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）：心脏作用电位的幅度和持续时间在很大程度上由电压门控的K+（KV）通道确定，在大多数心脏细胞中，多个具有不同时间和电压依赖性特性的KV电流共表达。提供了对功能性心肌KV通道多样性的潜在分子基础的重要见解，并鉴定了大量KV通道孔形成（1）和附件（2）亚基。此外，积累的证据表明，心肌KV通道起着大分子蛋白络合物的组成部分，包括（四个）KV 1和多个KV2亚基和调节蛋白，尽管配件和调节蛋白在控制功能细胞表面表达和性质较差的肌肉核心kv的特性中的作用是肌无知的kive蛋白的作用。该新的R21提案将检验一个新的假设：电压门控的Na+（NAV）通道附件（NAV2）亚基调节KV4在心室心肌细胞中的快速，瞬态外向KV（ITO，F）通道的功能表达，而不是调节电压的Na+（NA+（NA）。该假设反映了最新的生化发现，表明NAV21（SCN1B）和NAV22（SCN2B）亚基与天然KV4 KV4 KV4 KV通道大分子大分子蛋白复合物中的KV4 1亚基共沉淀。该提案中有两个相关的目标，这些目标将同时实现。具体而言，此处的研究将检验以下新的假设，即NAV21起作用，可以调节细胞表面表达和/或KV4编码的心肌ITO的特性，F通道（AIM＃1），而不是（AIM＃1），或者除了调节NAV NAV频道（AIM＃2）（AIM＃2）（AIM＃2）并直接确定NAV21在塑造Mocartial Exportial Posity Waver Provical Waver Prove Wavel（AIM＃2）中的作用。为了实现这些目的，使用针对性的基因“敲低”策略在体外（小鼠）心室心肌细胞中操纵内源性NAV21亚基的表达，而具有小的干扰RNA（siRNAS）的“敲低”策略，并且这些操作对ITO，f（and and）的细胞表面表达对这些操作的功能后果将是F（and f（nav）和NAV（NAV）。并行实验将在构成SCN1B（NAV21）基因座的靶向破坏的小鼠（SCN1B - / - ）中分离的肌细胞上完成。预计这些研究将为控制大分子分子KV通道复合物的表达和功能的机制提供新的和根本上重要的见解。此外，此处的研究结果将指导未来的研究，重点是描述与心肌膜兴奋性动态调节有关的分子，细胞和全身机制以及与SCN1B突变有关的心脏兴奋性的危险。 公共卫生相关性：电压门控钾（KV）通道控制心肌动作电位的高度和持续时间，并重要地贡献了正常心律的产生。在许多遗传和获得的心脏病中观察到KV通道表达和/或特性的变化，这些变化可能会带来深远的生理后果，包括增加潜在威胁生命的心律不齐的风险。尽管积累的证据表明，心肌KV通道充当大分子蛋白络合物的组成部分，包括孔形成（1）亚基和各种附件（2）亚基（2）亚基，影响通道稳定性，贩运和/或性质，目前对附属亚基在物理学监管中的角色几乎不知所措，kv in kv of kv of kv of kv ins of k.v consectiact card card card card card card card sv sv sv sv sv sv sv sv sv的疾病。该新的研究计划利用分子遗传学策略来操纵渠道亚基，重点是定义NAV2（SCNXB）附属亚基在调节心肌兴奋性中的生理作用，并在测试NAV2辅助量的新型假设中，而不是测试NAV2辅助量的新型假设。门控Na+（NAV）通道。这些研究将提供有关NAV2亚基在心肌中的生理作用以及控制心肌膜兴奋性的分子机制的新的重要见解。