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。它们是药物和天然毒素的主要靶点，这些毒素可以改变电活动，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，但我们对支撑钠通道功能的生物物理机制的了解仍然存在很大差距。人们对结构重排和驱动门控转变的潜在作用力知之甚少，这些转变允许通道短暂地打开，以响应膜电位的变化。由于我们无法孤立地研究激活过程，以及缺乏热力学工具来实验测量复杂蛋白质中的分子力，严格约束结构模型的开发一直受到阻碍。对结构域特定毒素的光谱和功能研究表明，结构域IV在钠通道中的电压依赖性移动比其他三个结构域慢。这个项目的中心目标是测试这一假设，即电压门控钠通道中的异步门控是由于每个区域内负责机电耦合的分子力的差异而产生的。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，以独立于模型的方式提取特定位置的相互作用能量。这一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结构相关的良好约束的定量描述。这些研究将在失活缺陷突变背景下进行，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，我们将建立一个良好约束的电压门控钠通道激活动力学模型。这些研究有望揭示与钠通道门控相关的不同特征，这些特征通常由于快速进入失活状态而被掩盖在野生型通道中。在特定的目标2中，我们将确定真核细胞钠通道激活门控的分子机制。这些实验将测试S6段允许进入这些通道中的毛孔的概念。最后，在具体目标3中，我们将利用广义相互作用能量分析(GIA)来探索电压相关离子通道中电压传感器和孔域相互作用的分子基础。这项拟议的研究有望揭示电压依赖性钠通道激活过程中构象变化背后的分子作用力。

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.