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膜蛋白，可传导 离子沿着电化学梯度下降以转换化学、机械和光学 信号转化为电信号。在这个提案中，我们利用了一系列创新工具来破译 TRP 离子通道两个亚家族 TRPV1-2 和 TRPM2 中的变构门控机制。 TRP 通道以其多模式门控而闻名，其中刺激模式多种多样， 热、冷、离子、脂质、营养素、其他蛋白质和各种天然产物（例如， 辣椒素、薄荷醇）进行变构整合以确定中心离子的活性 导电孔。每种刺激方式调节传感的构象能量学 模块。传感模块依次调节构象能量和电导 的毛孔。感测模块​​可以耦合至孔、彼此耦合或两者耦合。 TRP 通道提供了一个理想的系统来破译变构构象能量学 产生蛋白质功能，因为它们受到许多刺激方式的调节，并且我们有 对其结构域和传感之间的对应关系有深刻的理解 模块。该提案的目标是首次测量构象 TRP 通道传感域的能量及其与孔和彼此之间的耦合 解决 TRP 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。我们最近的 结合荧光寿命成像显微镜 (FLIM) 和贴片技术的技术进步 钳电生理学测量孔中的构象能量和传感 模块同时承诺快速实现这一目标。

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.