A gap junction-independent role for Connexin43 in cardiac electrophysiology

描述(由申请人提供)：这是一份竞争性续期申请的修订版RO1-GM57691-13。自从它成立以来(1999年)，我们专注于调节连接蛋白43(Cx43)形成的缝隙连接的机制。对于这个应用，我们采取相反的角度，并看看Cx43作为其他分子络合物的调节器的能力。在其经典定义中，连接蛋白43(Cx43)的功能是形成缝隙连接。 虽然这种描述当然是正确的，但它不排除Cx43发挥其他作用的可能性，独立于缝隙连接气孔形成的作用。在这里，我们认为a)Cx43羧基末端结构域中的特定氨基酸调节钠通道的功能，b)这种调节发生在“周围神经丛”的范围内，即缝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正常心律所必需的。在目标1下，我们将描述Cx43表达缺失(Cx43-CKO)或Cx43最后五个氨基酸缺失后(Cx43D378stop线)小鼠的心脏电生理特征。这两只小鼠都出现了室颤和猝死。一个根本的区别是，突变D378停止不能阻止缝隙连接斑块的形成。我们认为，Cx43是其他离子通道正常运行所必需的。在目标2中，我们将定义与钠通道复合体相关的周围神经丛的结构，以及保留Cx43的378-382区域以控制分子间距离的重要性。我们实施了现代成像/记录技术(扫描离子电导显微镜、SICM引导的膜片钳、断层扫描电子显微镜、直接随机重建显微镜、邻近结扎分析)和新的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理解的新假说。总体而言，我们的实验挑战了流行的概念，即在结构正常的心脏中，Cx43依赖的心律失常只是细胞间缝隙连接通道丧失的结果。

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.