TOXIN: NA-CHANNEL INTERACTIONS AND DRUG DESIGN

毒素：NA 通道相互作用和药物设计

• 批准号: 6340578
• 负责人: KENNETH BLUMENTHAL
• 金额: $ 27.98万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6340578
• 关键词: Anthozoa; cardiotonic agents; drug design /synthesis /production; electrophysiology; fluorescent dye /probe; marine toxins; neurotoxins; nuclear magnetic resonance spectroscopy; site directed mutagenesis; sodium channel; toxicant interaction; voltage gated channel

DESCRIPTION (Verbatim from applicant's abstract): Polypeptide neurotoxins have historically provided invaluable reagents for both purification and structural and pharmacologic characterization of voltage-sensitive cation channels. One such family of toxins, elaborated by sea anemones and exemplified in this proposal by Anthopleurin-B (ApB) from Anthopleura xanthogrammica has been demonstrated to greatly delay inactivation of voltage-dependent Na+ channel isoforms from heart, skeletal muscle, and brain (in order of binding affinity) by binding to toxin site III. ApB has been cloned and expressed in bacteria in the p.i.'s lab, and a number of functionally essential residues, including Arg-12, Leu-18, and Trp-33, identified by electrophysiologic and biochemical analyses of designed mutant toxins. By applying thermodynamic mutant cycle analysis to mutated forms of each protein, we have also demonstrated a direct interaction between Lys-37 of ApB and Asp-1612 of the cardiac Na-channel, thus confirming the S3-S4 extracellular loop of channel domain IV as a primary component of site III. Among the anemone toxins, ApB has the highest known affinity for cardiac Na-channels, and we have shown it to display an approximate 100fold preference for the cardiac over the neuronal isoform. In this application, we propose to (1) identify additional ApB residues essential for defining a high-affinity binding epitope, as well as residues important for determining channel isoform specificity of ApB; (2) fill out the terra incognita of the channel binding site, using both chimeric channels and thermodynamic mutant cycle analysis of mutated toxin:channel pairs; (3) characterize the role played by orientation of a flexible loop containing Residues 9-19 of ApB in defining isoform selectivity; and (4) solve the solution structure of a complex containing recombinant ApB and a synthetic peptide which recapitulates the sequence of the S3-S4 linker of channel domain IV. The results of these studies will provide important information on the 3-dimensional structure of the voltage-sensitive Na-channel. In addition, since ApB has been demonstrated in a number of whole animal studies to serve as a powerful positive inotropic agent, identification of additional atomic details regarding its interaction with the sodium channel should open the door to development of new cardiotonic peptidomimetics.

描述（逐字研究来自申请人的摘要）：多肽神经毒素具有 历史上为纯化和结构提供了宝贵的试剂 电压敏感阳离子通道的药理表征。一 这种毒素家族，由海葵详细阐述并在此示例 Anthopleura Xanthogrammica的Anthopleurin-B（APB）提出的建议已 证明电压依赖性Na+通道极大地延迟失活 来自心脏，骨骼肌和大脑的同工型（按结合亲和力顺序） 通过与毒素位点III结合。 APB已克隆并在细菌中表达 P.I.的实验室和许多功能上必不可少的残基，包括 通过电生理和生化鉴定的Arg-12，Leu-18和TRP-33 设计突变毒素的分析。通过应用热力学突变循环 分析每种蛋白质的突变形式，我们还展示了一个直接的 APB的LYS-37与心脏NA通道的ASP-1612之间的相互作用 确认通道域IV的S3-S4细胞外环为主要 站点III的组成部分。在海葵毒素中，APB的知名度最高 对心脏NA通道的亲和力，我们已经证明了它显示 对于神经元同工型而言，心脏比心脏偏爱大约100倍。在 该应用程序，我们建议（1）确定其他APB残基必不可少的 定义高亲和力结合表位，以及残基对 确定APB的通道同工型特异性； （2）填写台 使用嵌合通道和 突变毒素的热力学突变循环分析：通道对； （3） 表征通过包含灵活环的方向起作用的作用 APB的残基9-19在定义同工型选择性方面； （4）解决 包含重组APB和合成的复合物的溶液结构 概述了通道域S3-S4接头序列的肽 iv。这些研究的结果将提供有关 电压敏感的Na通道的3维结构。此外，自从 在许多全动物研究中已经证明了APB作为A 强大的阳性肌力剂，识别其他原子细节 关于它与钠通道的相互作用，应打开门 开发新的心脏肽肽仪。