Dysfunction in Ankyrin-based Pathways and Human Arrythmia

基于锚蛋白的通路功能障碍和人类心律失常

• 批准号: 7882729
• 负责人: Peter J. Mohler
• 金额: $ 14.16万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-02-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882729
• 关键词: Action Potentials; Adaptor Signaling Protein; Adult; Affinity; Ankyrins; Arrhythmia; Binding; Cardiac; Cardiac Myocytes; Cells; Cerebellum; Data; Disease; Functional disorder; Gap Junctions; Goals; Heart; Human; Inherited; Intercalated disc; Ion Exchange; Knock-out; Laser Scanning Confocal Microscopy; Lead; Length; Long QT Syndrome; Membrane; Molecular; Mus; Mutation; Myocardial Contraction; Neurons; Pathway interactions; Physiological; Play; Positioning Attribute; Property; Protein Isoforms; Proteins; Rattus; Research; Role; SCN2A protein; Site; Sudden Death; Sudden infant death syndrome; Syndrome; Testing; Ventricular; base; in vivo; intermolecular interaction; loss of function; mutant; research study; trafficking; voltage

Voltage-gated Nav channel Nav1.5 (encoded by SCN5A) initiates rapid depolarization of the cardiac action potential and is essential for normal cardiac conduction. Human SCN5A mutations may lead to cardiac arrhythmia and sudden death. Nav1.5 function is determined by its channel properties as well as its cellular localization. The identity of the cellular pathway(s) required for Nav1.5 localization at excitable membranes in heart is an important and currently unresolved question. Our long-term goals are to elucidate the cellular pathway(s) and molecular determinants underlying cardiac Nav1.5 targeting. Our specific hypothesis is that ankyrin-G (a membrane adaptor protein) is required for Nav1.5 targeting to intercalated disc and T-tubule membrane domains. We base this hypothesis on previous observations that 1) targeted knockout of ankyrin-G in mouse cerebellum blocks targeting of Nav1.6 and 1.2 in neurons, 2) ankyrin-G binds Nav1.2 through a 9 residue motif on Nav1.2 loop 2, 3) this motif is required Nav1.2 targeting in neurons, and 4) Nav1.5 contains a nearly identical sequence in loop 2. Additionally, our preliminary results support the interaction and co-localization of Nav1.5 and ankyrin-G in heart, and suggest that Nav1.5 requires ankyrin-G-binding for targeting and normal physiological function in humans. Based on these observations, the experiments in this proposal will test a role for an ankyrin-G- based pathway for Nav1.5 targeting in heart. We predict that these experiments will supply the first evidence for a cellular pathway required for cardiac Nav1.5 targeting, and provide in vivo evidence for a new class of human Na 'channelopathies' due to abnormal Nav1.5 targeting. The specific aims are to: 1) Determine the structural requirements for ankyrin-G/Nav1.5 interactions and test human Nav1.5 (SCN5A) disease mutants for ankyrin-G loss-of-binding. 2) Evaluate the requirement of ankyrin-G for Nav1.5 expression, targeting, and function in heart. 3) Characterize the ankyrin-G pathway for Nav1.5 targeting in cardiomyocytes including identification of cellular intermediates in Nav1.5 targeting pathway and identification/characterization of ankyrin-G-interacting proteins for effects on ankyrin-G/ Nav1.5 localization and expression.

电压门控 Nav 通道 Nav1.5（由 SCN5A 编码）启动心脏快速去极化 动作电位，对于正常的心脏传导至关重要。人类SCN5A突变可能导致 心律失常和猝死。 Nav1.5 功能由其通道属性以及 它的细胞定位。 Nav1.5 兴奋定位所需的细胞通路的身份 心脏膜是一个重要且目前尚未解决的问题。我们的长期目标是 阐明心脏 Nav1.5 靶向的细胞途径和分子决定因素。我们的 具体假设是 Nav1.5 靶向需要锚蛋白-G（一种膜接头蛋白） 闰盘和 T 管膜域。我们的假设基于之前的观察 1) 小鼠小脑中锚蛋白-G 的靶向敲除会阻断神经元中 Nav1.6 和 1.2 的靶向， 2) 锚蛋白-G 通过 Nav1.2 环上的 9 个残基基序结合 Nav1.2 2, 3) 该基序是 Nav1.2 所必需的 靶向神经元，4) Nav1.5 在环 2 中包含几乎相同的序列。此外，我们的 初步结果支持 Nav1.5 和锚蛋白-G 在心脏中的相互作用和共定位，并且 表明 Nav1.5 需要锚蛋白-G 结合来实现靶向和正常生理功能 人类。基于这些观察，本提案中的实验将测试锚蛋白-G-的作用 基于 Nav1.5 的心脏靶向途径。我们预测这些实验将提供第一个 心脏 Nav1.5 靶向所需的细胞途径的证据，并提供体内证据 由于异常 Nav1.5 靶向导致的新型人类 Na“通道病”。具体目标是：1） 确定锚蛋白-G/Nav1.5 相互作用的结构要求并测试人类 Nav1.5 (SCN5A) 锚蛋白-G 结合丧失的疾病突变体。 2) 评估 Nav1.5 对锚蛋白-G 的要求 expression, targeting, and function in heart. 3) 表征 Nav1.5 靶向的锚蛋白-G 通路 在心肌细胞中，包括鉴定 Nav1.5 靶向途径中的细胞中间体和 鉴定/表征锚蛋白-G 相互作用蛋白对锚蛋白-G/Nav1.5 的影响 本地化和表达。