ENZYMOLOGY OF SINGLE NA-CHANNELS IN ARTIFICIAL BILAYERS

这项提案是一项致力于 离子通道的生化和生物物理联合分析 调节可兴奋组织的电活动。具体目标 目前的项目包括体外实验，旨在研究 骨骼肌动作电位下的钠通道。这个 采用的主要方法是直接电记录 通过单个钠通道的电流已被合并到 平面脂质双分子膜。此方法已由 首席调查员在前一年的前期工作中研究 胍毒素对巴曲霉毒素修饰的钠通道的阻断作用 大鼠骨骼肌质膜。学习的一个主要优势 巴曲霉毒素修饰钠通道是电压依赖的 这种毒素可以消除该通道的失活。这允许 电压依赖的激活和阻断剂的作用 便于在恒压下的稳态条件下研究。 使用这项技术，通道进入离子的通道的结构将 被特定的毒素探测到，这种毒素作用于细胞外侧，还 由胍类化合物和其他局部麻醉剂发挥作用 海峡的两边。电压依赖门控将通过以下方式进行研究 稳态电流波动随时间变化的统计分析 电压。为研究结构和机制而设计的其他实验 包括对变异的毒素不敏感钠通道的研究，对 通道多肽(S)通过特异性蛋白降解和化学标记，以及 中性和负电荷的脂类对门控的影响， 传导和阻断行为。该项目将推进基本 对Na-通道的机制及其在心脏中的行为的认识 有局麻药存在。此外，申请的必要方法 其他重要离子通道的技术也将得到发展。这个 这个项目与人类健康的可能关系包括神经学 疾病、麻醉和神经系统生理学。

This proposal is the initial project of a research program dedicated to the combined biochemical and biophysical analysis of ion channels which regulate the electrical activity of excitable tissues. The specific goals of the present project include in vitro experiments designed to investigate the Na-channel underlying the action potential of skeletal muscle. The primary method to be employed is the direct electrical recording of currents through individual Na-channels that have been incorporated into planar lipid bilayers. This method has been established and used by the principal investigator during a previous year of preliminary work to study guanidinium-toxin blockade of batrachotoxin-modified Na-channels from the plasma membrane of rat skeletal muscle. A primary advantage of studying the batrachotoxin-modified Na-channel is that voltage-dependent inactivation of the channel is eliminated by this toxin. This permits voltage-dependent activation and the effects of blocking agents to be conveniently studied under steady-state conditions at constant voltage. Using this technique, the structure of the channel's entryway to ions will be probed by specific toxins which act from the extracellular side and also by guanidinium compounds and other local anesthetics which may act from either side of the channel. Voltage-dependent gating will be studied by statistical analysis of steady-state current fluctuations as a function of voltage. Other experiments designed to investigate structure and mechanism include studies of variant toxin-insensitive Na-channels, mapping of the channel polypeptide(s) by specific proteolysis and chemical labeling, and the effect of neutral versus negatively charged lipids on gating, conduction and blocking behavior. This project will advance basic understanding of the mechanism of the Na-channel and its behavior in the presence of local anesthetics. In addition, methods necessary for applying the technique to other important ion channels will be developed. The possible relationships of this project to human health include neurological diseases, anesthesia and nervous system physiology.