Cell Swelling, Heat, and Chemical Agonists Use Distinct Pathways for the Activation of the Cation Channel TRPV4

TRPV4 is a Ca2+- and Mg2+-permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca2+-dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5′,6′-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase $A_2\>(PLA_2)$ and cytochrome P450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4α-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4α-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA2-dependent formation of AA, and its subsequent metabolization to 5′, 6′-epoxyeicosatrienoic acid by means of a cytochrome P450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA2- and cytochrome P450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.