STRUCTURE-FUNCTION OF THE CARDIAC SODIUM CHANNEL

心脏钠通道的结构与功能

• 批准号: 6527642
• 负责人: HARRY A FOZZARD
• 金额: $ 33.72万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-29 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527642
• 关键词: Xenopus oocyte; binding sites; cardiac myocytes; cardiotoxin; cell line; gene mutation; membrane permeability; mutant; protein isoforms; protein structure function; sodium channel

DESCRIPTION (the applicant's description verbatim): The long-term purpose of this project is to understand cardiac Na+ channel function at the molecular level, and to use the understanding to develop strategies for control of lethal arrhythmias. This project has three interdependent goals for the next five years: 1) resolving the molecular configuration of the Na+ channel permeation path/selectivity region, including the sites for local anesthetic drug binding, 2) examining the roles of charged vestibule residues in permeation and selectivity, and 3) determining the functional abnormalities resulting from naturally occurring channel mutants, especially alpha-subunit interactions. The Na+ channel is a major participant in most serious or lethal arrhythmias, and it is the target of some of our most powerful antiarrhythmic drugs. Considerable progress has been made in identifying the molecular structure of the outer vestibule (outer third of the permeation path) by effects of point mutations on permeation and selectivity and by determining the complimentary surface for binding of the pore-blocking toxins. Our unifying structural hypothesis is that the Na+ channel is related through evolution to the structurally determined KcsA bacterial channel, and that it spore motif of a helix teepee and a selectivity motif of helix-turn-strand is applicable to the Na+ channel. We propose to extend the complimentary interaction surface for outer vestibule toxins to identify the structure of the "turrets" in the outer path, perhaps also attempting to resolve the isoform selectivity of u-conotoxin in the process. These important carboxyls in the outer vestibule will be examined by mutation and pH titration for their contribution to dehydration of the permeating ions. The residues facing the inner pore will be identified by sequential mutation of the residues to cysteine, with access determined by methanesulfonate derivative interaction. Their roles in local anesthetic drug binding will be determined, in order to find all of the molecular parts of the drug binding site. State-dependent access of these residues will be examined to determine the changes in the site with channel gating. Each experimental step is developed based on the best molecular model we can develop, and in turn the model is improved as new experimental information is obtained in these experiments or published by others.

描述（申请人的逐字描述）：长期目的 本项目旨在从分子水平了解心脏Na+通道的功能， 水平，并利用理解制定战略，控制致命的 心律不齐该项目在未来五年内有三个相互依存的目标 年：1）解析Na+通道渗透的分子构型 路径/选择性区域，包括局部麻醉药物结合的位点， 2)检查带电前庭残留物在渗透中的作用， 选择性，以及3）确定由以下原因引起的功能异常： 天然存在的通道突变体，尤其是α-亚基相互作用。的 Na+通道是大多数严重或致死性心律失常的主要参与者， 它是我们一些最有效的抗疟疾药物的目标。 在鉴定其分子结构方面取得了相当大的进展， 外前庭（渗透路径的外三分之一）通过点的影响 突变的渗透性和选择性，并通过确定互补的 用于结合孔阻塞毒素的表面。我们的统一结构 假设是Na+通道通过进化与 结构确定的KcsA细菌通道，和它的孢子基序， 螺旋圆锥形和螺旋-转角-链的选择性基序适用于 Na+通道。我们建议扩展互补的相互作用表面， 外前庭毒素，以确定在外部的“炮塔”的结构 路径，也许也试图解决u-芋螺毒素的异构体选择性 在这个过程中。这些重要的羧基在外部前庭将 通过突变和pH滴定检查它们对脱水的贡献， 渗透的离子。将通过以下方法识别面向内孔的残留物： 将残基顺序突变为半胱氨酸，通过 甲磺酸衍生物相互作用。它们在局麻药中的作用 结合将被确定，为了找到所有的分子部分的 药物结合位点。这些残留物的状态依赖访问将被检查， 确定具有通道门控的站点中的变化。每个实验步骤 是基于我们所能开发的最好的分子模型开发的，反过来， 随着新的实验信息的获得， 实验或由他人发表。