Abstract
Regulation of the heat- and capsaicin-activated transient receptor potential vanilloid 1 (TRPV1) channel by phosphoinositides is complex and controversial. In the most recent TRPV1 cryo-EM structure, endogenous phosphatidylinositol (PtdIns) 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)P2] being a well-established positive regulator of TRPV1. Here we show that in the presence of PtdIns(4,5)P2 in excised patches, PtdIns, but not PtdIns(4)P, partially inhibited TRPV1 activity at low, but not at high capsaicin concentrations. This is consistent with PtdIns acting as a competitive vanilloid antagonist. However, in the absence of PtdIns(4,5)P2, PtdIns partially stimulated TRPV1 activity. We computationally identified residues, which are in contact with PtdIns, but not with capsaicin in the vanilloid binding site. The I703A mutant of TRPV1 showed increased sensitivity to capsaicin, as expected when removing the effect of an endogenous competitive antagonist. I703A was not inhibited by PtdIns in the presence of PtdIns(4,5)P2, but it was still activated by PtdIns in the absence of PtdIns(4,5)P2 indicating that inhibition, but not activation by PtdIns proceeds via the vanilloid binding site. In molecular dynamics simulations, PtdIns was more stable than PtdIns(4,5)P2 in this inhibitory site, whereas PtdIns(4,5)P2 was more stable than PtdIns in a previously identified, nonoverlapping, putative activating binding site. Our data indicate that phosphoinositides regulate channel activity via functionally distinct binding sites, which may explain some of the complexities of the effects of these lipids on TRPV1.
Highlights
Underlie the increased sensitivity to heat in inflammation
We found that transient receptor potential vanilloid 1 (TRPV1) activity shows a decrease after patch excision in the presence of 0.5 μM capsaicin in the patch pipette (Fig. 1A), which is consistent with our earlier findings (15)
We found that the D509A mutant had reduced rectification compared with WT TRPV1 and the I703 mutant when activated by capsaicin (Fig. S5E) or by low pH (Fig. S7E), especially at low stimulation strengths
Summary
Underlie the increased sensitivity to heat in inflammation. genetic deletion of this channel in mice essentially eliminated thermal hyperalgesia (2). Phosphoinositides, especially phosphatidylinositol 4,5bisphosphate [PtdIns(4,5)P2], are general regulators of many ion channels (3), including members of the transient receptor potential (TRP) family (4). Negative regulatory effects of this lipid have been proposed for some TRP channels, for example TRPV3 (5). Phosphoinositides modulate TRPV1 activity in a complex manner; both positive and negative effects of these lipids have been proposed (10). It was first suggested that PtdIns(4,5)P2 negatively regulates TRPV1 and that relief from this inhibition plays a major role in sensitization of the channel upon PLC activation by proinflammatory receptor stimulation (11). When the purified channel was incorporated into planar lipid bilayers, the capsaicin- and heat-induced activity of TRPV1 depended on the presence of PtdIns(4,5)P2, or PtdIns(4)P, demonstrating a direct effect on the channel (15, 17)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
