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，其在细胞膜上起重要作用。 各种生理和病理过程，从神经元兴奋性和肌肉收缩， 自身免疫中风和癌症它们都有一个共同的结构模块，即电压感应模块。 结构域（VSD），负责响应于 膜电位我们和其他团体以前的研究表明，虽然大多数VSD都是如此， 不导电的离子，它们可能由于突变而变得泄漏。突变的离子渗透性VSD或 质子导致严重的遗传疾病，如低钾性周期性麻痹， 心律失常伴扩张型心肌病电压门控通道Hv 1的VSD，在 另一方面，是固有的质子传导性，这种性质是通道的许多关键 生理功能。这项研究的长期目标是阐明VSD如何传导离子， 质子，它们的活性是如何调节的，以及它们如何被阻断以用于治疗 目的在这里，我们将重点关注Hv 1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VSD介导的Hv 1质子传导的机制是 对Hv 1活性的小分子抑制剂了解甚少，并且存在未满足的需求。我们有 先前发现了一类作为Hv 1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核心内的芳香相互作用， 用来制造更好的药物来抑制Hv 1的活性。在目标1中，我们建议使用 电生理学测量和非天然氨基酸取代，以检查这些 相互作用有助于Hv 1阻断和电压依赖性激活。的主要问题之一 限制我们对质子选择性渗透的理解的是对通道的不充分描述， 基于经典力学的模拟方法的质子相互作用。在aim 2中，我们将使用量子 在经过验证的Hv 1结构模型上进行力学/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 在Hv 1渗透途径中。已知hv 1功能在细胞中受到严格调控。但是， 我们知道这个规则是如何实现的。我们最近发现了一种新的渠道模式， 调节介导的机械应力，这可以提供一个解释的过度活跃， 先前在缺血性卒中条件下的小胶质细胞中描述了hv 1。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 Hv 1机械敏感性的机制。

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.