TRPV4 cation channel activation by cytochrome P450-mediated derivatives of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), constitute a major mechanisms of endothelium-derived vasodilatation. Besides, TRPV4 mechano/osmosensitivity depends on phospholipase A2 (PLA2) activation and subsequent production of AA and EETs. However, the lack of evidence for a direct interaction of EETs with TRPV4 together with claims of EET-independent mechanical activation of TRPV4 has cast doubts on the validity of this mechanism. We now report: 1) The identification of an EET-binding pocket that specifically mediates TRPV4 activation by 5′,6′-EET, AA and hypotonic cell swelling, thereby suggesting that all these stimuli shared a common structural target within the TRPV4 channel; and 2) A structural insight into the gating of TRPV4 by a natural agonist (5′,6′-EET) in which K535 plays a crucial role, as mutant TRPV4-K535A losses binding of and gating by EET, without affecting GSK1016790A, 4α-phorbol 12,13-didecanoate and heat mediated channel activation. Together, our data demonstrates that the mechano- and osmotransducing messenger EET gates TRPV4 by a direct action on a site formed by residues from the S2-S3 linker, S4 and S4-S5 linker.

The research is supported by awards from the Spanish Ministry of Economy and Competitiveness (Grants SAF2015-69762-R to M.A.V. and J.M.F.-F., and MDM-2014-0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), and FEDER Funds (Fondo Europeo de Desarrollo Regional). M.I.-S. holds a “Juan de la Cierva-Formación” Fellowship funded by the Spanish Ministry of Economy and Competitiveness. FGN acknowledge the support of FONDECYT Grant 1170733 and The Centro Interdisciplinario de Neurociencia de Valparaíso (CINV) is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economía, Fomento y Turismo. R.V.S. is funded by CONICYT PCHA/Doctorado Nacional 2013-21130631 fellowship