INACTIVATION GATING OF SODIUM CHANNELS

钠通道是动作电位快速去极化的基础 大多数可兴奋的细胞，参与神经传导，自愿 肌肉收缩，心脏兴奋-收缩偶联，以及离子 经络相关的肌肉疾病 它们作为心脏的受体 抗骨质疏松剂，用于干预骨骼 肌肉源性周期性麻痹，以及抗惊厥剂。 因此，在本发明中， 这些重要的兴奋性蛋白质对于正常的生理功能是必需的。 行为和治疗干预的重要目标。 有 大量证据表明Na+通道门控之间存在相互依赖性 以及局部麻醉剂样药物造成的通道阻滞。 但目前 关于通道门控的分子决定因素知之甚少， 药理学 本提案的长期目标是确定 人Na+通道的两个主要蛋白亚基（α和β）的作用 β 1亚基）以及它们的相互作用如何调节通道门控， 药理学 Na=通道的特定区域（氨基酸结构域） 将通过蛋白质工程和重组DNA方法进行操纵 目的是确定它们在通道门控（打开和关闭）中的作用， 失活），与β 1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 在非洲爪蟾卵母细胞或哺乳动物细胞中表达的通道钳。 一个主要的战略将是利用自然的功能和 不同Na+通道的结构多样性来指导实验。 三 将研究不同的人Na+通道;人心脏Na+通道， 人骨骼肌Na+通道，hSkm 1;和一个新发现的 从人心室克隆的hNav2.1。 使用的一个理由是 这些通道虽然高度保守， 与序列差异相结合， 操纵蛋白质中的重要结构域。 需要了解状态依赖性药物阻滞和通道门控模式 变化是根本。 Na+通道是开始最简单的系统 这些研究在分子水平上。 它提供了重要的 成功的蛋白质结构-功能研究的优势。 离子通道 有一个功能签名（单通道电流），可以 测量具有出色的，功能相关的时间分辨率， 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， 将有助于识别与通道门控和结合有关的蛋白质结构域 治疗相关的药理学试剂。

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.