NOVEL MECHANISMS LINKING SCN1B TO CARDIAC EXCITABILITY

描述（由申请人提供）：心脏动作电位的幅度和持续时间主要由电压门控K+（Kv）通道决定，并且在大多数心脏细胞中，共同表达具有不同时间和电压依赖性特性的多种Kv电流。大量Kv通道孔形成（1）和辅助（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见解。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个）Kv 1和多个Kv 2亚基以及调节蛋白，尽管对辅助蛋白和调节蛋白在控制心肌Kv通道功能性细胞表面表达和特性中的作用知之甚少。这个新的R21提案将测试新的假设，电压门控Na+（Nav）通道辅助（Nav 2）亚基调节心室肌细胞中Kv 4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。该假设反映了最近的生物化学发现，其表明Nav 21（SCN 1b）和Nav 22（SCN 2b）亚基与天然Kv 4编码的Kv通道大分子蛋白复合物中的Kv 41亚基共免疫沉淀。这项建议有两个相关的目标，将同时进行。具体而言，本文的研究将测试新的假设，即Nav 21的功能是调节细胞表面表达和/或Kv 4编码的心肌Ito，f通道的性质（目的#1），而不是调节Nav通道（目的#2），或者除了调节Nav通道之外（目的#2），并直接确定Nav 21在塑造心肌动作电位波形中的作用。(aim#2）。为了实现这些目标，内源性Nav 21亚基的表达将在体外（小鼠）心室肌细胞中使用靶向基因“敲低”策略与小干扰RNA（siRNA）进行操作，并将确定这些操作对Ito，f（和Nav）通道的性质和细胞表面表达的功能后果。将对从携带Scn 1b（Nav 21）基因座靶向破坏的小鼠（Scn 1b-/-）中分离的肌细胞完成平行实验。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，重点是描绘参与心肌膜兴奋性的动态调节和与SCN 1b突变相关的心脏兴奋性紊乱的分子，细胞和全身机制。 公共卫生相关性：电压门控性钾通道控制心肌动作电位的高度和持续时间，对正常心律的产生有重要作用。在许多遗传性和获得性心脏病中观察到Kv通道表达和/或特性的变化，并且这些变化可能具有深刻的生理后果，包括增加潜在危及生命的心律失常的风险。尽管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，包括孔形成（1）亚基和各种影响通道稳定性、运输和/或特性的辅助（2）亚基，但目前对辅助亚基在心肌细胞Kv通道生理调节中的作用知之甚少。利用分子遗传学策略在体内和体外操纵通道亚基，该新的研究计划专注于定义Nav 2（SCNxb）辅助亚基在调节心肌细胞兴奋性中的生理作用，并测试Nav 2辅助亚基调节Kv通道而不是调节电压门控Na+（Nav）通道或除此之外调节Kv通道的新假设。这些研究将为Nav 2亚基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性的重要见解。

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.