A single residue contributes to phosphoinositide regulation of TRPV1 across two binding sites

The ion channel TRPV1 (transient receptor potential vanilloid subtype I) is expressed in the peripheral nervous system where it mediates heat sensation and pain signaling. Although an inhibitory binding site for phosphoinositides was identified at the vanilloid site in TRPV1 structures, a mechanism for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to both inhibit and potentiate TRPV1 activity is lacking. This gap in knowledge led us to examine structural and sequence overlap between TRPV1 and the PI(4,5)P₂ binding site of TRPV5. In an adjacent space to the S6 helix, we identify a second PI(4,5)P₂ binding site in TRPV1, which we term the "front porch." On the one hand, we obtained electrophysiological evidence with the TRPV1-H410R mutant that PI(4,5)P₂ associates with TRPV1 at the front porch site through an electrostatic interaction to potentiate channel activity. However, the charge reversal in TRPV1-H410D conferred increased capsaicin sensitivity, which was not expected if loss of electrostatic interaction with PI(4,5)P₂ negatively impacts channel function. Importantly, capsaicin-activated currents from TRPV1-H410D experience significant current run-up, consistent with the slow displacement of a lipid in the vanilloid site by capsaicin. Lastly, we tested TRPV1-H410D sensitivity to PI(4,5)P₂ with the pseudojanin rapamycin-phosphatase (PJ) system, which converts the membrane pool of PI(4,5)P₂ to phosphatidylinositol (PI). Depletion of PI(4,5)P₂ with the PJ system in wild-type channels induces (1) a robust increase in baseline activity as channels are released from inhibition and (2) loss of current potentiation, driven by PI(4,5)P₂ dissociation from the front porch, both of which were absent in TRPV1-H410D. We hypothesize that plasma membrane TRPV1-H410D maintains PI rather than PI(4,5)P₂ in its vanilloid site and does not interact with PI(4,5)P₂ in the front porch site. Integrating our discoveries that H410 maintains contacts with PI(4,5)P₂ at two distinct sites in a mutually exclusive manner, we present a cohesive model for phosphoinositide inhibition and potentiation of TRPV1.