Mechanisms of permeation and gating of voltage-sensing domains

电压传感域的渗透和门控机制

• 批准号: 8854101
• 负责人: Francesco Tombola
• 金额: $ 28.19万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8854101
• 关键词: Acids; Action Potentials; Address; Affinity; Arginine; Arrhythmia; Autoimmune Diseases; Autoimmune Process; Binding; Biological Process; C-terminal; Calcium; Calcium Channel; Cardiovascular Diseases; Cardiovascular system; Cell membrane; Cells; Chronic; Coiled-Coil Domain; Coupling; Development; Dimerization; Disease; Enzymes; Epilepsy; Fertility; Fluorescence; Generations; Genetic; Goals; Homeostasis; Immune response; Individual; Infection; Inflammation; Inflammatory; Integral Membrane Protein; Ion Channel; Ions; Laboratories; Lead; Light; Malignant Neoplasms; Mediating; Membrane; Membrane Potentials; Molecular; Molecular Probes; Muscle; Muscle Contraction; Mutagenesis; Mutation; NADPH Oxidase; Neurons; Paralysed; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Point Mutation; Potassium; Potassium Channel; Probability; Process; Production; Property; Protein C; Proteins; Protons; Reactive Oxygen Species; Regulation; Research; Role; Scanning; Signal Transduction; Sodium; Sodium Channel; Stroke; Structure; Techniques; Tryptophan; Work; Yeasts; base; channel blockers; guanidinium; inhibitor/antagonist; insight; male; member; molecular dynamics; neurotransmission; novel; protein function; screening; simulation; small molecule; sperm cell; tool; voltage; voltage gated channel; yeast two hybrid system

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.

描述（由申请人提供）：电压传感域（VSD）是跨膜蛋白模块，可检测细胞膜内传播的电信号。包含这些结构域的离子通道和酶在许多生物过程中发挥着关键作用，从神经元和肌肉中动作电位的产生，到感染和炎症期间活性氧 (ROS) 的调节。含有 VSD 的蛋白质的功能障碍或错误表达与许多疾病有关，例如癫痫、周期性麻痹、心律失常、癌症和自身免疫性疾病。一些 VSD 在生理条件下跨膜传导离子。由于突变，另一些在病理条件下变得离子渗透。本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一般机制的关系。该研究重点关注电压门控质子通道 Hv1，这是一种缺乏电压门控钠、钾和钙通道典型孔结构域的蛋白质，可通过其 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、门控和通道调制的机制仍然存在许多悬而未决的问题。在这项研究中，我们计划通过使用一种将电生理学和荧光技术与诱变扫描和分子动力学模拟相结合的方法来回答其中一些问题。具体来说，我们的目标是：1）通过使用我们实验室最近开发的扰动分析新技术来确定VSD的哪些部分构成质子孔和门，2）探索质子通过VSD的渗透机制与质子门之间的关系。电压传感机制，使用新的 Hv1 阻断剂作为分子探针，3) 研究辅助蛋白的亚基耦合和门控调节机制。拟议的研究将显着扩展我们对 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 过量产生问题，并将为 VSD 突变如何导致疾病提供新的见解。