We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25 °C activated currents and increased [Ca²⁺]_i in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4αPDD (α-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance. Deletion of the three N-terminal ankyrin binding domains of TRPV4 abolished current activation cells by heat in HEK293. In inside-out patches, TRPV4 could not be activated by heat but still responded to the ligand 4αPDD. In MAEC, the same channel is activated under identical conditions as in the HEK expression system. Our data indicate that TRPV4 is a functional temperature-sensing channel in native endothelium, that is likely involved in temperature-dependent Ca²⁺ signaling. The failure to activate TRPV4 channels by heat in inside-out patches, which responded to 4αPDD, may indicate that heat activation depends on the presence of an endogenous ligand, which is missing in inside-out patches.

TRPV4 is a Ca ²⁺ - and Mg ²⁺ -permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca ²⁺ -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 PLA ₂ -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, PLA ₂ - and cytochrome P450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4α-Phorbol 12,13-didecanoate (4α-PDD) induced an increase in intracellular Ca²⁺ concentration, [Ca²⁺]_i, in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca²⁺]_i, 4α-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca²⁺-permeable withP _Ca/P _Na = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca²⁺]_i but was ∼50 times less effective than 4α-PDD. EC₅₀ for Ca²⁺ increase and current activation was nearly identical (pEC₅₀ ∼ 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4α-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca²⁺]_i; an increase in [Ca²⁺]_i inhibits the channel with an IC₅₀ of 406 nm. Ruthenium Red at a concentration of 1 μm completely blocks inward currents at −80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.