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

摘要 离子通道是具有门控跨膜孔的完整的膜蛋白，它通过 离子沿其电化学梯度向下传递化学、机械和光学 将信号转换成电信号。在这项提案中，我们利用一系列创新工具来破译 Trp离子通道的两个亚家族，TRPV1-2和TRPM2的变构门控机制。 TRP频道以其多模式选通而闻名，其中刺激模式多种多样 热、冷、离子、脂类、营养素、其他蛋白质和各种天然产物(例如， 辣椒素、薄荷醇)变构整合，以确定中心离子的活性- 传导毛孔。每种刺激形式都调节着一种感觉的构象能量学 模块。传感模块反过来调节构象能量和电导 毛孔的。传感模块可以耦合到孔、彼此耦合或两者都耦合。Trp 通道提供了一个解释变构构象能量学的理想系统 产生蛋白质功能，因为它们受许多刺激形式的调节，而我们有 对它们的结构域和感知之间的对应关系有重要的理解 模块。这项提议的目标是首次测量构象 色氨酸通道敏感结构域的能量学及其与孔道和相互间的耦合 解决色氨酸通道生物学中的紧迫问题。我们的长期愿景是了解 离子通道中变构构象转变的一般主题。我们最近 结合荧光寿命成像显微镜(FLIM)和贴片的技术进步- 用于测量孔内构象能量的钳子电生理学和传感器 同时，模块承诺朝着这一目标快速发展。

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.