The ion channel TRPV1 is expressed in the peripheral nervous system where it mediates heat sensation and pain signaling. Intense research has achieved a broad appreciation of how TRPV1 functions, but how lipids contribute to its regulation remains an open topic after two decades. Although a primary binding site for phosphoinositides was identified in TRPV1 structures, a mechanism for PI(4,5)P2 to both inhibit and facilitate TRPV1 activity is lacking. This gap in knowledge led us to examine structural and sequence overlap between the PI(4,5)P2 binding site of TRPV5 and TRPV1 to identify a second site, which includes H410. On the one hand, we obtained electrophysiological evidence with H410 mutants that PI(4,5)P2 associates with TRPV1 through H410 to increase channel function. On the other hand, the charge reversal in H410D conferred a gain of function, which was unexpected if disruption of PI(4,5)P2 interactions cripple function. Importantly, H410D channels experience significant capsaicin dependent current run-up, which is consistent with a tightly associated phosphoinositide lipid that partially occupies the vanilloid binding site. Lastly, we tested TRPV1-H410D sensitivity to PI(4,5)P2 manipulations by depleting the lipid with a rapamycin-phosphatase system. Depletion of PI(4,5)P2 with rapamycin had no effect, with currents in TRPV1-H410D expressing cells showing no sign of disinhibition at low activity or current reduction under saturating capsaicin conditions. Based on these findings, we hypothesize that plasma membrane TRPV1-H410D has disproportionate PI rather than PI(4,5)P2 in its primary phosphoinositide site. Our conclusion addresses maturation of TRPV1 in the cell with a model that relies on H410 to coordinate the exchange of PI in the primary site via a second, activating, “front porch” site that interacts favorably with PI(4,5)P2.
Senning et al. (Sun,) studied this question.