A single residue determines regulation of TRPV1 by phosphoinositides

Abstract

The ion channel TRPV1 is expressed in the peripheral nervous system where it mediates heat sensation and pain signaling. How lipids contribute to TRPV1 regulation remains an open topic. Although an inhibitory binding site for phosphoinositides was identified at the vanilloid site in TRPV1 structures, a mechanism for phosphatidylinositol-2-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 a space located adjacent to the turn from S6 to TRP helices, we identify a second, possible PI(4,5)P2 binding site in TRPV1, which we term the "front porch" site and includes residue H410. 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. On the other hand, the charge reversal in H410D conferred an increase in channel activity, which was not expected if the electrostatic interaction with PI(4,5)P2 is lost and channel function is crippled. Importantly, capsaicin activated currents from H410D experience significant current run-up, consistent with the slow displacement by capsaicin of a tighter-bound lipid in the vanilloid site, which structures suggest is phosphoinositide (PI). Lastly, we tested TRPV1-H410D sensitivity to PI(4,5)P2 manipulations by dephosphorylating the membrane pool of PI(4,5)P2 to PI with the Pseudojanin rapamycin-phosphatase (PJ) system. 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. Based on these findings 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. We integrated our discoveries that H410 interacts with PI(4,5)P2 at two distinct sites in a mutually exclusive manner and present a cohesive model for phosphoinositide inhibition and potentiation in TRPV1, addressing a maturation process for TRPV1 that relies on H410 to coordinate the exchange of PI for PI(4,5)P2. Statement of SignificanceA lingering question that hangs over TRPV1 research is how the lipid PI(4,5)P2 regulates the function of this channel. We devised a study based on structural homology between TRPV5 and TRPV1 to identify critical contact points between the lipid and a putative PI(4,5)P2 binding site in TRPV1, which we probed using mutagenesis experiments. The results we obtained lay the groundwork for how a two-site binding mechanism of PI(4,5)P2 couples to TRPV1 gating and address important questions about the channels maturation and association with phosphoinositides as it is transferred through membrane compartments with different phosphoinositide compositions. We conclude that TRPV1 expressed and purified from different compartments may preserve the phosphoinositide character of that compartment (PI in the endoplasmic reticulum, PI(4,5)P2 in the plasma membrane) and bestow on structures a state that is not necessarily relevant to the biological function of the channel in the plasma membrane.