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)的一种新机制。 根据所获得的见解，指导并开发治疗心律失常的新方法。我们已经报道了 Na通道的NaV1.5亚基集中在Cx43(Gja1)缝隙连接(GJ)边缘 插入的盘状纳米结构域被称为周围网络。钠离子通道浓度高，钙离子浓度低。 周围处的膜宽度与结构单元的理论预测一致，该结构单元可能 支持触觉传导。经检验的假设是，钠通道复合体的β亚单位是 这个单位的核心组织元素：启示录。我们的前提是β-1(Scn1b)的黏附功能 促进间盘内膜间接触的特殊区域的形成。这 设想的安排是使跨相互作用的钠通道能够在心肌细胞之间形成，促进 AP的细胞间传导。此外，我们提出，维持脑组织黏附的激动剂可以 制定新的治疗方法，以减轻心肌梗死后致心律失常的传导缺陷。在……里面 支持这一假设的数据来自超分辨率、电子显微镜和免疫电子显微镜 而智能膜片钳研究表明，NaV1.5和β1亚基以及活性钠通道 集中在周围神经。此外，我们还开发了一种新的β-1抑制剂-19个氨基酸的模拟物 β-1的粘附区(β-adp1)。在电池-衬底阻抗传感分析中，βadp1引起 异源过表达β-1的细胞间黏附丧失。将β-adp1注入心脏导致 剂量依赖性：(1)附属器裂隙增宽，与β-1脱粘一致；(2)传导减慢； (3)室性心律失常发生率增加。重要的是，虽然抑制β1黏附似乎可以 对全细胞钠电流无影响，SPC显示GJ相关钠通道选择性减少 活动。最后，显示了合理设计的β-1介导的反式相互作用激动剂的初步数据。 (DBL-β腺苷二磷酸)促进β1过表达细胞之间的黏附。要检验以下假设：(1)β1是 和(2)β-1介导的黏附是一种 抗心律失常靶点：目标1将确定β1和Cx43 GJS对假想触觉的要求 机制。编码Scn1b和Gja1零等位基因的小鼠，以及β1跨黏附抑制因子βadp1， 将被用于研究假想的启示体的结构和自主功能。目标2将 β-1黏附对心肌细胞钠通道活性、重构和AP传导的影响 单层。GJ-菌斑形成，但功能不健全的Cx43突变体(L90V)将用于策略 研究触觉机制对不依赖GJ耦合的传导的贡献。目标3将 基于我们的第一代激动剂DBL-βADP开发稳定β1黏附的有效分子并进行测试 这些化合物在急性心肌梗死前心律失常环境中的抗心律失常效果。

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.