Mechanisms of Permeation and Gating of Voltage-Sensing Domains

项目摘要 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，从神经元兴奋性和肌肉收缩， 与自身免疫、中风和癌症有关。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域以响应 膜电位。我们和其他组织之前的研究表明，尽管大多数VSD 如果不是传导离子，它们可能会因为突变而变得泄漏。突变的VSD对离子或 质子导致严重的遗传疾病，如低血钾性周期性麻痹，以及 扩张型心肌病合并心律失常。电压门控通道Hv1的VSD在 另一方面，本质上是质子导电的，这一特性是通道许多 生理功能。这项研究的长期目标是阐明VSD是如何传导离子和 质子，它们的活动是如何被调节的，以及它们如何在药物上被阻断以用于治疗 目的。在这里，我们将重点关注Hv1通道，这是一种治疗多种疾病的新兴药物靶点， 包括癌症和中风。VSD介导的Hv1质子传导的机制是 人们对Hv1活性的小分子抑制剂知之甚少，而且还没有得到满足。我们有 之前发现了一类充当Hv1阻滞剂的化合物，并表征了它们的结合 环境。我们确定了通道VSD核心内的芳香相互作用可能是 被利用来创造更好的药物来抑制Hv1的活性。在目标1中，我们建议使用 电生理测量和非天然氨基酸替代来检查这些 相互作用有助于阻断Hv1和电压依赖的激活。主要问题之一是 限制我们对质子选择性渗透的理解是对通道的不充分描述。 基于经典力学的质子相互作用模拟方法。在目标2中，我们将使用量子 Hv1结构模型的力学/分子力学模拟 质子传导VSD的合理设计以获取质子运动的详细信息 在Hv1渗透途径内。已知的是，HV1功能在细胞内受到严格调控。但是，很少 都知道这一规定是如何实现的。我们最近确定了一种新的渠道模式 机械应力介导的调节，这可以为多动提供解释 之前描述的在缺血性卒中条件下的小胶质细胞中的HV1。在目标3中，我们将使用 电生理学、高速压力钳刺激和靶向诱变来确定 Hv1的机械敏感性机制。

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.