NOVEL MECHANISMS LINKING SCN1B TO CARDIAC EXCITABILITY

连接 SCN1B 与心脏兴奋性的新机制

• 批准号: 8020039
• 负责人: JEANNE M. NERBONNE
• 金额: $ 19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-03 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8020039
• 关键词: Action Potentials; Affect; Arrhythmia; Biochemical; Cardiac; Cardiac Myocytes; Cell surface; Cells; Complex; Data; Electrophysiology (science); Future; Gene Targeting; Generations; Heart Diseases; Height; In Vitro; Inherited; Investigation; Ion Channel; Kv channel-interacting protein 2; Life; Link; Macromolecular Complexes; Membrane; Molecular; Molecular Genetics; Multiprotein Complexes; Mus; Muscle Cells; Mutation; Myocardial; Myocardium; Physiological; Potassium; Property; Proteins; Regulation; Research; Risk; Role; Shapes; Small Interfering RNA; Testing; Time; Ventricular; base; density; genetic regulatory protein; heart rhythm; in vivo; insight; novel; programs; public health relevance; research study; trafficking; voltage

DESCRIPTION (provided by applicant): The amplitudes and durations of cardiac action potentials are largely determined by voltage-gated K+ (Kv) channels, and in most cardiac cells, multiple Kv currents with distinct time- and voltage-dependent properties are co-expressed. Important insights into the potential molecular basis of functional myocardial Kv channel diversity were provided with the identification of large numbers of Kv channel pore-forming (1) and accessory (2) subunits. In addition, accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising (four) Kv 1 and multiple Kv2 subunits and regulatory proteins, although the roles of accessory and regulatory proteins in controlling the functional cell surface expression and the properties of myocardial Kv channel are poorly understood. This new R21 proposal will test the novel hypothesis that voltage-gated Na+ (Nav) channel accessory (Nav2) subunits regulate the functional expression of Kv4-encoded fast, transient outward Kv (Ito,f) channels in ventricular myocytes rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. This hypothesis reflects recent biochemical findings demonstrating that the Nav21 (SCN1b) and Nav22 (SCN2b) subunits co- immunoprecipitate with Kv4 1 subunits in native Kv4-encoded Kv channel macromolecular protein complexes. There are two related aims in this proposal, and these will be pursued in parallel. Specifically, the studies here will test the novel hypothesis that Nav21 functions to regulate the cell surface expression and/or the properties of Kv4-encoded myocardial Ito,f channels (aim #1) rather than, or in addition to, regulating Nav channels (aim #2) and determine directly the role of Nav21 in shaping myocardial action potential waveforms.(aim #2). To achieve these aims, the expression of the endogenous Nav21 subunit will be manipulated in (mouse) ventricular myocytes in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of Ito,f (and Nav) channels will be determined. Parallel experiments will be completed on myocytes isolated from mice (Scn1b-/- ) harboring a targeted disruption of the Scn1b (Nav21) locus. It is anticipated that these studies will provide new and fundamentally important insights into the mechanisms that control the expression and the functioning of macromolecular Kv channel complexes. In addition, the results of the studies here will guide future investigations focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of myocardial membrane excitability and in the derangements in cardiac excitability linked to mutations in SCN1b. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels control the heights and durations of myocardial action potentials and contribute importantly the generation of normal cardiac rhythms. Changes in Kv channel expression and/or properties are observed in a number of inherited and acquired cardiac diseases, and these changes can have profound physiological consequences, including increasing the risk of potentially life-threatening cardiac arrhythmias. Although accumulating evidence suggests that myocardial Kv channels function as components of macromolecular protein complexes, comprising pore-forming (1) subunits and a variety of accessory (2) subunits that affect channel stability, trafficking and/or properties, very little is presently known about the roles of accessory subunits in the physiological regulation of Kv channels in cardiac myocytes. Exploiting molecular genetics strategies to manipulate channel subunits in vivo and in vitro, this new research program is focused on defining the physiological role(s) of the Nav2 (SCNxb) accessory subunits in regulating the excitability of cardiac myocytes and on testing the novel hypothesis that the Nav2 accessory subunits function to regulate Kv channels rather than, or in addition to, regulating voltage- gated Na+ (Nav) channels. These studies will provide new and fundamentally important insights into the physiological roles of Nav2 subunits in the myocardium and into the molecular mechanisms controlling myocardial membrane excitability.

描述（由申请人提供）：心脏动作电位的幅度和持续时间在很大程度上是由电压门控的K+ (Kv)通道决定的，在大多数心脏细胞中，具有不同时间和电压依赖特性的多个Kv电流共同表达。通过鉴定大量Kv通道成孔(1)和辅助(2)亚基，对心肌功能Kv通道多样性的潜在分子基础有了重要的认识。此外，越来越多的证据表明，心肌Kv通道作为大分子蛋白复合物的组成部分发挥作用，包括(4)Kv 1和多个Kv2亚基和调节蛋白，尽管辅助蛋白和调节蛋白在控制功能性细胞表面表达和心肌Kv通道特性中的作用尚不清楚。这项新的R21提案将验证新的假设，即电压门控Na+ (Nav)通道附属（Nav2）亚基调节心室肌细胞中kv4编码的快速、瞬时外向Kv （Ito,f）通道的功能表达，而不是调节电压门控Na+ (Nav)通道。这一假设反映了最近的生化研究结果，表明在天然Kv4编码的Kv通道大分子蛋白复合物中，Nav21 （SCN1b）和Nav22 （SCN2b）亚基与Kv4 1亚基共免疫沉淀。这项建议有两个相关的目标，这两个目标将并行进行。具体来说，这里的研究将验证Nav21的功能是调节细胞表面表达和/或kv4编码的心肌Ito，f通道（目的1）的特性，而不是，或者除了调节Nav通道（目的2），并直接确定Nav21在塑造心肌动作电位波形中的作用。(目标# 2)。为了实现这些目标，内源性Nav21亚基的表达将在体外（小鼠）心室肌细胞中使用小干扰rna （sirna）靶向基因“敲低”策略进行操作，并确定这些操作对Ito，f（和Nav）通道的特性和细胞表面表达的功能后果。平行实验将在从小鼠（Scn1b-/-）分离的肌细胞上完成，这些肌细胞含有Scn1b （Nav21）位点的靶向破坏。预计这些研究将为控制大分子Kv通道复合物的表达和功能的机制提供新的和根本性的重要见解。此外，这里的研究结果将指导未来的研究，重点是描述心肌膜兴奋性动态调节以及与SCN1b突变相关的心脏兴奋性紊乱的分子、细胞和系统机制。