ENZYMOLOGY OF SINGLE NA-CHANNELS IN ARTIFICIAL BILAYERS

电压依赖性钠通道是一类膜蛋白 负责快速激活和失活，内向钠- 可兴奋细胞快速动作电位的基础电流。 该项目的长期目标是促进 了解这类离子的结构和机理 渠道 该项目这一阶段的具体目标侧重于 三个研究领域：（1）生物化学分析和 一个不寻常的水的功能意义的调查- 一种存在于牛蛙骨骼中石房蛤毒素的可溶性受体 肌肉. (2)结合的分子基础分析 胍类毒素如石房蛤毒素和河豚毒素 各种钠通道亚型上的受体位点。 (3)分析 各种无机和无机盐阻断钠通道的机制 有机阳离子，特别强调高亲和性阻断 河豚毒素不敏感的Na通道， 局部麻醉剂作用的多个部位的可能性。 （3 H）石房蛤毒素的特异性结合将被用作 纯化青蛙骨骼肌中的新毒素受体， 可以是河豚毒素不敏感的Na- 频道 对纯化受体的生化研究将被用于 以确定其与已知钠通道的结构关系 proteins. 重构为平面脂质双层将是 试图作为一种方法，以可能的钠通道功能， 这个受体。 不同的蛙毒素激活的Na通道 结合到平面脂质双层中的亚型将用作 体外模型系统，以研究在单个 渠道水平。 毒素阻断事件的统计分析将 提供配体-受体的详细动力学信息 在河豚毒素结合位点的相互作用。 类似的方法 将用于研究钠通道阻断的机制 从犬心脏和大鼠骨骼肌的锌，局部 麻醉药、抗惊厥药和抗惊厥药。 一个 高亲合力Zn ~（2+）生理意义的评价 还将通过膜片钳实验尝试阻断 表达毒素不敏感Na通道的培养细胞。 该项目的结果应进一步基本了解 钠通道的生物化学和药理学，并可能找到最终 在肌肉骨骼、心血管和神经系统中的应用 疾病

Voltage-dependent Na-channels are a class of membrane proteins responsible for the rapidly activating and inactivating, inward Na- current that underlies the fast action potential of excitable cells. The long-term objective of this project is to advance understanding of the structure and mechanism of this class of ion channels. The specific aims of this phase of the project focus on three areas of research: (1) Biochemical analysis and investigation of the functional significance of an unusual water- soluble receptor for saxitoxin that is present in bullfrog skeletal muscle. (2) Analysis of the molecular basis for binding of guanidinium toxins such as saxitoxin and tetrodotoxin to a specific receptor site on various Na-channel subtypes. (3) Analysis of the mechanisms of block of Na-channels by diverse inorganic and organic cations, with particular emphasis on high-affinity block of tetrodotoxin-insensitive Na-channels by Zn2+ and on the possibility of multiple sites of action of local anesthetics. Specific binding of (3H) saxitoxin will be used as an assay in the purification of a novel toxin receptor in frog skeletal muscle that may be a water-soluble form of a tetrodotoxin-insensitive Na- channel. Biochemical studies on the purified receptor will be used to establish its structural relationship to known Na-channel proteins. Reconstitution into planar lipid bilayers will be attempted as an approach to the possible Na-channel function of this receptor. Batrachotoxin-activated Na-channels of various subtypes incorporated into planar lipid bilayers will be used as an in vitro model system to study blocking mechanisms at the single channel level. Statistical analysis of toxin blocking events will provide detailed kinetic information on ligand-receptor interactions at the tetrodotoxin binding site. A similar approach will be used to investigate the mechanism of block of Na-channels from canine heart and rat skeletal muscle by Zn2+, local anesthetics, antiarrhythmic and anticonvulsant drugs. An evaluation of the physiological significance of high-affinity Zn2+ block will also be attempted by patch-clamp experiments on cultured cells that express toxin-insensitive Na-channels. The results of this project should further basic understanding of Na-channel biochemistry and pharmacology and may find ultimate applications in musculoskeletal, cardiovascular and neurological diseases.