Novel Mechanisms of Cardiac Ion Channel Regulation

心脏离子通道调节的新机制

• 批准号: 8966027
• 负责人: Robin M Shaw
• 金额: $ 42.5万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2018-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966027
• 关键词: Actins; Action Potentials; Adult; Affect; American; Arrhythmia; Award; Biochemical; Biological Phenomena; Biology; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cause of Death; Cell Communication; Cell membrane; Cells; Cessation of life; Code; Connexin 43; Critical Pathways; Cytoskeleton; Data; Disease; Endoplasmic Reticulum; Exons; Genes; Goals; Golgi Apparatus; Health; Heart; Heart Contractilities; Heart Diseases; Heart failure; Hour; Human; Individual; Intercalated disc; Ion Channel; Knowledge; L-Type Calcium Channels; Laboratories; Lead; Life Cycle Stages; Location; Malignant - descriptor; Mechanics; Medical; Membrane; Membrane Protein Traffic; Methods; Microfluidics; Microscopy; Microtubules; Molecular; Morbidity - disease rate; Movement; Mus; Muscle Cells; Outcome; Pathologic; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; Recruitment Activity; Regulation; Research; Rest; Scaffolding Protein; Serine; Site; Sorting - Cell Movement; Specificity; Stress; Structure; Syndrome; System; Testing; Therapeutic Intervention; Translation Initiation; Translations; United States; United States National Institutes of Health; Ventricular Arrhythmia; Vesicle; Work; base; cellular targeting; disability; experience; gap junction channel; improved; insight; interest; mortality; novel; novel diagnostics; novel marker; novel therapeutic intervention; novel therapeutics; protein kinase C epsilon; research study; sudden cardiac death; therapeutic development; tool; trafficking

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.

描述(申请人提供)：心血管疾病仍然是美国人死亡和残疾的主要原因。死亡通常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。衰竭心脏中离子通道功能的改变也限制了心脏的收缩能力，进一步恶化了心力衰竭，并导致心律失常和死亡的恶性循环。单个离子通道的半衰期约为几个小时，而心力衰竭时离子通道功能改变的一个主要原因是改变的运输。改变的贩运意味着通道没有被传递到心肌细胞质膜上正确的亚区(正向贩运)，或者被错误地移除(反向贩运或内化)。改变的正向和反向贩运是大多数心脏疾病的组成部分。然而，膜蛋白的正向和反向转运机制，特别是心脏离子通道的机制，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。在这场竞争性的更新中，我们将确定三条具有强大初步数据的新途径。感兴趣的通道是负责细胞间通讯的缝隙连接通道Cx43和L钙通道(α亚基Cav1.2)，后者负责触发细胞内钙瞬变，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。第一个目标是确定替代翻译是否是Cx43在健康和疾病中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6个AUG帧序列在Cx43(GJA1)外显子内启动翻译。我们的数据表明，截短的Cx43亚型至少是Cx43离开内质网和高尔基体所必需的。目的2是确定肌动蛋白细胞骨架如何参与定向通道传递。我们的研究表明，由于与肌动蛋白有关，大多数细胞内的Cx43运动缓慢或根本不运动。我们已经开发了一种新的基于微流体的细胞模式系统来量化肌动蛋白的动力学及其对离子通道运输的贡献，并发现肌动蛋白在提供Cx43和Cav1.2递送的特异性方面对于与微管一起工作是重要的。目的#3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依赖磷酸化的内化机制，以及是否涉及替代翻译的产物。