INACTIVATION GATING OF SODIUM CHANNELS

钠通道是动作电位快速去极化的基础 最易兴奋的细胞，并参与神经传导，自愿 肌肉收缩、心肌兴奋收缩偶联以及离子 经络相关的肌肉疾病。它们是心脏的受体 抗心律失常药物，用于干预骨骼发作的药物 肌源性周期性麻痹，以及抗惊厥药物。因此， 这些重要的兴奋性蛋白是正常生理所必需的。 并且是治疗干预的重要靶点。的确有 钠通道门控之间相互依赖的大量证据 并被局部麻醉剂阻断通道。然而，目前 对通道门控和通道的分子决定因素知之甚少 药理学。这项提案的长期目标是确定 人钠通道两个主要蛋白质亚基(α和α)的作用 β1亚基)以及它们的相互作用如何调制通道门控和 药理学。钠离子通道的特定区域(氨基酸区) 将通过蛋白质工程和重组DNA方法进行操作 目标是确定他们在通道门控中的角色(打开和 失活)、与Beta1亚基的相互作用以及药物结合。 方法包括膜片钳和高速切开卵母细胞电压。 在非洲爪哇卵母细胞或哺乳动物细胞中表达的通道钳。 一项主要战略将是利用自然的功能性和 不同钠通道的结构多样性，以指导实验。三 不同的人钠通道将被研究；人的心脏钠通道， Hh1；人骨骼肌钠通道，hSkm1；以及一个新发现的 从人的脑室克隆的通道，hNav2.1。使用的一个理由是 这些渠道是，尽管高度保守，功能差异 结合序列差异提供了识别和识别 操纵蛋白质中的重要区域。 需要了解状态依赖的药物阻断和通道门控模式 变革是根本性的。钠通道是开始时最简单的系统 这些在分子水平上的研究。它提供了重要的 成功进行蛋白质结构功能研究的优势。离子通道 具有功能签名(单通道电流)，可以 以出色的、与功能相关的时间分辨率测量 单个蛋白质分子的水平。结果会有所改善。 了解这些新发现的人类蛋白质的功能和 将有助于识别与通道门控和结合相关的蛋白质结构域 与治疗相关的药理药剂。

Sodium channels underlie the rapid depolarization of action potentials in most excitable cells and are involved in nervous conduction, voluntary muscle contraction, cardiac excitation-contraction coupling, as well as ion channel linked muscle diseases. They serve as receptors for cardiac antiarrhythmic agents, agents used to intervene in episodes of skeletal muscle derived periodic paralysis, as well as anticonvulsant agents. Thus, these important excitability proteins are essential to normal physiological behavior and are important targets for therapeutic intervention. There is considerable evidence for an inter-dependence between Na+ channel gating and channel block by local anesthetic-like agents. However at present little is known about the molecular determinants of channel gating and pharmacology. The long term aims of this proposal are to determine the role of the two principal protein subunits of human Na+ channel (alpha and beta1 subunits) and how their interactions modulate channel gating and pharmacology. Specific regions of the Na= channel (amino acid domains) will be manipulated through protein engineering and recombinant DNA methods with the goal of identifying their role in channel gating (opening and inactivation), interactions with the beta1 subunit, and in drug binding. The methods include patch clamp and high speed cut-open oocyte voltage clamp of channels expressed in Xenopus oocytes or in mammalian cells. A major strategy will be to capitalize on the natural functional and structural diversity of distinct Na+ channels to guide experiments. Three distinct human Na+ channels will be studied; the human cardiac Na+ channel, hH1; the human skeletal muscle Na+ channel, hSkm1; and a newly identified channel cloned from human ventricle, hNav2.1. One rationale for using these channels is that although highly conserved, functional differences combined with sequence differences provide clues for identifying and manipulating important domains in the protein. The need to understand state dependent drug block and channel gating mode changes is fundamental. The Na+ channel is the simplest system to begin these investigations at the molecular level. It offers significant advantages for successful protein structure-function studies. Ion channels have a functional signature (the single channel current) that can be measured with excellent, and functionally relevant temporal resolution at the level of a single protein molecule. The results will improve understanding of the function of these newly identified human proteins and will help identify protein domains involved with channel gating and binding of therapeutically relevant pharmacological agents.