Structural basis of modulation of Na+ channels by local anesthetics

描述（由申请人提供）：电信号传导构成中枢神经系统中主要的通讯方式之一，电压依赖性钠通道负责启动电脉冲。Na+通道存在三种功能状态，取决于跨膜电压：关闭，开放和失活。导致不完全失活的Na+通道突变与各种疾病有关，包括先天性长QT综合征、全身性癫痫和肌强直。局部麻醉剂是一类开放通道阻滞剂，用于治疗某些通道相关疾病，并被认为可使通道稳定在失活状态。我实验室的长期目标是使用结构方法来理解Na+通道门控及其调节的物理基础。在这项研究中，我们提出解决一个基本问题：如何像局部麻醉剂的分子结合到通道孔修改离子通道的电压依赖性门控行为。我们将使用荧光记录的位点特异性标签沿着与电生理测量研究局部麻醉剂的构象变化与电压敏感的Na+通道S4段的影响。我们建议研究a）局部麻醉剂对单个S4段动力学的影响，B）局部麻醉剂对单个S4段结构的影响，c）确定S4段和局部麻醉剂在孔处结合之间偶联的分子基础，以及d）确定在失活状态下稳定通道是否有利于局部麻醉剂结合。这些实验将被解释在最近阐明的原型电压门控离子通道（KV 1.2）的结构，以了解Na+通道门控及其调制局部麻醉剂的结构基础。公共卫生相关性：为了开发更好的药物来治疗离子通道相关疾病，有必要了解离子通道功能的结构基础。本文提出的研究利用一种相对新颖的结构方法来研究Na+通道的动力学及其受局部麻醉剂的调节。这项研究将促进人类的健康和福祉，有助于开发下一代离子通道药物，以特定的方式调节通道功能。

DESCRIPTION (provided by applicant): Electrical signaling constitutes one of the primary means of communication in the central nervous system with the voltage-dependent sodium channels being responsible for initiating electrical impulses. Na+ channels exist in three functional states depending on transmembrane voltage: closed, open and inactivated. Mutations of Na+ channels that lead to incomplete inactivation has been linked to various disease conditions including congenital long QT syndrome, generalized epilepsy and muscle myotonia. Local anesthetics are a class of open channel blockers that are used to treat some channel-associated conditions and are believed to stabilize the channel in the inactivated state. The long term goal of my laboratory is to use structural approaches to understand the physical basis of gating of Na+ channels and their modulation. In this study, we propose to address a fundamental question: How do molecules like local anesthetics that bind to the channel pore modify the voltage-dependent gating behavior of ion channels. We will use fluorescence recordings of site-specific labels along with electrophysiological measurements to study the effect of local anesthetic on the conformational changes associated with voltage-sensing S4 segments of Na+ channels. We propose to study a) the effect of local anesthetic on the dynamics of individual S4 segments, b) the effect of local anesthetic on structure of the individual S4 segments, c) determine the molecular basis of coupling between S4 segments and local anesthetic binding at the pore, and d) determine if stabilizing the channel in the inactivated state favors local anesthetic binding. These experiments will be interpreted in light of the recently elucidated structure of a prototypical voltage-gated ion channel (Kv 1.2) to understand the structural basis of Na+ channel gating and its modulation by local anesthetics. PUBLIC HEALTH RELEVANCE: In order to develop better drugs to treat ion channel associated disease conditions, it becomes necessary to understand the structural underpinnings of ion channel function. The research proposed here utilizes a relatively novel structural approach to study the dynamics of the Na+ channel and its modulation by local anesthetics. This research will advance human health and well-being by contributing to the development of next generation of ion channel drugs that will modulate the channel function in a specified manner.