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: 9240299
• 负责人: Francesco Tombola
• 金额: $ 32.22万
• 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 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9240299
• 关键词: Address; Affinity; Arginine; Arrhythmia; Autoimmunity; Binding; Binding Sites; Brain Injuries; Cell membrane; Cell physiology; Cells; Charge; Chemosensitization; Chimera organism; Closure by clamp; Collaborations; Complex; Conflict (Psychology); Data; Development; Dilated Cardiomyopathy; Disease; Drug Targeting; Electrophysiology (science); Environment; Event; Gated Ion Channel; Goals; Hereditary Disease; Human Activities; Hydrophobicity; Hyperactive behavior; Hypokalemic periodic paralysis; Immune system; Ion Channel Gating; Ions; Iowa; Ischemic Stroke; Lead; Light; Malignant Neoplasms; Measurement; Mechanical Stress; Mechanics; Mediating; Membrane; Membrane Lipids; Membrane Potentials; Membrane Proteins; Methods; Microglia; Modality; Modeling; Molecular; Molecular Conformation; Motor; Movement; Muscle Contraction; Mutagenesis; Mutate; Mutation; NADPH Oxidase; Neuroprotective Agents; Pathologic; Pathologic Processes; Pathway interactions; Patients; Permeability; Pharmaceutical Preparations; Pharmacology; Physiological; Physiological Processes; Play; Process; Property; Proteins; Protons; Quantum Mechanics; Regulation; Research Project Grants; Role; Scanning; Side; Site; Speed; Stimulus; Stretching; Stroke; Structural Models; Swelling; Testing; Therapeutic; Universities; Vestibule; Work; anti-cancer therapeutic; base; cancer cell; cell motility; cell type; chemical bond; design; dynein light chain; inhibitor/antagonist; mechanical force; molecular mechanics; neuronal excitability; pH Homeostasis; pressure; respiratory; response; simulation; small molecule; small molecule inhibitor; sperm cell; unnatural amino acids; voltage; voltage gated channel

Project Summary Voltage-gated ion channels are a diverse group of membrane proteins that play significant roles in a variety of physiological and pathological processes, from neuronal excitability and muscle contraction, to autoimmunity, stroke, and cancer. They all share a common structural module, the voltage-sensing domain (VSD), responsible for turning on and off an effector domain in response to changes in membrane potential. Previous studies from us and other groups have shown that, while most VSDs do not conduct ions, they can become leaky as a result of mutations. Mutated VSDs permeable to ions or protons are responsible for serious genetic disorders, such as hypokalemic periodic paralysis, and cardiac arrhythmias with dilated cardiomyopathy. The VSD of the voltage-gated channel Hv1, on the other hand, is inherently proton-conductive and this property is key to the channel's many physiological functions. The long-term goal of this study is to elucidate how VSDs conduct ions and protons, how their activity is regulated, and how they can be blocked pharmacologically for therapeutic purposes. Here, we will focus on the Hv1 channel, an emerging drug target for a variety of diseases, including cancer and stroke. The mechanism underlying VSD-mediated proton conduction in Hv1 is poorly understood and there is an unmet need for small-molecule inhibitors of Hv1 activity. We have previously discovered a class of compounds that act as Hv1 blockers and characterized their binding environment. We identified aromatic interactions within the core of the channel's VSD that could be harnessed to create better drugs to suppress Hv1 activity. In aim 1, we propose to use electrophysiological measurements and unnatural amino acid substitutions to examine how these interactions contribute to Hv1 block and voltage-dependent activation. One of the main problems limiting our understanding of proton-selective permeation is the inadequate description of channel- proton interactions by simulation methods based on classic mechanics. In aim 2, we will use quantum mechanics/molecular mechanics simulations on a validated Hv1 structural model in combination with the rational design of a proton-conducting VSD to obtain detailed information on how protons move within the Hv1 permeation pathway. Hv1 function is known to be tightly regulated in the cell. But, little is known about how this regulation is achieved. We have recently identified a new modality of channel regulation mediated by mechanical stress, which can provide an explanation for the hyperactivity of Hv1 previously described in microglia under conditions of ischemic stroke. In aim 3, we will use electrophysiology, high-speed pressure clamp stimulation, and targeted mutagenesis to determine the mechanism of Hv1 mechanosensitivity.

项目摘要 电压门控离子通道是一组多样的膜蛋白，在A中起着重要作用 来自神经元兴奋性和肌肉收缩的生理和病理过程多种多样， 自身免疫，中风和癌症。它们都共享一个通用的结构模块，即电压感应 域（VSD），负责打开和关闭效应子域，以应对变化 膜电位。我们和其他小组的先前研究表明，尽管大多数VSD都这样做 不进行离子，它们可能由于突变而变得泄漏。突变的VSD渗透到离子或 质子负责严重的遗传疾病，例如降低性周期性瘫痪和 心律不齐，心肌病。电压门控通道HV1的VSD 另一方面，本质上是质子导导的，此属性是频道许多人的关键 生理功能。这项研究的长期目标是阐明VSD如何进行离子和 质子，如何调节其活性，以及​​如何在药理上阻塞它们以进行治疗 目的。在这里，我们将重点关注HV1通道，HV1通道是各种疾病的新兴药物目标， 包括癌症和中风。 HV1中VSD介导的质子传导的基础机制为 知识渊博，对HV1活性的小分子抑制剂的需求未满足。我们有 以前发现了一类用作HV1阻滞剂的化合物，并表征了它们的结合 环境。我们确定了频道VSD核心内的芳族相互作用 利用以创建更好的药物来抑制HV1活性。在AIM 1中，我们建议使用 电生理测量和不自然的氨基酸取代 相互作用有助于HV1块和电压依赖性激活。主要问题之一 限制我们对质子选择性渗透的理解是对通道的描述不足 基于经典力学的模拟方法质子相互作用。在AIM 2中，我们将使用量子 在经过验证的HV1结构模型上的力学/分子力学模拟与 质子导电VSD的合理设计，以获取有关质子如何移动的详细信息 在HV1渗透途径中。已知HV1功能在细胞中受到严格调节。但是，很少 知道如何实现这一法规。我们最近确定了一种新的渠道方式 由机械应力介导的调节，这可以为多动症的多动症提供解释 在缺血性中风条件下，在小胶质细胞中描述的HV1。在AIM 3中，我们将使用 电生理学，高速压力夹刺激和靶向诱变，以确定 HV1机械敏感的机理。