Phosphoinositides regulate the TRPV1 channel via two functionally distinct binding sites

Regulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channel by phosphoinositides is controversial. In a recent cryoEM structure, an endogenous phosphoinositide was detected in the vanilloid binding site, and phosphoinositides were proposed to act as competitive vanilloid antagonists. This model is difficult to reconcile with phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P 2 ] being a well established positive regulator of TRPV1. To resolve this controversy, we propose that phosphoinositides regulate TRPV1 via two functionally distinct binding sites. Our molecular dynamics simulations show that phosphatidylinositol (PtdIns) is more stable in the vanilloid binding site, whereas a distinct site responsible for activation is preferentially occupied by PtdIns(4,5)P 2 . Consistently, we show that in the presence of PtdIns(4,5)P 2 in excised patches PtdIns partially inhibited TRPV1 activity induced by low, but not high capsaicin concentrations. In the absence of PtdIns(4,5)P 2 on the other hand, PtdIns partially stimulated TRPV1 activity presumably by binding to the activating site. Overall, our data resolve a major controversy in the regulation of TRPV1.