The Proximal C-Terminal Region of TRPV1 Controls Phosphoinositide Selectivity

Abstract

The lipid messenger PIP2 is a critical modulator of multiple membrane proteins, including TRPV1 ion channels. We have previously reported that PIP2 is an important cofactor for activation of TRPV1. A critical question to elucidate the molecular mechanism of activation is where in the channel is the binding site for PIP2. Here we report that a single amino-acid mutation, located in a region of the C-terminus proximal to the transmembrane domains of TRPV1, inverts selectivity of these channels for phosphoinositides by making PI(4)P a stronger activator than PIP2. An in vitro FRET-based binding assay shows this proximal site is capable of binding PIP2. In addition, the distal C-terminal region, previously proposed as a candidate site for PIP2 binding, is not required for PIP2 regulation. We also addressed a recent report suggesting that an integral membrane protein called Pirt acts as the PIP2 sensor for regulation of TRPV1. Pirt expression did not appear to alter TRPV1 apparent affinity for PIP2. In summary, these results implicate the C-terminus proximal site as a PIP2 interacting domain. More importantly, PIP2 binding to this proximal site is central to TRPV1 activation.