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

基本信息

批准号：
8816386
负责人：
Jose S Jalife
金额：
$ 48.99万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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/8816386
关键词：
Action Potentials Acute Affect Arrhythmia Atrial Fibrillation Barium Binding C-terminal Cardiac Cardiac Myocytes Caring Cell membrane Cells Code Complex Computer Simulation Confocal Microscopy Consensus Sequence DLG1 gene Disease Dissection Dystrophin-Associated Protein Complex 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 Maps Mediating Medical Membrane Membrane Protein Traffic Modification Molecular Mutagenesis Mutation Optics Pathway interactions Phenotype Play Population Post-Translational Protein Processing Process Property Proteins Proteomics Publishing Recycling Regulation Rest Role 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 cellular imaging density domain mapping fluorescence imaging genetic variant immunocytochemistry improved induced pluripotent stem cell intermolecular interaction inward rectifier potassium channel loss of function mutation membrane-associated guanylate kinase monolayer mutant patch clamp protein protein interaction protein purification protein transport public health relevance research study scaffold simulation syntrophin tool trafficking virtual voltage

项目摘要

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）：本提案重点关注内向整流钾通道蛋白 Kir2.1 和主要心脏钠通道 NaV1.5 的 ubunit 之间的分子相互作用的机制和强烈的电生理学后果。 NaV1.5 和 Kir2.1 通过大分子复合物内各自的 PDZ 结合域调节彼此的表面表达和功能，以控制心脏兴奋性。翻译后，至少部分涉及运输的相互调节和共同膜区室中两种通道蛋白的靶向以及内化，我们重点关注已知会破坏 NaV1.5 运输的遗传突变（布鲁格达综合症， BS）或 Kir2.1（Andersen-Tawil 综合征，ATS）我们推测，破坏一种通道蛋白类型（例如 NaV1.5）表达的突变会导致这种突变。也影响其他类型（例如，Kir2.1，通过干扰它们相互作用的共同大分子复合物，从而促进电生理表型和致心律失常电位。我们将测试以下三个主要假设：1）NaV1.5和Kir2。 1 蛋白质通道与大分子复合物中的共同伙伴发生 PDZ 结构域介导的相互作用，其膜稳定性 2) 大分子复合物的形成影响对照的顺行和/或逆行运输； Kir2.1 和 NaV1.5；以及 3) 表达影响蛋白质运输的 ATS 或 BS 突变的人诱导多能干细胞来源的心肌细胞 (hiPSC-CM) 将表现出兴奋性降低，反映出两种通道蛋白表达的改变，这应有助于我们建议将蛋白质组学（例如蛋白质纯化、酵母双杂交测定和相互作用域作图）和遗传（例如诱变）结合起来。和沉默）工具、高度共焦显微镜、活细胞成像、光漂白后的荧光恢复、膜片钳、光学图谱、异源系统中以及单个成熟的心室样 hiPSC-CM 和 hiPSC-CM 单层中的基因转移和沉默。还进行计算机模拟，以实现对 NaV1.5-Kir2.1 相互作用及其引起的电生理和心律失常变化的虚拟解剖和解释大分子复合物中的突变体。