Intermolecular Interactions of NaV1.5 and Kir2.1 In Ion Channel Diseases

NaV1.5 和 Kir2.1 在离子通道疾病中的分子间相互作用

• 批准号: 9173051
• 负责人: Jose S Jalife
• 金额: $ 47.46万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-11-14 至 2018-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9173051
• 关键词: Action Potentials; Acute; Affect; Arrhythmia; Atrial Fibrillation; Barium; Binding; Brugada syndrome; C-terminal; Cardiac; Cardiac Myocytes; Caring; Cell membrane; Cells; Closure by clamp; Code; Complement; Complex; Computer Simulation; Confocal Microscopy; Consensus Sequence; DLG1 gene; Dilated Cardiomyopathy; Disease; Dissection; Dystrophin-Associated Proteins; Electrophysiology (science); Endocytosis; Endoplasmic Reticulum; Fluorescence; Fluorescence Recovery After Photobleaching; Functional disorder; Gene Silencing; Gene Transfer; Genes; Genetic; Golgi Apparatus; Health; Heart Diseases; Heart failure; Human; Inherited; Ion Channel; Ion Channel Protein; Joints; Life; Link; Macromolecular Complexes; Mediating; Medical; Membrane; Modification; Molecular; Mutagenesis; Mutation; Optics; Pathway interactions; Phenotype; Play; Population; Post-Translational Protein Processing; Process; Property; Proteins; Proteomics; Publishing; Recycling; Regulation; Rest; Role; SCN1A protein; Scaffolding Protein; Sick Sinus Syndrome; Signal Transduction; Sodium Channel; Sudden infant death syndrome; Surface; Syndrome; System; Testing; Two-Hybrid System Techniques; Ventricular; Viral; Work; Yeasts; density; domain mapping; experimental study; fluorescence imaging; genetic variant; immunocytochemistry; improved; indium arsenide; induced pluripotent stem cell; intermolecular interaction; inward rectifier potassium channel; live cell imaging; loss of function mutation; membrane-associated guanylate kinase; monolayer; mutant; patch clamp; protein E; protein complex; protein protein interaction; protein purification; protein transport; public health relevance; scaffold; simulation; syntrophin; tool; trafficking; virtual; voltage

DESCRIPTION (provided by applicant): This proposal focuses on the mechanisms and electrophysiological consequences of the molecular interactions between the inward rectifier potassium channel protein Kir2.1 and the �ubunit of the major cardiac sodium channel NaV1.5. Our preliminary results strongly suggest that NaV1.5 and Kir2.1 modulate each other's surface expression and function through their respective PDZ binding domains within a macromolecular complex to control cardiac excitability. Such a dynamic reciprocity is post-translational, involving, at least in part, mutual regulation of trafficking and targeting of both channel proteins at common membrane compartments, as well as internalization. We focus on inheritable mutations that are known to disrupt trafficking of NaV1.5 (Brugada Syndrome, BS) or Kir2.1 (Andersen-Tawil Syndrome, ATS). We surmise that a mutation that disrupts the expression of one channel protein type (e.g., NaV1.5) will also affect the other type (e.g., Kir2.1 by disturbing the common macromolecular complex through which they interact, thus contributing to both the electrophysiological phenotype and arrhythmogenic potential. We will test the following three major hypotheses: 1) NaV1.5 and Kir2.1 protein channels undergo PDZ-domain mediated interactions with common partners in a macromolecular complex that controls their membrane stability; 2) macromolecular complex formation affects anterograde and/or retrograde trafficking of Kir2.1 and NaV1.5; and 3) human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CMs) expressing either ATS or BS mutations that affect protein trafficking will show reduced excitability reflecting altered expression of both channel proteins, which should contribute strongly to the inherited arrhythmia phenotype. We propose to combine proteomics (e.g., protein purification, yeast two-hybrid assay and interaction domain mapping) and genetic (e.g., mutagenesis and silencing) tools, confocal microscopy, live cell imaging, fluorescence recovery after photobleaching, patch clamping, optical mapping, gene transfer and silencing in heterologous systems, and in single, highly mature ventricular-like hiPSC-CMs and hiPSC-CM monolayers. We will also conduct computer simulations to enable the virtual dissection and interpretation of the electrophysiological and arrhythmogenic changes resulting from NaV1.5-Kir2.1 interactions and their mutants in a macromolecular complex.

描述(由申请人提供)：这项建议集中于内向整流钾通道蛋白Kir2.1和主要心脏钠通道NaV1.5的�亚单位之间的分子相互作用的机制和电生理后果。我们的初步结果强烈表明，NaV1.5和Kir2.1通过各自在大分子复合体中的PDZ结合结构域来调节彼此的表面表达和功能，以控制心脏的兴奋性。这种动态的相互作用是翻译后的，至少部分涉及两个通道蛋白在共同的膜室的运输和靶向的相互调节，以及内化。我们专注于已知能中断NaV1.5(Brugada综合征，BS)或Kir2.1(Andersen-Tawil综合征，ATS)贩运的可遗传突变。我们推测，破坏一种通道蛋白类型(例如NaV1.5)表达的突变也会影响另一种类型(例如Kir2.1)，因为它干扰了它们相互作用的常见大分子复合体，从而导致电生理表型和致心律失常的可能性。我们将检验以下三个主要假设：1)NaV1.5和Kir2.1蛋白通道与控制其膜稳定性的大分子复合体中的共同合作伙伴进行PDZ结构域介导的相互作用；2)大分子复合体的形成影响Kir2.1和NaV1.5的顺行和/或逆行运输；以及3)人诱导的多能干细胞来源的心肌细胞(hiPSC-CMS)表达ATS或BS突变，影响蛋白质运输，将显示出较低的兴奋性，反映两种通道蛋白表达的变化，这应该是遗传性心律失常的重要原因。我们建议将蛋白质组学(例如蛋白质纯化、酵母双杂交分析和相互作用结构域定位)和遗传(例如突变和沉默)工具、共聚焦显微镜、活细胞成像、光漂白后的荧光恢复、膜片钳、光学作图、基因转移和沉默结合在一起，在异源系统中，以及在单个、高度成熟的类似脑室的HiPSC-CMS和HiPSC-CM单层中。我们还将进行计算机模拟，以便能够虚拟解剖和解释由NaV1.5-Kir2.1相互作用和它们在大分子复合体中的突变体引起的电生理和致心律失常的变化。