Structural basis of modulation of Na+ channels by local anesthetics

描述(申请人提供)：电信号是中枢神经系统的主要通讯方式之一，电压依赖的钠通道负责启动电脉冲。根据跨膜电压的不同，钠离子通道存在三种功能状态：关闭、开放和失活。导致钠通道不完全失活的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。局麻药是一类开放的通道阻滞剂，用于治疗某些通道相关的疾病，被认为可以将通道稳定在失活状态。我的实验室的长期目标是使用结构方法来了解钠通道门控的物理基础及其调节。在这项研究中，我们提出了一个基本问题：像局部麻醉剂这样与通道孔结合的分子是如何改变离子通道的电压依赖门控行为的。我们将使用位置特异性标记的荧光记录和电生理测量来研究局麻药对与电压敏感的钠通道S4段相关的构象变化的影响。我们建议研究a)局麻药对单个S4节段动力学的影响，b)局麻药对单个S4节段结构的影响，c)确定S4节段与局部麻醉剂在孔处结合的分子基础，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。这些实验将根据最近阐明的典型电压门控离子通道(Kv 1.2)的结构来解释，以了解Na通道门控的结构基础及其被局麻药调节。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。这项研究采用了一种相对新颖的结构方法来研究钠通道的动力学以及局麻药对它的调节。这项研究将通过促进下一代离子通道药物的开发来促进人类的健康和福祉，这些药物将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.