Mechanisms of Gating in Voltage-dependent Sodium Channels

描述（申请人提供）：真核电压门控钠通道负责在神经元和大多数可兴奋细胞中启动和传播电脉冲。它们是改变电活动的药物和天然毒素的主要靶点，钠通道基因的突变与先天性长QT综合征、全身性癫痫和肌强直等疾病有关。尽管在了解钠通道在人体中的作用方面取得了很大进展，但我们对钠通道功能的生物物理机制的了解仍然存在重大差距。对于结构重排和驱动门控转变的潜在力量知之甚少，门控转变允许通道在响应膜电位的变化时短暂打开。由于我们无法孤立地研究激活过程和缺乏热力学工具来实验测量复杂蛋白质中的分子力，约束良好的结构模型的发展受到阻碍。特异结构域毒素的光谱和功能研究表明，钠通道中结构域IV的电压依赖性运动比其他三个结构域的运动要慢。本项目的中心目标是测试电压门控钠通道中的异步门控是由于每个域内负责机电耦合的分子力的差异而产生的假设。这一建议是基于我们最近的发现，这些发现导致了分析工具的发展，以一种与模型无关的方式提取特定位点的相互作用能量。该分析将与半胱氨酸可及性、电压钳荧光法和单通道记录研究相结合，以开发钠通道门控的良好约束结构相关定量描述。这些研究将在失活缺陷突变体背景下进行，以避免由于激活和失活过程重叠而产生的任何并发症。在具体目标1中，我们将为电压门控钠通道的激活建立一个约束良好的动力学模型。这些研究有望揭示与钠通道门控相关的独特特征，这些特征通常在野生型通道中由于快速进入失活状态而被掩盖。在具体目标2中，我们将确定真核钠通道中激活门控的分子机制。这些实验将验证S6片段允许进入这些通道中的孔隙的概念。最后，在具体目标3中，我们将使用广义相互作用能分析（GIA）来探索电压依赖离子通道中电压传感器和孔域相互作用的分子基础。提出的研究有望揭示在电压依赖性钠通道激活过程中构象变化背后的分子力。

DESCRIPTION (provided by applicant): The eukaryotic voltage-gated sodium channel is responsible for initiating and propagating electrical impulses in neurons and most excitable cells. They are the major targets of drugs and naturally occurring toxins that modify electrical activity and mutations of sodium channel genes have been linked to disease conditions such as congenital long QT syndrome, generalized epilepsy and muscle myotonia. Despite much progress in understanding the role of sodium channels in the human body, there remains a significant gap in our knowledge of the biophysical mechanisms that underpin sodium channel function. Very little is known about the structural rearrangements and the underlying forces that drive the gating transitions which allow the channels to open briefly in response to a change in membrane potential. Development of well-constrained structural models has been hampered both due to our inability to study the activation process in isolation and a lack of thermodynamic tools to experimentally measure molecular forces in complex proteins. Spectroscopic and functional studies with domain specific toxins have revealed that the voltage-dependent movement of domain IV in the sodium channel is slower than those of the other three domains. The central goal of this project is to test the hypothesis that asynchronous gating in voltage-gated sodium channels arises due to differences in molecular forces responsible for electromechanical coupling within each domain. This proposal is based on our recent findings that have led to the development of analytical tools to extract site-specific interaction energies n a model-independent fashion. This analysis will be combined with cysteine accessibility, voltage-clamp fluorimetry and single-channel recording studies to develop a well-constrained structurally relevant quantitative description of sodium channel gating. These studies will be conducted on an inactivation deficient mutant background to avoid any complications that arise due to overlap of the activation and inactivation process. In specific aim 1, we will develop a well-constrained kinetic model for activation of voltage-gated sodium channels. These studies are expected to reveal distinct features associated with sodium channel gating which are typically masked in the wild-type channels due to rapid entry into the inactivated state. In specific aim 2, we will determine the molecular mechanism of activation gating in eukaryotic sodium channels. These experiments will test the notion that the S6 segments grant access to the pore in these channels. Finally, in specific aim 3, we will probe the molecular basis of voltage-sensor and pore domain interactions in voltage-dependent ion channel using Generalized Interaction energy Analysis (GIA). The proposed studies are expected to shed new light on the molecular forces that underlie conformational changes during the activation of a voltage-dependent sodium channels.