic acid (5, 6-EET). ROS, reactive oxygen species. Created with Biorender.com. 5. Role of TRPV4 in Renal Physiology TRPV4 is an important molecule for kidney function since, as will be discussed, it regulates the balance of water in cells. Since TRPV4 was described in the year of 2000, it was shown to be abundantly-expressed in the kidney and to respond to hypotonicity [22] and several studies have focused on studying the role of this ion channel in the physiology of the renal system. This channel is absent in the early parts of the kidney tubule (where passive reabsorption of water occurs) but it is present in the distal convoluted tubule and further throughout the kidney. This means that TRPV4 expression is somewhat confined to tubule segments that do not exhibit apical water permeability where transcellular osmotic gradients can develop [152], except for the macula densa region that exhibits apical water permeability and where it is also expressed [47]. By being present in the basolateral membrane of kidney tubular epithelial cells, TRPV4 could respond to changes in osmolarity in the medullary interstitium, tracking local interstitial water balance [152]. In this sense, it has been demonstrated that when the osmolarity decreases in the renal medulla, ATP is released from epithelial cells in the thick ascending limb. When renal tubules are treated with a chemical agonist of TRPV4, ATP is released in the thick ascending limb under isotonic conditions, but if TRPV4 is knocked down then there is a decrease in ATP release under hypotonic conditions [153]. The conclusion is that ATP is released in the thick ascending limb when there is cell '" .pitwl ..1 c.n \. M..,n.m:.( chemicllOl !kili¡¡ callnJur, AI>·..,..,~_I.lc.1I - _~o l c.>'1 "'.""" ¡,.. .m ~Ibar .... drsfo.not_ M.d!arWcI~ ema b llo\il.C43 ◦C) and capsaicin, the pungent compound present in hot chili peppers that functions as a chemical agonist of the channel, eliciting pain-associated behaviors in animals and pain in humans [2]. Soon after, it was shown that TRPV1 is also activated by low extracellular pH (pH ≤ 5.9). It is important to consider that when several stimuli are present together, TRPV1 activation is potentiated [2]. Several more agonists for this channel have been described and have been reviewed elsewhere [3–11]; however, this review will discuss some of the structural details of TRPV1 and focus only on those agonists of an endogenous nature that have been described for this channel. 2. Structural Overview of the TRPV1 Channel The TRPV1 channel was not only the first mammalian TRP channel to be cloned [1] but it was also the first structure for a TRP channel to be resolved [12,13]. The first clues about TRPV1′s structure were obtained by relying on hydrophobicity analyses, suggesting that TRPV1 is a six-pass transmembrane protein (S1–S6) with a hydrophobic handle between S5 and S6 (pore region). The sequence alignment of the rodent TRPV1 protein showed that the long N-terminus contains multiple ankyrin repeats and a relatively short C-terminal region [1]. These intracellular domains are important scaffolds for interactions with other proteins and also contain Int. J. Mol. Sci. 2020, 21, 3421; doi:10.3390/ijms21103421 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3421 2 of 18 binding sites for compounds that regulate TRPV1 function (i.e., calmodulin or CaM and ATP binding sites located at the N- and C- termini) [14–16]. Remarkably, capsaicin detection is abrogated in birds; although these animals also express TRPV1 channels, activation of these channels is dependent on protons but not on binding of the vanilloid compound [17]. Specific structural differences between avian and mammalian TRPV1 proteins result in dissimilar responses to capsaicin, since this compound binds to specific amino acids near the S3 of the mammalian TRPV1 protein and this vanilloid-binding pocket is different from that of the avian protein [17]. This finding was confirmed in 2015 [18], establishing that the capsaicin pocket is a hydrophobic cavity constituted by residues Y512, S513, T551, and E571 in the human TRPV1 sequence. While the first two residues are conserved between species, T551 is different in rabbit and chicken, the two species known to be insensitive to the pungency of chili peppers. Hence, these fine structural differences between TRPV1 orthologues produce channels with distinct susceptibilities to capsaicin [18]. Unlike the capsaicin pocket, the binding site for protons is conserved between species and it is located extracellularly at the S5 linker (E600 and E648). Interestingly, protons interact with the E600 amino acid residue and potentiate TRPV1 activation by other stimuli such as temperature or capsaicin, while E648 specifically regulates activation by protons [19]. Although the proton binding sites are conserved between species, a recent study showed that chicken TRPV1 activation by protons produces smaller currents than mouse TRPV1 [20]. Moreover, the avian channel is resistant to the typical desensitization produced by repeated application of TRPV1 agonists (i.e., protons) [20]. A structural determinant of TRPV1′s desensitization is the interaction of CaM at sites located at the N- and C-terminal regions [14,15]. Interestingly, it has been concluded that differences in the CaM binding site found in the C-terminus between these TRPV1 orthologues confer resistance to desensitization by protons in the avian channel [20]. Several lines of evidence have revealed binding sites for several TRPV1-activating compounds [2,21–31]. During the last two decades, the TRPV1 channel research field has produced a large body of evidence on the regulation of this channel by multiple compounds. Conversely, the determination of a high-resolution structure for TRPV1 was slower due to the experimental difficulties to crystallize this type of protein. The first contribution of this kind was the crystallization of the isolated ankyrin repeats and determination of their structure by X-ray diffraction methods [16]. This analysis solved the structure of the six ankyrin repeats of TRPV1 with a resolution of 2.7 and 3.2 Å and showed the typical 33-amino acid motifs forming antiparallelα-helices followed by a finger loop [23]. These motifs generate surfaces available for interactions with the ankyrins from other proteins. Additionally, the TRPV1 ankyrin repeats showed an electron density corresponding to an ATP molecule bound to these structures 1–3 [16], which has been shown to positively regulate TRPV1 activation. Subsequently, the first 3D structure of the rat TRPV1 was determined by using electron cryomicroscopy (cryo-EM). The 19 Å, low-resolution structure of the full-length TRPV1 channel demonstrated the fourfold symmetry of this channel (similar to Kv channels), and the existence of two evident regions: the intracellular N- and C-terminal regions that resemble a basket domain and comprise ~70% of the total mass of the channel while the transmembrane region forms a compact and small domain [32]. Functional studies using fluorescence resonance energy transfer (FRET) showed that the N-termini surround the C-termini and the N-termini are farther away from the membrane than the C-termini [33]. Other findings related to the structural details of TRPV1 were mainly established by functional studies. For example, constrictions in the pore of TRPV1 were identified using an experimental strategy of accessibility to thiol-modifying agents [34] and important sites for the regulation of its gating properties were reported [35]. During the last years, our knowledge on the structure of TRPV1 was greatly enhanced by the obtainment of the first high-resolution structure (at a 3.4 Å resolution) through single-particle cryo-electron microscopy (cryo-EM) [12]. This TRPV1 structure showed the classic fourfold symmetry Int. J. Mol. Sci. 2020, 21, 3421 3 of 18 of this channel, and clearly resolved the transmembrane helices S1–S6, the TRP box, and the re-entrant loop between S5 and S6, corresponding to the pore region. Intracellular domains such as the N-terminal ankyrin repeats were also identified and the TRP box at the C-terminus was shown to be oriented towards the S4–S5 linker and the pre-helix-S1, suggesting an essential role for it in the allosteric modulation of TRPV1 channels [12]. The outer pore region resembles a wide funnel structure and the small selectivity filter was observed further down the channel and shown to contain a signature sequence (643-GMGD-646). The cryo-EM TRPV1 structure also showed a deep constriction site, near a previously suggested gate [34]. Several TRPV1 structures were obtained in these experiments. An “apo-state” of the channel was determined in the absence of agonist [34]. In addition to the “apo”-TRPV1 structure, there are two additional structures: one in the presence of agonists resiniferatoxin (RTX) together with the double-knot toxin (DkTx) and another in the presence of capsaicin [13]. RTX is a compound found in the “cactus-like” plant Euphorbia resinifera that functions as an agonist of TRPV1 by binding to the vanilloid pocket of the channel but that is more potent than capsaicin [11,36–38]. On the other hand, DkTx is a peptide toxin found in the venom of the Ornithoctonus huwena spider. This peptide contains two inhibitor-cysteine-knots (ICK) motifs called K1 and K2 lobes which interact with TRPV1 through several external aromatic residues [30]. The toxin stretches out into a space normally filled by lipids in the absence of the toxin constituted by S4, S6, and the pore helix, where it interacts with the lipid membrane [39] and gates the channel through a mechanism different to that of capsaicin [40]. The 3D structure with DkTx and RTX unveiled the full-open TRPV1 channel with a resolution of 3.8 Å. This reconstruction revealed that DkTx binds to the extracellular loops of the tetrameric channel and shows an electronic density in the vanilloid pocket suggesting that RTX is situated at this binding site [1]. Similarly, a weaker electronic density at the vanilloid pocket was observed in the reconstruction of the 3D TRPV1 structure with capsaicin (resolution at 4.2 Å), perhaps reflecting the fact that capsaicin displays less affinity to this site than RTX. Interestingly, these densities are in close proximity, although there is not a complete overlap, which suggests that these agonists bind to the same pocket but they do not interact with the same amino acids [13]. Ligand-bound TRPV1 structures provided information on the existence of different pore profiles in this channel. Firstly, the capsaicin-bound structures displayed clear expansion at the lower gate in comparison to the “apo”-state. Moreover, the full-open channel structure (DkTx/RTX-TRPV1) revealed that the ion pore conduction lacks any structural constriction [13]. The outer pore region is also rearranged in the full-open channel since a substantial change at the position of the helix pore confers inflexibility to this region. Consequently, the helix moves away from the central axis and transduces a conformational change at the loop between S5 and S6. This loop contains two glutamic acids (E600 and E648) which are essential to TRPV1 activation/potentiation by protons. The apo-TRPV1 state shows interaction between E600 and two contiguous amino acids (Y663 and D654), maintaining the channel in an inactivated state. However, the interaction of these amino acids is interrupted in the DkTx/RTX-TRPV1 structure, which results in an increase in the distance between these amino acids and maintains the channel in a fully conducting form [13]. These changes at the pore suggest high flexibility of this region and provide evidence to the hypothesis that each agonist can lead to a different gating mechanism. Finally, three high-resolution TRPV1 structures have been recently obtained combining the cryo-EM with the lipid nanodisc technology. These structures correspond to the apo channel and agonist- and antagonist-bound states [41]. To obtain these better resolution TRPV1 structures, the channels were embedded in a lipid bilayer-like environment provided by the, promoting a structural arrangement closer to the native environment and yielding density maps of higher qualities. In these nanodisc structures, the electronic densities of lipids interacting with the channel were well resolved and a tripartite complex of lipids–TRPV1–toxin was obtained at a 2.9 Å resolution. The elucidation of this structure determined that the two hydrophobic fingers of DkTx are inserted into the bilayer, where the aliphatic tail of a phospholipid interacts with the side chain of the W11 residue of finger 1 of DkTx and the negatively Int. J. Mol. Sci. 2020, 21, 3421 4 of 18 charged head of this lipid electrostatically interacts with residue R534 located at the S3-S4 extracellular loop [41]. In addition, hydrophobic interactions were shown to be established between a phenylalanine located in finger 2 of the toxin and the aliphatic tail of the lipid while the head group is coordinated by extracellular residues located at the pore helix (S629). These data suggest that the toxin induces molecular arrangements in TRPV1 in a phospholipid-dependent manner stabilizing the full-activated state of the channel [41]. Strikingly, the TRPV1 apo-state obtained at 3.2 Å resolution showed a phosphatidylinositol lipid associated to the vanilloid pocket, where the acyl chain is lengthened along S4 of one subunit and towards S5-S6 of the adjacent subunit. This lipid is displaced from the vanilloid pocket in the TRPV1 structure associated to RTX and this binding pocket is also occupied by capsazepine, a TRPV1 antagonist [41]. Since several of the endogenously produced molecules that will be here discussed have been shown or proposed to interact with the vanilloid-binding pocket, we will next describe how vanilloid molecules were shown to interact at this site in the cryo-EM structures. As mentioned above, the structure obtained in the presence of capsaicin alone was considered a “partially open” structure because the presence of this agonist only produced the opening of the lower gate and no changes at the region of the selectivity filter were evident in the presence of capsaicin [13]. The cryo-EM structures also confirmed previous mutagenesis studies that showed that residues Y511 (whose aromatic ring points toward the pocket and attracts the ligands), S512 in the S3, and M547 and T550 in the S4 are all important for vanilloid binding and that capsaicin and/or RTX are coordinated in a pocket above residue E570 in the S4-S5 linker of one subunit [17,42,43]. This E570 residue is proximal to residue L669 in the S6 of the neighboring subunits; hence, this vanilloid binding site could putatively affect gating by impacting on both regions, the S4-S5 linker and the S6. In summary, the idea is that the RTX or capsaicin can interact with residues in the S4-S5 linker and pull them away from the central pore, leading to the opening of the channel [12,13,41,44]. More recently, details on the stoichiometry of TRPV1 activation by capsaicin were provided by Liu and collaborators [45]. These authors used microfluorometry to measure intracellular Ca2+ increases in HEK293 cells that expressed linked tetrameric TRPV1 receptors with different subunit compositions that contained the capsaicin-insensitive S512F mutant and/or wild-type subunits. The authors covalently linked the subunits in order to produce all possible tetrameric compositions containing one single wild-type repeat and observed that the mutant channels could be partially opened by capsaicin and that the binding of two vanilloid molecules (i.e., two wild-type and two mutant subunits) is required in order to fully transduce capsaicin-dependent stimuli [45]. In summary, the last years of the TRPV1 research field have seen growth, enhanced by the pivotal information provided by high-resolution structures and along the way establishing cryo-EM as the best tool for structural analysis of several members of the TRP channel family. As will be detailed below, the last decade has also witnessed the discovery of several endogenously produced agonists (molecules listed in Table 1) of this channel, albeit interaction sites with the protein have been deciphered only for some of these molecules (Figure 1). Int. J. Mol. Sci. 2020, 21, 3421 5 of 18 Table 1. Abbreviations for compounds. Abbreviation Name AA Arachidonic acid AEA Anandamide A-GABA N-arachidonoyl GABA ALA α-Linolenic acid COX-1,-2 Cyclooxygenase-1,-2 CYP450 Cytochrome P450 D-GABA N-docosahexaenoyl GABA DHA Docosahexaenoic acid EETs Epoxyeicosatrienoic acids eLOX-3 Epidermis-type lipoxygenase 3 EPA Eicosapentaenoic acid HETEs Hydroxyeicosatetraenoic acids HXA3 Hepoxilin A3 HXB3 Hepoxilin B3 H2S Hydrogen sulfide LA Linoleic acid L-GABA N-linoleoyl GABA LPA Lysophosphatidic acid NAANs N-acyl amino acids/neurotransmitters NAEs N-acylethanolamines NaHS Sodium hydrosulfide NAPEs N-acylphosphatidylethanolamines OEA or NOE Oleoyl-ethanolamine PEA Palmitoylethanolamide PUFAs Polyunsaturated fatty acids 9-HODE 9-hydroxy-10E,12Z-octadecadienoic acid 9-oxoODE 9-oxo-10E,12Z-octadecadienoic acid 9, 10-DiHOME 9,10-dihydroxy-12Z-octadecenoic acid 9(10)-EpOME 9(10)-epoxy-12Z-octadecenoic acid 12(S)-HPETE 12(S)-hydroperoxyeicosatetraenoic acid 12, 13-DiHOME 12,13-dihydroxy-9Z-octadecenoic acid 12(13)-EpOME 12(13)-epoxy-9Z-octadecenoic acid 12/15 -LOX 12/15-lipoxygenase 13-HODE 13-hydroxy-9Z, 11E-octadecadienoic acid 13-oxoODE 13-oxo-9Z,11E-octadecadienoic acid 20-HEPE 20-hydroxyeicosapentaenoic acid 20-HETE 20-hydroxyeicosatetraenoic acid 22-HDoHE 22-hydroxydocosahexaenoic acid Int. J. Mol. Sci. 2020, 21, 3421 6 of 18Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 20 Figure 1. Structure of TRPV1 depicted with different agonists bound to various sites in the protein. Except for (A) capsaicin (that binds to residues Y512, S513, T551, and E571), all other agonists are endogenously produced: (B) 20-hydroxyeicosatetraenoic acid (20-HETE, which interacts with residue S502); (C) anandamide (which interacts with residues Y511, S512, and R591); (D) oxytocin (which interacts with residues E600, G602, Y631, and L635); and (E) lysophosphatidic acid (LPA, which binds to the K710 residue). The 3j5q PDB file that corresponds to the open structure (obtained with RTX and DkTx) of TRPV1 [41] was used. The black squares represent the regions of the channel where the different depicted endogenous agonists bind. 3. Endogenously Produced Agonists of TRPV1 3.1. Products Derived from Polyunsaturated Fatty Acids Fatty acids are large chains of monocarboxylic acids (8 to 22 carbons), the synthesis of which occurs through the successive addition of acetyl CoA. In mammals, fatty acids are found in their saturated form [46] while polyunsaturated fatty acids (PUFAs) are provided through diet. PUFAs are classified into two families: n-3 or ω-3 (α-linolenic acid or ALA; docosahexaenoic acid or DHA; eicosapentaenoic acid or EPA; also see Table 1 with abbreviations for all compounds mentioned) and n-6 or ω-6 (arachidonic acid, linoleic acid, γ-linolenic acid), according to the position of the first double bond present in their structures [47,48]. Table 1. Abbreviations for compounds. Abbreviation Name AA Arachidonic acid AEA Anandamide A-GABA N-arachidonoyl GABA ALA α-Linolenic acid COX-1,-2 Cyclooxygenase-1,-2 CYP450 Cytochrome P450 D-GABA N-docosahexaenoyl GABA DHA Docosahexaenoic acid EETs Epoxyeicosatrienoic acids eLOX-3 Epidermis-type lipoxygenase 3 EPA Eicosapentaenoic acid Figure 1. Structure of TRPV1 depicted with different agonists bound to various sites in the protein. Except for (A) capsaicin (that binds to residues Y512, S513, T551, and E571), all other agonists are endogenously produced: (B) 20-hydroxyeicosatetraenoic acid (20-HETE, which interacts with residue S502); (C) anandamide (which interacts with residues Y511, S512, and R591); (D) oxytocin (which interacts w th residues E600, G602, Y631, and L635); and (E) lysoph sphatidic acid (LPA, which binds to the K710 residue). The 3j5q PDB file that corresponds to the open structure (obtained with RTX and DkTx) of TRPV1 [41] was used. The black squares represent the regions of the channel where the different depicted endogenous agonists bind. 3. Endogenously Produced Agonists of TRPV1 3.1. Products Derived from Polyunsaturated Fatty Acids Fatty acids are large chains of monocarboxylic acids (8 to 22 ca bons), the synthesis of which occurs through the successive addition of acetyl CoA. In mammals, fatty acids are found in their saturated form [46] while polyunsaturated fatty acids (PUFAs) are provided through diet. PUFAs are classified into two families: n-3 or ω-3 (α-linolenic acid or ALA; docosahexaenoic acid or DHA; eicosapentaenoic acid or EPA; also see Table 1 with abbreviations for all compounds mentioned) and n-6 or ω-6 (arachidonic acid, linoleic acid, γ-linolenic acid), according to the position of the first double bond present i their structures [47,48]. In the body, specifically in the hepatocyte’s endoplasmic reticulum, they are transformed (elongated and desaturated) and form long-chain polyunsaturated fatty acids, starting from reactions orchestrated by malonyl CoA. In this way, linoleic acid (LA 18:2) serves as a precursor to several other molecules such as arachidonic acid (AA 20:4) [47]. Then, this is followed by hydroxylation or epoxidation reactions catalyzed by cytochrome P450 (CYP450) enzymes, resulting in the generation of hydroxyeicosatetraenoic acids (HETEs), such as 20-hydroxyeicosatetraenoic acid (20-HETE), or epoxieicosatrienoic acids (EETs) [49,50] (Figure 2). α-linolenic acid (ALA 18:3) produces eicosapentaenoic acid (EPA 20:5) and docosahexaenoic acid (DHA 22:6) [47], which are precursors of 20-hydroxyeicosapentaenoic acid (20-HEPE) and 22-hydroxyeicosapentaenoic acid (22-HDoHE) [50] (Figure 3). Int. J. Mol. Sci. 2020, 21, 3421 7 of 18 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 20 Figure 2. Products of linoleic acid (LA) that activate TRPV1. LA is the precursor of several long-chain polyunsaturated fatty acids including arachidonic acid, 9-HODE, and 13-HODE. All byproducts downstream of the lipooxygenase and cytochrome P450 (CYP450) pathway shown in this scheme have been proposed to activate TRPV1 [51,52]. α-linolenic acid (ALA 18:3) produces eicosapentaenoic acid (EPA 20:5) and docosahexaenoic acid (DHA 22:6) [47], which are precursors of 20-hydroxyeicosapentaenoic acid (20-HEPE) and 22- hydroxyeicosapentaenoic acid (22-HDoHE) [50] (Figure 3). In 2000, Hwang and collaborators proved that hydroxyeicosapentaenoic acid (12 (S)-HPETE, the (S)-enantiomer of 12-HPETE derived from AA, Figure 2) is capable of activating the TRPV1 channel [53] and this discovery led to further studies which investigated the effects of other PUFA-derived molecules on the activation of TRPV1, as mentioned below. It has also been shown that hepoxylin (HXA3 and HXB3; Figure 2), a product of arachidonic acid and 12(S)-HPETE, has the ability of triggering Ca2+ mobilization in a heterologous expression system (HEK cells) that stably expresses the TRPV1 and Transient Receptor Potential Ankyrin 1 (TRPA1) channels as well as in rat sensory neurons. It is worth mentioning that TRPA1 channels are also noxious stimuli-sensing proteins that are coexpressed with TRPV1 in some sensory neuron subpopulations, hence the importance of evaluating the effects of the above-mentioned agonists on the activity of both channels [54]. Notably, when applied to cells from both TRPV1 and TRPA1 knock- out (KO) animals or when antagonists of these channels (AMG9810 for TRPV1 or HC030031 for TRPA1) are applied, these effects are attenuated. Thus, these results lead to the conclusion that HXA3 promotes tactile allodynia and hyperalgesia mediated by the activation of TRPV1 and TRPA1 channels [55]. Figure 2. Products of linoleic acid (LA) that activat PV1. LA is the precursor of several lo g-chain polyunsaturated f tty cids including ar chido i cid, 9-HODE, and 13-HODE. All byproducts downstream of the lipooxygenase and cytochrome P450 (CYP450) pathway shown in this scheme have been proposed to activate TRPV1 [51,52]. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 20 Figure 3. α-linolenic acid (ALA, n-3) activates TRPV1. ALA is a precursor of EPA and EPA is transformed into 20-HEPE via ω-oxidation and into DHA through elongation reactions. 22-HDoHE is a polyunsaturated fatty acid which is derived from DHA through a ω-hydroxylation reaction catalyzed by the cytochrome P450 enzyme omega-hydroxylase. 20-HETE, which is also derived from AA, is capable of activating and sensitizing TRPV1 in humans and in mice [22]. According to Wen and collaborators, the activation might imply the direct binding of 20-HETE to residue S502 or conversely, considering that the site is fundamental for functional phosphorylation of TRPV1 [56,57], it could promote activation through mechanisms involving protein kinase A (PKA, which is dependent on cyclic adenosine monophosphate or cAMP) and/or protein kinase C (PKC, which is dependent on Ca2+). This, in turn, would lead to the opening of the channel in the absence of the direct binding of 20-HETE. Interestingly, it has also been reported that channel opening and its sensitization occur through actions of 20-HETE on different sites; while mutation of S502 affected only the opening of the channel, it did not affect its sensitization or its activation by capsaicin [22]. Other PUFAs such as 20-hydroxyeicosapentaenoic acid (20-HEPE, derived from EPA) and 22- hydroxydocosahexaenoic acid (22-HDoHE, derived from DHA) have been shown to be more efficient than 20-HETE for TRPV1 activation while expressed in HEK cells but they did not produce pain in a murine model [50]. Hwang et al. hypothesized that, since both molecules belong to the ω-3 family of fatty acids, due to their intrinsic structural features, these could either interact with a different binding site on the channel or bind with higher affinity than 20-HETE [50]. Since the beginning of the decade, 9- and 13-hydroxyoctadecadienoic acids (9-HODE and 13- HODE, derivatives of LA) were proposed as agonists of the TRPV1 channel. The presence of both molecules was detected in depolarized spinal cord neurons in rats [51], in skin biopsies of mice and rats when they were exposed to harmful heat >43 °C [52], and when LA was exogenously applied to DRG neurons [58]. In endogenous and heterologous expression systems, TRPV1 is activated by 9- and 13-HODE, as well as by its oxidized forms 9- and 13-oxoODE, causing mechanical allodynia [34], heat sensitivity in rodents [52], and inflammatory hyperalgesia induced by λ-carrageenan [58], a soluble polysaccharide isolated from sea plants that causes inflammation and pain [59]. The latter effects were not reproducible when neurons from TRPV1 KO mice were used or when the heterologous system was exposed to TRPV1 antagonists (i.e., I-RTX and capsazepine) or to PD146176, a 15-lipoxygenase blocker (enzyme responsible for metabolizing LA to HODEs, mainly 13-HODE; [60]), or when they were immunoneutralized [51,52,58]. Figure 3. α-linolenic acid (AL , n-3) activat PV1. LA is a p ecursor of EPA and EPA is transformed into 20-HEPE via ω-oxidation and into DHA through elongation reactions. 22- DoHE is a polyunsaturated fatty acid which is derived from DHA through a ω-hydroxylation reaction catalyzed by the cytochrome P450 enzyme omega-hydroxylase. In 2000, Hwang and collaborators proved that hydroxyeicosapentaenoic acid (12 (S)-HPETE, the (S)-enantiomer of 12-HPETE derived from AA, Figure 2) is capable of activating the TRPV1 channel [53] and this discovery led to further studies which in estigated the ffect of other PUFA-derived molecules on the activation of TRPV1, as mentioned below. It has also been shown that hepoxylin (HXA3 and HXB3; Figure 2), a product of arachidonic acid and 12(S)-HPETE, has the ability of triggering Ca2+ mobilization in a heterologous expression system (HEK cells) that stably expresses the TRPV1 and Transient Receptor Potential Ankyrin 1 (TRPA1) channels as well as in rat sensory neurons. It is worth mentioning that TRPA1 channels are also noxious Int. J. Mol. Sci. 2020, 21, 3421 8 of 18 stimuli-sensing proteins that are coexpressed with TRPV1 in some sensory neuron subpopulations, hence the importance of evaluating the effects of the above-mentioned agonists on the activity of both channels [54]. Notably, when applied to cells from both TRPV1 and TRPA1 knock-out (KO) animals or when antagonists of these channels (AMG9810 for TRPV1 or HC030031 for TRPA1) are applied, these effects are attenuated. Thus, these results lead to the conclusion that HXA3 promotes tactile allodynia and hyperalgesia mediated by the activation of TRPV1 and TRPA1 channels [55]. 20-HETE, which is also derived from AA, is capable of activating and sensitizing TRPV1 in humans and in mice [22]. According to Wen and collaborators, the activation might imply the direct binding of 20-HETE to residue S502 or conversely, considering that the site is fundamental for functional phosphorylation of TRPV1 [56,57], it could promote activation through mechanisms involving protein kinase A (PKA, which is dependent on cyclic adenosine monophosphate or cAMP) and/or protein kinase C (PKC, which is dependent on Ca2+). This, in turn, would lead to the opening of the channel in the absence of the direct binding of 20-HETE. Interestingly, it has also been reported that channel opening and its sensitization occur through actions of 20-HETE on different sites; while mutation of S502 affected only the opening of the channel, it did not affect its sensitization or its activation by capsaicin [22]. Other PUFAs such as 20-hydroxyeicosapentaenoic acid (20-HEPE, derived from EPA) and 22-hydroxydocosahexaenoic acid (22-HDoHE, derived from DHA) have been shown to be more efficient than 20-HETE for TRPV1 activation while expressed in HEK cells but they did not produce pain in a murine model [50]. Hwang et al. hypothesized that, since both molecules belong to the ω-3 family of fatty acids, due to their intrinsic structural features, these could either interact with a different binding site on the channel or bind with higher affinity than 20-HETE [50]. Since the beginning of the decade, 9- and 13-hydroxyoctadecadienoic acids (9-HODE and 13-HODE, derivatives of LA) were proposed as agonists of the TRPV1 channel. The presence of both molecules was detected in depolarized spinal cord neurons in rats [51], in skin biopsies of mice and rats when they were exposed to harmful heat >43 ◦C [52], and when LA was exogenously applied to DRG neurons [58]. In endogenous and heterologous expression systems, TRPV1 is activated by 9- and 13-HODE, as well as by its oxidized forms 9- and 13-oxoODE, causing mechanical allodynia [51], heat sensitivity in rodents [52], and inflammatory hyperalgesia induced by λ-carrageenan [58], a soluble polysaccharide isolated from sea plants that causes inflammation and pain [59]. The latter effects were not reproducible when neurons from TRPV1 KO mice were used or when the heterologous system was exposed to TRPV1 antagonists (i.e., I-RTX and capsazepine) or to PD146176, a 15-lipoxygenase blocker (enzyme responsible for metabolizing LA to HODEs, mainly 13-HODE; [60]), or when they were immunoneutralized [51,52,58]. Although a detailed mechanism for TRPV1′s direct activation by byproducts of LA has not been established, these reports suggest that this is a possibility. As mentioned by Patwardhan and collaborators in their 2009 study, several metabolites of linoleic acid could be contributing to the activation of the channel and acting in concert (entourage effect), as seen for other lipid families [51]. It has been shown that mutants of TRPV1 at positions 511 and 512, that are characterized by being able to respond to heat and pH but not to capsaicin, did exhibit Ca2+ mobilization in the presence of 9-HODE, suggesting that the binding site for this compound is different from that of capsaicin [52]. In 2016, a lipidomic profile of the rat’s spinal cord after burn injury was determined for the first time. In this study, abnormal levels of HODEs were not observed, although an increase in hydroxy- and epoxy- metabolites of linoleic acid, namely, 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME); 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME); 9(10)-epoxy-12Z-octadecenoic acid (9(10)-EpOME); and 12(13)-epoxy-9Z-octadecenoic acid (12(13)-EpOME), was reported in spinal cord tissue [61]. Then, activation of several TRP channels by 9,10-DiHOME, 12,13-DiHOME, 9(10)-EpOME, and 12(13)-EpOME was assessed using the patch-clamp technique and it was shown that these Int. J. Mol. Sci. 2020, 21, 3421 9 of 18 metabolites activated TRPV1 and TRPA1 but not TRPV2, TRPV3, TRPV4, or Transient Receptor Potential Melastatin 8 (TRPM8, a cold-sensing ion channel that also responds to compounds such as menthol and icilin) [61,62]. Moreover, in vivo experiments showed that four LA metabolites applied to the spinal cord induced allodynia, an effect that was partially blocked with the TRPV1 antagonist, AMG9810, or with HC030031 (a TRPA1 antagonist). Furthermore, allodynia produced by these LA metabolites was completely eliminated by using both antagonists at the same time [61]. 3.2. Endocannabinoids Endocannabinoids are endogenous ligands that activate CB1 and CB2 cannabinoid receptors and are derived from esters, ethers, and amides of PUFAs such as arachidonic acid [63]. Nonetheless, these molecules possess a chemical structure that allows many of them to activate other types of proteins, including TRPV1 [64]. 3.2.1. N-Acyl Amides N-acyl amides are one of the main groups of simple lipids with structures consisting of a fatty acid (acyl group) attached to a simple amine by an amide link where amines can be substituted by amino acids or their byproducts, if they can be conjugated with a fatty acid [65]. N-acyl amides are considered “orphan lipids”, since no specific receptor for these molecules has been discovered; nevertheless, there is evidence of their ability to bind to different endocannabinoid receptors including CB1 and CB2, to G-protein-coupled receptors (GPCRs) 18 and 55 (GPR18 and 55), and to some members of the TRP ion channel family [65]. In 2014, Bradshaw and collaborators reported different N-acyl amides as activators of TRP ion channels (TRPV1-V4). Using an internal library of 70 N-acyl amides and measurements of changes in intracellular Ca2+ levels through fluorescent ratiometric indicators (i.e., Fura-2 AM), these authors identified several agonists of the TRPV1 channel [66] (Figure 4).Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 1 of 20 Figure 4. N-acyl amides with agonist effects on TRPV1. The family of N-acyl GABA molecules that are structurally related is shown in the top panel. The middle and lower panels depict the structures of other N-acyl amides that arise from different families and that do not necessarily share functional groups but also activate TRPV1. Figure 4. N-acyl amides with agonist effects on TRPV1. The family of N-acyl GABA molecules that are structurally related is shown in the top panel. The middle and lower panels depict the structures of other N-acyl amides that arise from different families and that do not necessarily share functional groups but also activate TRPV1. Int. J. Mol. Sci. 2020, 21, 3421 10 of 18 Other molecules such as N-acyl GABA and similar compounds such as N-docosahexaenoyl GABA (D-GABA), N-linoleoyl GABA (L-GABA), and N-arachidonoyl GABA (A-GABA) also resulted in Ca2+ mobilization into the cell in a concentration-dependent fashion. In addition to N-acyl GABA and its derivatives, five other N-acyl amides that were more efficient in activating TRPV1 were identified: N-docosahexaenoyl ethanoyl serine, N-docosahexaenoyl glycine, N-docosahexaenoyl aspartic acid, N-docosahexaenoyl ethanolamide, and N-linoleoyl ethanolamide [66] (Figure 4). In summary, by testing several biologically active lipids, the authors concluded that long-chain unsaturated acyl amides function as TRPV1 activators. Nonetheless, work still needs to be performed to determine the mechanisms by which these molecules modulate the function of TRPV1 and establish binding sites for these in the ion channel. 3.2.2. N-Acylethanolamines N-Acylethanolamines (NAEs) are lipids which, in most cases, originate as products of N-acyl-phosphatidylethanolamine (NAPE) hydrolysis by the specific D-NAPE phospholipase [67]. They are characterized by the presence of a variable length fatty acid chain linked to an ethanolamine through an amine and are classified based on the number of carbons they possess and on the level of saturation of the acyl chain [68]. Anandamide (AEA), an endocannabinoid and an NAE, was the first endogenous agonist described for TRPV1 and it is characterized by also binding to the vanilloid pocket [69,70], as capsaicin does. Moreover, several studies have revealed that AEA’s production is accompanied by the generation of other NAEs, particularly palmitoylethanolamide PEA (C16:0) and oleoylethanolamine OEA (C18:1) [71]. NAEs have been shown to be effectors of TRPV1 activity (Figure 4). For example, N-oleoyl ethanolamine (18:1 NOE or OEA) gives rise to TRPV1 currents in cells previously sensitized with PKC [30,31] and intraperitoneal OEA administration leads to visceral pain behavior, which is not observed in TRPV1-null mice [72], supporting a nociceptive role for OEA through the activation of TRPV1 ion channels [73]. PEA was shown to be present in egg yolk, soybean lecithin, and peanut oil since the 1950s [74] and has since been described to exhibit analgesic and anti-inflammatory properties [75]. Structurally, it is similar to anandamide and other endocannabinoids and shares the same synthesis and degradation routes as these [76]. Despite its classification as an endocannabinoid, PEA is weakly linked to the function of the CB1 and CB2 receptors [69]; however, in the last decade, it has been suggested that it interacts with other receptors such as PPARα (Peroxisome Proliferator-Activated Receptor alpha) and TRPV1 [77,78]. In 2008, a link between TRPV1 and PEA was suggested when it was shown that the application of PEA to the sciatic nerve of rats had an antihyperalgesic effect, which was partially reduced with capsazepine [79]. Other experiments demonstrated that PEA induced dose-dependent vasodilation of endothelial mesenteric arteries (and that it also potentiated the effect of AEA, although PEA is even less potent than OEA at activating TRPV1), a phenomenon which was absent when TRPV1 was inhibited with the antagonist SB36679 [80,81]. In synthesis, these experiments suggested a possible interaction between PEA and TRPV1. A few years later, PEA was proposed to activate TRPV1 in a somatic cell hybrid cell line composed of rat embryonic DRG neurons with a mouse neuroblastoma cell line (F11) since it was shown to increase intracellular Ca2+ concentrations in a dose-dependent fashion that was blunted if TRPV1 antagonists such as capsazepine and SB36679 were applied. The authors suggested that this activation occurred through a mechanism that involved PPARα, since other experiments showed that PEA-induced TRPV1 currents were inhibited by 50% with GW-6471, a PPARα antagonist, an effect that was not observed when TRPV1 was activated by capsaicin [82]. Nonetheless, in contrast with the mechanism proposed above, it was also found that PEA activated transiently transfected TRPV1 in CHO (Chinese hamster ovary) cells, where patch-clamp experiments Int. J. Mol. Sci. 2020, 21, 3421 11 of 18 suggested that TRPV1 was activated and desensitized by PEA in a similar way to what occurs with capsaicin [82]. Finally, the synergistic effects of PEA and tramadol (an opioid analgesic) on pain-like behavior have also been assessed using the formalin test. Experiments have shown that a more efficient effect on analgesia can be achieved for PEA if it is used together with tramadol and that this effect depends upon the activities of the CBRs, TRPV1, and the PPARα [83]. 3.3. N-acyl Amino Acids/Neurotransmitters N-acyl amino acids (NAANs) are lipidic compounds with different headgroup moieties (such as glycine, dopamine, or GABA) that are conjugated to long-chain fatty acids and which can potentially participate in the modulation of the function of membrane proteins such as GPCRs, ion channels, and transporters. More than 70 endogenous NAANs with potential roles in the vasculature and the nervous and immune systems have been identified [84]. Recently, the presence of TRPV1 in the soma of pyramidal neurons of the prelimbic cortex of the prefrontal cortex (PFC) has been reported [85]. This finding is interesting since the activation of TRPV1 produces an increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) in the substantia gelatinosa of the rat spinal cord and in the substantia nigra [86,87]. It has been shown that TRPV1 is activated by N-arachidonoyl taurine (NAT) [88]. Moreover, it has been demonstrated that when capsaicin and NAT activate TRPV1, the compounds do not modify the amplitude of the sEPSC but rather increase their frequency by 150–175% (as compared to the control group) before desensitization occurs, effects that are prevented when capsazepine is applied [85]. Hence, these data suggested a role for TRPV1′s activity in the generation of sEPSC and in modifying the excitatory glutamatergic transmission. 3.4. Oxytocin Oxytocin is a hormone that is produced in the paraventricular and supraoptic nuclei of the hypothalamic nucleus, as well as in the intermediate accessory nuclei [89], and that stimulates the contraction of the uterine smooth muscle and the induction of lactancy, and regulates complex social behavior [90] by binding to its canonical G-protein-coupled oxytocin receptor (OXTR). Recently, by using the PLC inhibitor U773122, it has been shown that oxytocin activates TRPV1 independently of the OXTR and that it induces the entrance of Ca2+ in a heterologous TRPV1 expression system and in F11 cells as well as in artificial bilayers. The direct effect of oxytocin on TRPV1 was confirmed with knock-out mice where the activity of the hormone is null. Computational predictions were performed to obtain specific mutants, which were experimentally tested to show that oxytocin interacts with TRPV1 through L635 and F649, suppressing pain by the activation and then desensitization of the channel [91]. 3.5. Hydrogen Sulfide Hydrogen sulfide (H2S) is an endogenous gaseous molecule that is a subproduct of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), which use L-cysteine as their main substrate. H2S functions as an important cytoprotective and exhibits antioxidant, anti-inflammatory, antiapoptotic, and muscle relaxant properties. Since the middle of the last decade, it was described that H2S affects TRPV1′s activity and this, in turn, has consequences on the function of certain organs leading to contraction of the urinary bladder of rats and of the respiratory tract of guinea pigs [29,92] as well as affecting secretion from the colon of guinea pigs and humans through the local release of substance P after activation of TRPV1 [93]. Sodium hydrosulfide (NaHS), a product of half-neutralization of H2S with sodium hydroxide, induces a biphasic effect on the spontaneous contraction of the duodenal longitudinal muscle strips of rats. Specifically, at 1–2 min after NaHS (1 mM) administration, there was an increase in the contractility of the muscle (excitatory effect) that returned to basal levels within 5 min and, following this excitatory Int. J. Mol. Sci. 2020, 21, 3421 12 of 18 effect, there was a long-lasting (more than 30 min) inhibition of the contractility that depended on the actions of TRPV1 present in afferent nerve fibers, the tachykinin receptor 1 (TACR1), also known as neurokinin 1 (NK1) receptor, and of potassium channels from the smooth muscle [94]. Another study showed that NaHS promotes gastric acid secretion which is attenuated by antagonists of TRPV1 (i.e., capsazepine), of the NK1 receptor (i.e., L703606), and of the nuclear factor NF-κB (i.e., pyrrolidine dithiocarbamate), suggesting that activation of TRPV1 channels from the sensory nerve endings by H2S is partly responsible for this phenomenon [95]. Both of the studies mentioned above proposed that H2S activates TRPV1 channels from sensory nerve endings promoting the release of substance P (an undecapeptide that functions as a neurotransmitter and a modulator of pain perception by altering cellular signaling pathways) and subsequent activation of its NK1 receptor, that is expressed in a cell-specific manner in the digestive system such as in the smooth muscle, interstitial cells of Cajal, the endothelium of blood vessels, white blood cells, fibroblasts, and neurons [96,97]. 3.6. Glycerophospholipids Up to here we have discussed that TRPV1 is an ion channel that is activated by several agonists and that it undergoes characteristic structural changes in the presence of different activators. In this regard, it is worthwhile to note that TRPV1 is also activated by some glycerophospholipids. Our group has previously reported that TRPV1 directly interacts with lysophosphatidic acid (LPA, [31]). LPA, similarly to TRPV1, has been extensively linked to the generation of chronic neuropathic pain. For several years, it was proposed that this phenomenon was only due to the interaction of LPA with its specific GPCRs [98–101], modulating the activity of several ion channels [5,102–104]. Most effects of LPA on other channels are related to how it regulates the expression levels of these proteins [105] and reports of direct interactions of LPA with ion channels in general are limited [104,106,107]. We have previously shown that LPA produces acute pain in mice and that, in HEK293 cells transfected with TRPV1, currents could be induced when LPA was applied to membrane patches expressing the ion channel and identified a site of interaction for this phospholipid, residue K710 in the C-terminus of TRPV1 [31]; this was later confirmed by another group in a study where TRPV1 was expressed in lipid bilayers and activated by LPA [108]. We also showed that activation of TRPV1 by compounds similar in structure to LPA was sensitive to the presence of a single unsaturation, an aliphatic chain of at least 18 carbons and the presence of a negatively charged group (i.e., phosphate); other combinations of these features failed to effectively activate the channel [109]. In a more recent study, we emphasized the biophysical details with which LPA promotes larger macroscopic currents than those promoted by saturating capsaicin concentrations [110]. However, this result could be explained by several mechanisms that included LPA producing an increase in the number of ion channels in the membranes of cells and an increase in the membrane negative surface charge resulting in the accumulation of positively charged ions near the pore mouth of TRPV1, a phenomenon termed “pore dilation” in which the permeability to large ions occurs in the presence of prolonged exposures to agonists [111] and/or a change in the single-channel conductance. By performing several experiments, we ruled out all of the first three possibilities and showed that LPA could produce an increase in the single-channel conductance and that this phenomenon depended upon the presence of the K710 residue in the TRP box of TRPV1 [110]. 4. Conclusions TRPV1 is a polymodal protein with several important functions that include its role in nociception. This ion channel has proven to be an extraordinary example of functional and structural flexibility. Several agonists have been identified for TRPV1 and the presence and activity of this ion channel have been described in several organs. In this review, we have summarized findings related to new endogenous agonists for TRPV1 and the resolution of the fine details of its structure. It is clear that Int. J. Mol. Sci. 2020, 21, 3421 13 of 18 more work is still needed because, for several agonists, fine mechanisms of action have not yet been meticulously clarified. An example of what we can learn from studying how agonists affect TRPV1′s function is that we found that LPA, a molecule that exhibits increased levels during certain inflammation processes and diseases, is widely associated with pain generation, and that binds to the C-terminus of TRPV1, induces a proposed different conformational open state with a distinct single-channel conductance to that obtained in the presence of capsaicin. This larger single-channel conductance results in larger macroscopic currents in comparison to those produced by saturating concentrations of capsaicin and this could be physiologically relevant because it would enable the neurons that express this channel to depolarize more efficiently during certain pathophysiological situations. Author Contributions: M.B.-A. did the bibliographic research and took the lead in writing the manuscript; E.J.-G. helped with writing and producing figures; S.L.M.-L. and T.R. conceived the original idea and supervised the work. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Dirección General de Asuntos del Personal Académico (DGAPA)-Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) IN200720; Consejo Nacional de Ciencia y Tecnología (CONACyT) A1-S-8760 and Secretaría de Educación, Ciencia, Tecnología e Innovación del Gobierno de la Ciudad de México SECTEI/208/2019 to T.R and by DGAPA-PAPIIT IN206819 and Estímulos a Investigaciones Médicas Miguel Alemán Valdés to S.L.M.-L. Conflicts of Interest: The authors declare no conflict of interest. References 1. Caterina, M.J.; Schumacher, M.A.; Tominaga, M.; Rosen, T.A.; Levine, J.D.; Julius, D. The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 1997, 389, 816–824. [CrossRef] [PubMed] 2. Tominaga, M.; Caterina, M.J.; Malmberg, A.B.; Rosen, T.A.; Gilbert, H.; Skinner, K.; Raumann, B.E.; Basbaum, A.I.; Julius, D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998, 21, 531–543. 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