Dysfunction in Ankyrin-based Pathways and Human Arrythmia

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，对正常的心脏传导是必不可少的。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定位所需的细胞通路(S)在可兴奋状态下的同一性 心脏的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(S)和分子决定因素。我们的 具体的假设是，锚蛋白-G(一种膜适配器蛋白)是NaV1.5靶向到 插入盘状区和T管膜区。我们把这一假设建立在先前观察的基础上。 1)靶向敲除小鼠小脑中的Ankyrin-G阻断了Nav1.6和1.2在神经元中的靶向， 2)Ankyrin-G通过Nav1.2环上的9个残基与Nav1.2结合，3)该基序是必需的Nav1.2 定位于神经元，以及4)NaV1.5在环路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Ankyrin-G在心脏中的相互作用和共定位，以及 提示NaV1.5需要Ankyrin-G结合来靶向和正常的生理功能 人类。在这些观察的基础上，本方案中的实验将测试Ankyrin-G- 基于NAV1.5在心脏靶向的途径。我们预测，这些实验将提供第一个 证明心脏NAV1.5靶向所需的细胞途径，并提供体内证据 由于NaV1.5靶向异常，人类出现了新的人类钠“通道病”。具体目标是：1) 确定Ankyrin-G/NaV1.5相互作用的结构要求并测试人NaV1.5(SCN5A) Ankyrin-G结合缺失的疾病突变体。2)评估锚蛋白-G对NaV1.5的要求 在心脏中表达、靶向和功能。3)鉴定针对NaV1.5靶向的Ankyrin-G途径 在心肌细胞中，包括鉴定NAV1.5靶向通路的细胞中间体和 锚蛋白-G相互作用蛋白的鉴定及其对锚蛋白-G/NaV1.5的影响 本土化和表现力。

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.