Structural basis of modulation of Na+ channels by local anesthetics

摘要 电信号是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 根据跨膜电压处于三种功能状态：关闭、打开和失活。突变 导致不完全失活的 Na 通道与多种疾病有关，包括 先天性长QT综合征、全身性癫痫和肌强直。局部麻醉药是一类 开放通道阻滞剂，用于治疗一些与通道相关的疾病，并被认为可以稳定病情 通道处于非激活状态。我实验室的长期目标是使用结构方法 了解 Na 通道门控及其调制的物理基础。在这项研究中，我们建议 解决一个基本问题：像局部麻醉剂这样与通道孔结合的分子如何改变 离子通道的电压依赖性门控行为。我们将使用特定位点的荧光记录 标签以及电生理测量来研究局部麻醉剂对 与 Na 通道电压感应 S4 片段相关的构象变化。我们建议学习 a) 局部麻醉剂对各个 S4 节段动态的影响，b) 局部麻醉剂对 各个 S4 片段的结构，c) 确定 S4 片段之间偶联的分子基础 和局部麻醉剂在孔处的结合，以及 d) 确定是否使通道稳定在失活状态 有利于局部麻醉剂的结合。这些实验将根据最近阐明的 原型电压门控离子通道 (Kv 1.2) 的结构，以了解 Na 通道的结构基础 门控及其局部麻醉剂的调节。叙事 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。这里提出的研究利用了 研究 Na 通道动力学及其局部调制的相对新颖的结构方法 麻醉剂。这项研究将通过促进人类健康和福祉的发展 下一代离子通道药物将以特定方式调节通道功能。

SUMMARY 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. NARRATIVE 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.