A gap junction-independent role for Connexin43 in cardiac electrophysiology

描述（由申请人提供）：这是申请的修订版本，用于资助RO 1-GM 57691 -13的竞争性续期。自其成立以来（1999年），我们专注于调节缝隙连接蛋白43（Cx43）形成的机制。对于这个应用，我们采取相反的角度，并查看Cx43作为其他分子复合物的调节剂的能力。在其经典定义中，连接蛋白43（Cx43）的功能是形成间隙连接。 虽然这种描述当然是正确的，但它并不排除Cx43发挥其他作用的可能性，这些作用与间隙连接孔形成的作用不同。在这里，我们提出：a）Cx43羧基末端结构域内的特定氨基酸调节钠通道的功能，B）这种调节发生在“perinexus”的范围内，即间隙连接斑块周围的区域，以及c）Cx43介导的细胞电生理调节是适当的心律所必需的。在目标1下，我们将描述Cx43表达缺失（Cx43-CKO）或Cx43最后5个氨基酸缺失（Cx43 D378 stop线）后小鼠的心脏电生理特征。这两种小鼠均出现心室纤颤和猝死。一个根本的区别是突变D378 stop不能阻止间隙连接斑块的形成。我们建议，Cx43是必要的其他离子通道的正常功能。在目标2中，我们将定义与钠通道复合物相关的perinexus的结构，以及保留Cx43的378-382区域以控制分子之间距离的重要性。我们实现了现代成像/记录技术（扫描离子电导显微镜，SICM引导膜片钳，断层扫描电子显微镜，直接随机重建显微镜，邻近连接测定）和新型动物模型的独特组合，以解决直接影响我们对心律失常分子机制的理解的新假设。总的来说，我们的实验挑战了流行的概念，即在结构正常的心脏中，Cx43依赖性心律失常仅是细胞之间间隙连接通道丢失的结果。

DESCRIPTION (provided by applicant): This is the revised version of an application for competitive renewal of grant RO1-GM57691-13. Since its inception (in 1999), we focused on the mechanisms that regulate gap junctions formed by Connexin43 (Cx43). For this application, we take the reverse angle, and look at the ability of Cx43 to be the regulator of other molecular complexes. In its classical definition, the function of Connexin43 (Cx43) is to form gap junctions. While this description is certainly correct, it does not exclude the possibility that Cx43 exerts other actions, separate from that of gap junction pore formation. Here, we propose that a) specific amino acids within the Cx43 carboxyl terminal domain modulate the function of sodium channels, b) this regulation occurs within the confines of the "perinexus," that is, the area surrounding a gap junction plaque, and c) Cx43-mediated regulation of cell electrophysiology is necessary for proper cardiac rhythm. Under Aim 1, we will characterize the cardiac electrophysiological profile of mice after loss of Cx43 expression (Cx43-CKO), or after deletion of the last five amino acids of Cx43 (line Cx43D378stop). Both of these mice present with ventricular fibrillation and sudden death. A fundamental difference is that mutation D378stop does not prevent formation of gap junction plaques. We propose that Cx43 is necessary for proper function of other ion channels. In aim 2, we will define the structure of the perinexus in relation to the sodium channel complex, and the importance of preservation of region 378-382 of Cx43 to control the distance between molecules. We implement a unique combination of modern imaging/recording techniques (scanning ion conductance microscopy, SICM-guided patch clamp, tomographic electron microscopy, direct stochastic reconstruction microscopy, proximity ligation assays), and novel animal models, to address new hypotheses that directly impact our understanding of the molecular mechanisms of cardiac arrhythmias. Overall, our experiments challenge the prevailing concept that, in a structurally normal heart, Cx43-dependent arrhythmias are only consequent to the loss of gap junction channels between cells.