Conformational Energetics and Heterogeneity to Reveal Gating Mechanisms of TRPV and TRPM Ion Channels

ABSTRACT Ion channels are integral membrane proteins with gated transmembrane pores that conduct ions down their electrochemical gradients to transduce chemical, mechanical, and optical signals into electrical signals. In this proposal, we leverage a host of innovative tools to decipher allosteric gating mechanisms in two subfamilies of TRP ion channels, TRPV1-2 and TRPM2. TRP channels are famous for their multimodal gating whereby stimulus modalities as diverse as heat, cold, ions, lipids, nutrients, other proteins, and a variety of natural products (e.g., capsaicin, menthol) are allosterically integrated to determine the activity of a central ion- conducting pore. Each stimulus modality regulates the conformational energetics of a sensing module. The sensing modules in turn regulate the conformational energetics and conductance of the pore. The sensing modules may be coupled to the pore, to each other, or both. TRP channels provide an ideal system in which to decipher how allosteric conformational energetics produce protein function because they are regulated by many stimulus modalities, and we have significant understanding of the correspondence between their structural domains and sensing modules. The goal of this proposal is to measure, for the first time, the conformational energetics of TRP channel sensing domains and their coupling to the pore and to each other to solve pressing questions in TRP channel biology. Our long-term vision is to understand the general themes that underlie allosteric conformational transitions in ion channels. Our recent technical advances combining fluorescence lifetime imaging microscopy (FLIM) and patch- clamp electrophysiology to measure conformational energetics in the pore and a sensing module simultaneously promise rapid progress toward this goal.