Dysfunction in Ankyrin-based Pathways and Human Arrythmia

电压门控Nav通道Nav1.5（由SCN 5A编码）启动心脏快速去极化 动作电位，并且是正常心脏传导所必需的。人类SCN 5A突变可能导致 心律失常和猝死。Nav1.5功能由其通道特性以及 它的细胞定位。Nav1.5定位于可兴奋的神经元所需的细胞途径的身份 心脏的膜是一个重要的和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 具体的假设是，锚蛋白-G（一种膜接头蛋白）是Nav1.5靶向 闰盘和T-小管膜结构域。我们根据先前的观察结果提出了这一假设 1）在小鼠小脑中靶向敲除锚定蛋白-G，阻断神经元中靶向Nav1.6和1.2， 2)锚定蛋白G通过Nav1.2环2上的9个残基序结合Nav1.2，3）该基序是Nav1.2所需的 在神经元中靶向，和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anke-G在心脏中的相互作用和共定位， 这表明Nav1.5需要结合锚定蛋白-G来靶向和正常的生理功能， 人类基于这些观察，本提案中的实验将测试锚-G- 基于心脏中Nav1.5靶向的途径。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， 由于异常的Nav1.5靶向而引起的一类新的人类Na '通道病'。具体目标是：1） 确定锚定蛋白-G/Nav1.5相互作用的结构要求并测试人Nav1.5（SCN 5A） 疾病突变体的抗-G结合丧失。2)评估Nav1.5对ankle-G的要求 在心脏中的表达、靶向和功能。3)表征Nav1.5靶向的锚定-G途径 包括Nav1.5靶向途径中细胞中间体的鉴定， 鉴定/表征锚蛋白G相互作用蛋白对锚蛋白G/ Nav1.5的影响 定位和表达。

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.