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.

项目概要 电压门控离子通道是一组不同的膜蛋白，在 各种生理和病理过程，从神经元兴奋性到肌肉收缩， 自身免疫、中风和癌症。它们都共享一个共同的结构模块，即电压传感 域（VSD），负责打开和关闭效应器域以响应 膜电位。我们和其他团体之前的研究表明，虽然大多数 VSD 确实 不传导离子，它们可能会因突变而泄漏。突变的 VSD 可渗透离子或 质子会导致严重的遗传性疾病，例如低钾性周期性麻痹，以及 心律失常合并扩张型心肌病。电压门控通道 Hv1 的 VSD， 另一方面，本质上具有质子传导性，这种特性是通道许多功能的关键 生理功能。本研究的长期目标是阐明 VSD 如何传导离子和 质子，如何调节它们的活性，以及​​如何通过药理学阻断它们以进行治疗 目的。在这里，我们将重点关注 Hv1 通道，这是一种针对多种疾病的新兴药物靶点， 包括癌症和中风。 Hv1 中 VSD 介导的质子传导的机制是 人们对此知之甚少，并且对 Hv1 活性的小分子抑制剂的需求尚未得到满足。我们有 先前发现了一类充当 Hv1 阻断剂的化合物并表征了它们的结合 环境。我们确定了通道 VSD 核心内的芳香族相互作用，这可能是 利用它来创造更好的药物来抑制 Hv1 活性。在目标 1 中，我们建议使用 电生理测量和非天然氨基酸取代来检查这些 相互作用有助于 Hv1 阻断和电压依赖性激活。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充分 通过基于经典力学的模拟方法来实现质子相互作用。在目标 2 中，我们将使用量子 对经过验证的 Hv1 结构模型进行力学/分子力学模拟 质子传导 VSD 的合理设计，以获得质子如何移动的详细信息 Hv1 渗透途径内。已知 Hv1 功能在细胞中受到严格调控。但是，一点点 了解该规定是如何实现的。我们最近确定了一种新的渠道模式 由机械应力介导的调节，这可以解释 先前描述了缺血性中风条件下小胶质细胞中的 Hv1。在目标 3 中，我们将使用 电生理学、高速压力钳刺激和定向诱变以确定 Hv1 机械敏感性的机制。