Abstract
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.
Highlights
Intracellular lipid metabolites are important modulators of TRPV4 gating: Phosphatidylinositol 4,5-bisphosphate (PIP2) binding to a stretch of positive charges within the N-tail of each cannel subunit is required for TRPV4 activation by hypotonicity and heat[11] while EETs derived from AA promote TRPV4 opening[13]
To gain structural insights into the potential direct interaction of 5′,6′-epoxyeicosatrienoic acid (5′,6′-EET) with the TRPV4 channel we combined in silico molecular docking with molecular dynamics (MD) simulations
Based on the most frequent interactions, i.e., residues showing more than 60% of time occupancy, MD simulation identified a 5′,6′-EET binding site in the TRPV4-WT system conformed by residues from S2-S3 linker (K535, F549 and Q550), S4 (Y591) and S4-S5 linker (R594) (Fig. 1a–c, f and Supplementary Fig. 3) that were predicted by the in silico docking
Summary
Intracellular lipid metabolites are important modulators of TRPV4 gating: Phosphatidylinositol 4,5-bisphosphate (PIP2) binding to a stretch of positive charges within the N-tail of each cannel subunit is required for TRPV4 activation by hypotonicity and heat[11] while EETs derived from AA promote TRPV4 opening[13]. EETs appear to act as messengers that mediate TRPV4 activation in response to either hypoosmotic shock[33] or mechanical stimulation[8, 34]. In this regard, PLA2 is activated by hypotonic and mechanical stimulation[35, 36] but no direct measurements of EETs have been reported in response to these stimuli. Autocrinally released EETs promote TRPV4-mediated Ca2+ entry in endothelial cells[21] that stimulates the activity of small and intermediate conductance Ca2+-gated K+ (SKCa and IKCa) channels, causing endothelial-dependent vascular relaxation[38,39,40]. We combine molecular simulations along with binding assays and functional studies to provide strong evidences supporting that EET-induced TRPV4 gating is due to direct EET binding to a crevice formed by helical segments S1 through S4 of each TRPV4 subunit, with a critical role of the K535 residue located at the S2-S3 linker in the stabilization of the ligand position
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