NOVEL MECHANISMS LINKING SCN1B TO CARDIAC EXCITABILITY

描述(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由电压门控K+(Kv)通道决定，在大多数心肌细胞中，具有不同时间和电压依赖特性的多Kv电流共同表达。大量Kv通道亚基的形成(1)和附属(2)亚基的鉴定为功能性心肌Kv通道多样性的潜在分子基础提供了重要的见解。此外，越来越多的证据表明，心肌Kv通道是大分子蛋白复合体的组成部分，包括(4个)Kv1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细胞表面功能表达和心肌Kv通道特性方面的作用尚不清楚。这一新的R21建议将检验这一新的假设，即电压门控Na+(Nav)通道附件(NAV2)亚单位调节Kv4编码的快速、瞬时外向Kv(Ito，f)通道的功能表达，而不是或除了调节电压门控Na+(Nav)通道之外。这一假说反映了最近的生化研究结果，表明在天然的Kv4编码的Kv通道大分子蛋白复合体中，Nav21(SCN1b)和Nav22(SCN2b)亚基与Kv4 1亚基共免疫沉淀。这项提议有两个相互关联的目标，这两个目标将同时实现。具体地说，这里的研究将测试新的假设，即Nav21功能调节Kv4编码的心肌Ito，f通道的细胞表面表达和/或特性(目标#1)而不是或除了调节NAV通道(目标2)，并直接确定Nav21在形成心肌动作电位波形中的作用(目标2)。为了达到这些目的，我们将利用小干扰RNA(SiRNAs)的靶向基因“敲除”策略在体外培养的小鼠心室肌细胞中操纵内源性Nav21亚单位的表达，并将确定这些操纵对Ito、f(和Nav)通道的性质和细胞表面表达的功能影响。平行实验将在从含有靶向破坏Scn1b(Nav21)基因的小鼠(Scn1b-/-)分离的心肌细胞上完成。预计这些研究将为控制大分子Kv通道复合体的表达和功能的机制提供新的和根本上重要的见解。此外，这里的研究结果将指导未来的研究，重点是描绘与心肌膜兴奋性的动态调节有关的分子、细胞和系统机制，以及与SCN1b突变相关的心脏兴奋性的紊乱。 与公众健康相关：电压门控钾(Kv)通道控制心肌动作电位的高度和持续时间，并对正常心律的产生起重要作用。在许多遗传性和获得性心脏病中可以观察到Kv通道表达和/或特性的变化，这些变化可以产生深刻的生理后果，包括增加潜在威胁生命的心律失常的风险。虽然越来越多的证据表明，心肌Kv通道是大分子蛋白复合体的组成部分，包括影响通道稳定性、转运和/或特性的成孔(1)亚基和各种辅助(2)亚基，但目前对辅助亚基在心肌细胞Kv通道的生理调节中的作用知之甚少。这一新的研究计划利用分子遗传学策略在体内和体外操纵通道亚基，重点在于确定NAV_2(SCNxb)辅助亚基在调节心肌细胞兴奋性中的生理作用(S)，并检验NAV_2辅助亚基调节Kv通道而不是或除了调节电压门控Na+(Nav)通道之外的功能的新假说。这些研究将为NAV2亚单位在心肌中的生理作用和控制心肌膜兴奋性的分子机制提供新的和根本上重要的见解。

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.