Novel Mechanisms of Cardiac Ion Channel Regulation

描述（由申请人提供）：心血管疾病仍然是美国死亡和残疾的主要原因。死亡最常见的是室性心律失常的直接结果，而室性心律失常又是离子通道功能改变的结果，影响动作电位和细胞间通讯。衰竭心脏中改变的离子通道功能也限制了心脏的收缩力，进一步恶化心力衰竭和心律失常和死亡的恶性循环。单个离子通道的半衰期大约为几个小时，在衰竭的心脏中改变离子通道功能的主要原因是改变了运输。改变的运输意味着通道没有被递送到心肌细胞质膜上的正确子域（正向运输），或者被不正确地去除（反向运输或内化）。改变的正向和反向运输是大多数心脏疾病的组成部分。然而，膜蛋白，特别是心脏离子通道的正向和反向运输机制仍然知之甚少。 我的实验室探索心脏离子通道运输的基本机制。在这次竞争性更新中，我们将确定三种新的途径，每种途径都有强大的初步数据。感兴趣的通道是负责细胞间通讯的连接蛋白43间隙连接通道和L型钙通道（α亚基Cav1.2），后者负责触发心跳间细胞内钙瞬变，启动并帮助确定每次心脏收缩的强度。所探讨的三条途径中的每一条都是本提案的一个单独的具体目标。 目的#1是确定替代翻译是否是健康和疾病中Cx43自动调节前向流量的机制。我们在人类心脏中有证据表明，多达六个符合读框的AUG序列在Cx43（GJA 1）外显子内启动翻译。我们的数据表明，截短的Cx43亚型是必要的，至少Cx43退出内质网和高尔基体。 目的#2是确定肌动蛋白细胞骨架如何有助于定向通道传递。我们的研究表明，由于与肌动蛋白的结合，大多数细胞内Cx43移动缓慢或根本不移动。我们已经开发了一种新的基于微流体的细胞图案化系统来量化肌动蛋白动力学及其对离子通道运输的贡献，并且发现肌动蛋白对于与微管一起提供Cx43和Cav1.2递送的特异性是重要的。 目的#3是阐明Cx43从质膜内化的翻译后级联反应。我们有证据表明，Cx43 C-末端的丝氨酸373磷酸化需要先于丝氨酸368磷酸化。我们的新数据将探索磷酸化依赖的内化机制以及是否涉及替代翻译的产物。

DESCRIPTION (provided by applicant): Cardiovascular disease remains the primary cause of death and disability in the United States. Death is most commonly a direct result of ventricular arrhythmia which, in turn, is the consequence of alternations in ion channel function, affecting action potentials and cell-cell communication. Altered ion channel function in failing hearts also limits the heart's contractility, further worsening heart failure and the vicious cycle towards arrhythmia and death. Individual ion channels have half-lives that are on the order of hours, and a major cause of altered ion channel function in failing hearts is altered trafficking. Altered trafficking implies that the channels are not delivered to the correct subdomain on the cardiomyocyte plasma membrane (forward trafficking), or are removed incorrectly (reverse trafficking or internalization). Altered forward and reverse trafficking are components of most cardiac disorders. However the mechanisms of forward and reverse trafficking of membrane proteins, especially of cardiac ion channels, remain poorly understood. My laboratory explores the basic mechanisms of cardiac ion channel trafficking. In this competitive renewal, we will identify three novel pathways with strong preliminary data for each. The channels of interest are the Connexin43 gap junction channel responsible for cell-cell communication, and the L-type Calcium Channel (alpha subunit Cav1.2), which is responsible for triggering the beat-to- beat intracellular calcium transient which initiates and helps determine the strength of each cardiac contraction. Each of the three pathways explored is a separate specific aim of this proposal. Aim #1 is to determine if alternative translation is a mechanism of Cx43 auto regulation of forward traffic in health and disease. We have evidence in human heart that as many as six in frame AUG sequences initiate translation within the Cx43 (GJA1) exon. Our data indicate the truncated Cx43 isoforms are necessary at least for Cx43 exit from the endoplasmic reticulum and Golgi apparatus. Aim #2 is to determine how the actin cytoskeleton contributes to directed channel delivery. Our studies indicate that the majority of intracellular Cx43 is moving slowly or not moving at all, due to association with actin. We have developed a novel microfluidics based cell patterning system to quantify actin dynamics and its contribution to ion channel trafficking and are finding that actin is important to working with microtubules in providing specificity of Cx43 and Cav1.2 delivery. Aim #3 is to elucidate the post-translation cascade by which Cx43 is internalized from the plasma membrane. We have evidence that serine 373 phosphorylation on the Cx43 C-terminus needs to precede serine 368 phopsphorylation. Our new data will explore mechanism of phosphorylation dependent internalization and whether the products of alternative translation are involved.