The Role of the Sodium Channel Beta Subunit in Cardiac Conduction

摘要：本项目旨在描述心脏动作电位（AP）的新机制 传导和开发新的方法来治疗心律失常的基础上获得的见解。我们已经报道 Na+通道的Nav1.5亚基集中在Cx43（Gja 1）间隙连接（GJ）边缘， 称为perinexus的夹层盘纳米结构域。高浓度的Na+通道和狭窄的内部通道， 在perinexus的膜宽度与理论预测的结构单元一致， 支持视神经传导。所检验的假设是Na+通道复合物的β亚基是 这个单位的核心组织元素：厄菲斯。我们的前提是，β1（Scn 1b）的粘附函数 促进在插盘内形成膜间接触的专门区域。这 设想这种布置能够使反式相互作用的Na+通道在肌细胞之间形成， AP的细胞间传导。此外，我们提出，激动剂，维持ephapse粘附， 构成减轻心肌梗塞后的致心律失常传导缺陷的新疗法。在 支持的假设，数据提供了从超分辨率，电子和免疫电子显微镜 和智能膜片钳（SPC）研究表明，Nav1.5和β1亚基，以及活性Na+通道， 集中在perinexus。此外，我们还开发了一种新的β1抑制剂--一种19个氨基酸的模拟物 β1粘附结构域（β adp 1）。在电细胞基质阻抗传感试验中，β adp 1引起 异源过表达β1的细胞之间的粘附丧失。向心脏内注入β ADP 1， 剂量依赖性：（1）与β1去粘附一致的会阴裂增宽;（2）传导减慢; （3）室性心律失常发生率增加。重要的是，虽然β1粘附的抑制似乎 SPC对全细胞Na+电流无影响，但对GJ相关Na+通道有选择性抑制作用 活动最后，初步的数据显示了一个合理设计的激动剂β1介导的反式相互作用 (dbl-βADP），其促进β1过表达细胞之间的粘附。为了检验假设：（1）β1是 β1-介导的粘附是一种抑制细胞膜上Na+通道活性的机制。 抗肿瘤靶点：目标1将确定假设的肝纤维化对β1和Cx43 GJ的需求。 机制编码Scn 1b和Gja 1无效等位基因以及β1跨膜粘附抑制剂β adp 1的小鼠， 将被用于研究的结构和自主功能的假设ephapse。目标2将 测定β1粘附对心肌细胞Na+通道活性、重构和AP传导的作用 单层。将在策略中使用GJ空斑形成但功能不全的Cx43突变体（L90 V） 以研究不依赖于GJ耦合的肝纤维化机制对传导的贡献。目标3将 基于我们的第一代激动剂dbl-βadp开发稳定β1粘附的有效分子，并测试 这些化合物在急性心肌梗塞的促心律失常情况下的抗心律失常功效。

ABSTRACT: This project seeks to characterize a novel mechanism of cardiac action potential (AP) conduction and develop new approaches to treat arrhythmias based on the insights gained. We have reported that the Nav1.5 subunit of the Na+ channel is concentrated at the Cx43 (Gja1) gap junction (GJ) edge in an intercalated disk nanodomain called the perinexus. The high concentration of Na+ channels and narrow inter- membrane width at the perinexus are consistent with theoretical predictions of a structural unit that may support ephaptic conduction. The hypothesis tested is that the β subunit of the Na+ channel complex is the central organizing element of this unit: The ephapse. Our premise is that the adhesion function of β1 (Scn1b) promotes the formation of specialized regions of inter-membrane contact within intercalated disks. This arrangement is envisaged as enabling trans-interacting Na+ channels to form between myocytes, facilitating cell-to-cell conduction of AP. Furthermore, we propose that agonists that maintain ephapse adhesion could constitute new therapies to mitigate arrhythmogenic conduction defects following myocardial infarction. In support of the hypothesis, data is provided from super-resolution, electron, and immuno-electron microscopy and smart patch clamp (SPC) studies that Nav1.5 and β1 subunits, as well as active Na+ channels, are concentrated in the perinexus. Moreover, we have developed a novel β1 inhibitor - a 19 amino acid mimetic of the adhesion domain of β1 (βadp1). In electric cell-substrate impedance sensing assays, βadp1 caused loss of adhesion between cells heterologously over-expressing β1. Infusion of βadp1 into hearts resulted in dose-dependent: (1) Widening of the perinexal cleft consistent with β1 de-adhesion; (2) Conduction slowing; and (3) Increased ventricular arrhythmia incidence. Importantly, while inhibition of β1 adhesion appeared to have no effect on whole-cell Na+ currents, SPC revealed a selective decrease in GJ-associated Na+ channel activity. Finally, preliminary data is shown for a rationally designed agonist of β1-mediated trans-interaction (dbl-βadp) that promotes adhesion between β1 over-expressing cells. To test the hypotheses that: (1) β1 is required for stabilizing trans-interacting Na+ channels at the ephapse; and (2) β1-mediated adhesion is an anti-arrhythmic target: Aim 1 will determine the requirement of β1 and Cx43 GJs for the hypothesized ephaptic mechanism. Mice encoding Scn1b and Gja1 null alleles, together with the β1 trans-adhesion inhibitor βadp1, will be used in studies of the structure and autonomous function of the hypothesized ephapse. Aim 2 will determine the role of β1 adhesion on Na+ channel activity and remodeling and AP conduction in myocyte monolayers. A GJ-plaque-forming, but functionally incompetent Cx43 mutant (L90V) will be used in a strategy to study the contribution of the ephaptic mechanism to conduction independent of GJ coupling. Aim 3 will develop efficient molecules that stabilize β1 adhesion based on our first-generation agonist dbl-βadp and test the anti-arrhythmic efficacy of these compounds in the pro-arrhythmic setting of acute myocardial infarction.