MOLECULAR PHARMACOLOGY OF VOLTAGE-SENSITIVE NA+ CHANNELS

电压敏感 NA 通道的分子药理学

• 批准号: 3309728
• 负责人: EDWARD G MOCZYDLOWSKI
• 金额: $ 22.79万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3309728
• 关键词: Anura; Gastropoda; action potentials; antiarrhythmic agent; anticonvulsants; batrachotoxins; drug metabolism; enzyme mechanism; ligands; lipid bilayer membrane; local anesthetics; membrane activity; membrane model; membrane potentials; membrane proteins; sarcolemma; saxitoxin; sodium channel; striated muscles; tetrodotoxin; tritium; zinc

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.

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Na- 电流是可兴奋细胞快速动作电位的基础。 这个项目的长期目标是推进 对这类离子的结构和机理的理解 频道。该项目这一阶段的具体目标侧重于 三个领域的研究：(1)生化分析和 一种不寻常的水的功能意义的研究- 牛蛙骨骼中存在的肉毒素可溶性受体 肌肉。(2)结合的分子基础分析 鸟粪类毒素如岩藻毒素和河豚毒素对特定的 不同钠通道亚型上的受体位置。(3)分析了 不同无机和亚硝酸盐对钠通道的阻断机制 有机阳离子，特别强调高亲和力的嵌段 河豚毒素不敏感钠离子通道对锌离子的影响 局麻药有多个作用部位的可能性。 (~3H)石杉毒素的特异性结合将作为一种分析方法。 蛙骨骼肌中一种新的毒素受体的纯化 可能是河豚毒素不敏感的Na-的一种水溶性形式 频道。将对纯化的受体进行生化研究 建立其与已知钠通道的结构关系 蛋白质。重组成平面脂质双层将是 作为对可能的钠通道功能的一种探讨 这个感受器。巴曲霉毒素激活的多种钠离子通道 结合到平面脂质双层中的亚型将被用作 体外模型系统研究单核细胞阻断机制 频道级别。毒素阻断事件的统计分析将 提供有关配体-受体的详细动力学信息 河豚毒素结合部位的相互作用。类似的方法 将用于研究钠通道的阻断机制 从犬心脏和大鼠骨骼肌中提取的锌离子，局部 麻醉药、抗心律失常药和抗惊厥药。一个 高亲和力锌离子生理意义的评价 膜片钳实验也将尝试阻断 培养的细胞表达毒素不敏感的钠通道。 这一项目的成果应该进一步加深对 NA通道的生物化学和药理学，可能会发现 在肌肉骨骼、心血管和神经病学中的应用 疾病。