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: 8162229
• 负责人: Francesco Tombola
• 金额: $ 28.53万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8162229
• 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; Screening procedure; Signal Transduction; Sodium; Sodium Channel; Stroke; Structure; Techniques; Tryptophan; Work; Yeasts; base; channel blockers; guanidinium; inhibitor/antagonist; insight; male; member; molecular dynamics; novel; protein function; 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. PUBLIC HEALTH RELEVANCE: Biological processes as diverse as neuronal signaling, muscle contraction, the immune response, and the heartbeat depend on the proper function of proteins containing voltage-sensing domains (VSDs). Some of these proteins conduct ions through their VSD under physiological or pathological conditions, and this project aims at determining the mechanisms underlying such ion conduction. The results of this study will help develop new drugs that reduce production of reactive oxygen species involved in chronic inflammation and cardiovascular diseases, and will provide new insights on how mutations of VSD-containing proteins lead to disease.

描述（由申请人提供）：电压感应域（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学过程中起关键作用，从神经元和肌肉的作用电位到感染和炎症期间活性氧（ROS）的调节。含有VSD的蛋白质的故障或明显与许多疾病有关，例如癫痫，周期性瘫痪，心律不齐，癌症和自身免疫性疾病。一些VSD在生理条件下在整个膜上进行离子。由于突变，其他人在病理条件下成为离子笼。这项研究的长期目标是阐明通过VSD及其与电压传感的一般机制的关系及其与其关系的基本机制。该研究的重点是电压门控质子通道HV1，这种蛋白质缺乏电压门控钠，钾和钙通道的典型孔结构域，并通过其VSD进行质子。最近的工作已经开始揭示HV1的结构组织，但是关于质子渗透，门控和调节机制的许多开放性问题仍然存在。在这项研究中，我们计划通过使用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。具体而言，我们的目的是：1）通过使用最近在我们的实验室中开发的新型扰动分析技术来确定VSD的哪些部分构成质子孔和门，2）使用VSD的机制与电压传感机理，使用新的HV1阻断器作为分子探针和3）的机制来探索质子渗透机制与分子探针的机制，并将其与分子进行改造。蛋白质。拟议的研究将大大扩展我们对VSD如何感知膜电位，导致离子并通过附加蛋白与细胞内过程相互作用的理解。这项工作还将为HV1抑制剂的发展铺平道路，这些抑制剂可用于解决几种心血管和炎症性疾病的典型ROS生产过量，并将为VSD突变如何导致疾病提供新的见解。 公共卫生相关性：像神经元信号传导，肌肉收缩，免疫反应和心跳一样多样化的生物过程取决于含有电压感应域（VSD）的蛋白质的正常功能。这些蛋白质中的一些通过其生理或病理条件下通过其VSD进行离子，该项目旨在确定这种离子传导的机制。这项研究的结果将有助于开发新药，以减少参与慢性炎症和心血管疾病的活性氧的产生，并就含有VSD的蛋白质突变导致疾病的新见解。