Mechanisms of permeation and gating of voltage-sensing domains

描述(申请人提供)：电压敏感结构域(VSD)是跨膜蛋白模块，检测在细胞膜内传播的电信号。含有这些结构域的离子通道和酶在许多生物过程中起着关键作用，从神经元和肌肉中动作电位的产生，到感染和炎症过程中活性氧物种(ROS)的调节。含有VSD的蛋白的功能障碍或错误表达与许多疾病有关，如癫痫、周期性瘫痪、心律失常、癌症和自身免疫性疾病。一些VSD在生理条件下将离子传导到膜上。另一些则是在病理条件下，由于突变而变成离子。这项研究的长期目标是阐明通过VSD进行离子传导的机制及其与电压传感的一般机制的关系。这项研究的重点是电压门控质子通道Hv1，这是一种蛋白质，缺乏电压门控钠、钾和钙通道的典型孔域，并通过其VSD传导质子。最近的工作已经开始揭示Hv1的结构组织，但关于质子渗透、门控和通道调节的机制仍有许多悬而未决的问题。在这项研究中，我们计划使用一种结合电生理学和荧光技术的方法来回答其中一些问题，以进行诱变扫描和分子动力学模拟。具体地说，我们的目标是：1)利用我们实验室最近发展起来的一种新的微扰分析技术来确定VSD的哪些部分构成了质子孔和门；2)利用新的Hv1阻滞剂作为分子探针，探索质子通过VSD的渗透机制与电压敏感机制之间的关系；3)研究辅助蛋白对亚基偶联和门控调节的机制。这项拟议的研究将极大地扩展我们对VSD如何感知膜电位、传导离子以及通过辅助蛋白与细胞内过程相互作用的理解。这项工作还将为Hv1抑制剂的开发铺平道路，该抑制剂可用于解决几种心血管和炎症性疾病中典型的ROS过度生产问题，并将为VSD突变如何导致疾病提供新的见解。

DESCRIPTION (provided by applicant): Voltage-sensing domains (VSDs) are transmembrane protein modules that detect electrical signals propagating within cell membranes. Ion channels and enzymes containing these domains play key roles in many biological processes, from the generation of the action potential in neurons and muscles, to the regulation of reactive oxygen species (ROS) during infection and inflammation. Malfunction or misexpression of VSD-containing proteins is associated with numerous diseases, such as epilepsy, periodic paralysis, cardiac arrhythmia, cancer and autoimmune disorders. Some VSDs conduct ions across the membrane under physiological conditions. Others become ion permeant under pathological conditions, as a result of mutations. The long-term goal of this study is to elucidate the mechanism underlying ion conduction through the VSD and its relationship to the general mechanism of voltage sensing. The study focuses on the voltage-gated proton channel Hv1, a protein that lacks the pore domain typical of voltage-gated sodium, potassium, and calcium channels and conducts protons through its VSD. Recent work has begun to unveil the structural organization of Hv1, but many open questions remain about the mechanisms of proton permeation, gating, and modulation of the channel. In this study we plan to answer some of these questions by using an approach that combines electrophysiological and fluorescence techniques to mutagenesis scanning and molecular dynamics simulations. Specifically, we aim at: 1) determining which parts of the VSD make up the proton pore and gate by using a novel technique of perturbation analysis recently developed in our laboratory, 2) exploring the relationship between the mechanism of proton permeation through the VSD and the mechanism of voltage sensing, using new Hv1 blockers as molecular probes, and 3) investigating the mechanisms of subunit coupling and gating modulation by accessory proteins. The proposed research will significantly expand our understanding of how VSDs sense the membrane potential, conduct ions, and interact with intracellular processes via accessory proteins. The work will also pave the way to the development of Hv1 inhibitors that can be used to address ROS overproduction typical of several cardiovascular and inflammatory disorders and will provide new insights on how mutations of VSDs lead to disease.