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（由适用提供）：该提案重点介绍了内向整流器钾通道蛋白KIR2.1与主要心脏钠通道NAV1.5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通过大分子复合物中各自的PDZ结合域调节对方的表面表达和功能，以控制心脏兴奋性。这种动态互惠是翻译后的，至少部分涉及对两种通道蛋白在常见膜室中的运输和靶向的相互调控，以及内在化。我们专注于已知会破坏NAV1.5（Brugada综合征，BS）或Kir2.1（Andersen-Tawil综合征，ATS）的遗传突变。我们浏览了一个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，通过干扰它们相互作用的常见的大分子复合物，从而通过它们相互作用，从而有助于电生理生理学表型和心律失常的潜在。 PDZ域介导的与共同伴侣的相互作用在控制其膜稳定性的大分子复合物中； 2）大分子复合物的形成会影响Kir2.1和NAV1.5的顺行和/或逆行运输； 3）人类诱导的多能干细胞衍生的心肌细胞（HIPSC-CMS）表达影响蛋白质运输的ATS或BS突变，将显示出反映两种通道蛋白表达的改变的兴奋反射率，这应该对遗传性心律失常表型有很大贡献。我们还将进行计算机模拟，以实现由NAV1.5-KIR2.1相互作用及其突变体在大分子复合物中产生的电生理和心律失常变化的虚拟解剖和解释。