Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
J Mol Cell Cardiol. 2020 Jun;143:71-84. doi: 10.1016/j.yjmcc.2020.04.004. Epub 2020 Apr 21.
Gap junction (GJ) channels formed by Cx45 exist in nodal cells in the heart where the action potential propagation is the slowest. The cellular mechanisms of slow propagation speed (or longer junctional delay) in nodal cells could be a combination of several factors, including lack of voltage-gated sodium channels, smaller cell size, and a lower GJ coupling conductance of Cx45. Compared to other cardiac GJs, Cx45 GJs possess not only the lowest unitary channel conductance, but also the highest extent and the fastest kinetics of the transjunctional voltage-dependent gating (V-gating) together with a slow recovery. These unique gating properties could make Cx45 GJs more vulnerable for dynamic uncoupling to a much lower coupling level, especially when junctional delay is lengthened and/or the heart rate is elevated. The molecular mechanisms determining the V-gating properties of Cx45 (a connexin belongs to γ group) GJs have not been studied. Previous functional studies on the amino terminal (NT) domain chimeras or point variants of other connexins belong to α or β group showed that their NT domains played an important role in determining their V-gating properties. The crystal and cryo-electron microscope structures of homologous connexin GJs showed that the NT domain lines the GJ pore, a position that could serve a role in V-sensing and gating. We hypothesize that the residues in the NT domain of Cx45 are important for its V-gating properties. Protein sequence alignment of human Cx45 NT domain with the connexins in the α and β groups revealed that the second and the eighth residues in Cx45 are different from most of these connexins. We generated a total of 14 variants on these two residues and studied their ability to form functional GJs and their V-gating properties in model cells. Our results revealed an important role of these two residues on fast V-gating kinetics and formation of morphological and functional GJ channels. In contrast, no V-gating change was observed on a GFP tagged Cx45 at its carboxyl terminus.
缝隙连接 (GJ) 通道由 Cx45 形成，存在于心脏的节点细胞中，而动作电位的传播速度最慢。节点细胞中较慢的传播速度（或更长的连接延迟）的细胞机制可能是多种因素的组合，包括缺乏电压门控钠通道、细胞尺寸较小以及 Cx45 的 GJ 偶联电导较低。与其他心脏 GJ 相比，Cx45 GJ 不仅具有最低的单位通道电导，而且还具有最高的跨连接电压依赖性门控 (V-门控) 程度和最快的动力学，同时恢复缓慢。这些独特的门控特性使 Cx45 GJ 更容易动态解偶联到更低的偶联水平，尤其是在连接延迟延长和/或心率升高时。确定 Cx45 (一种属于γ组的连接蛋白) GJ 的 V-门控特性的分子机制尚未得到研究。先前对其他属于α或β组的连接蛋白的氨基末端 (NT) 结构域嵌合体或点突变体的功能研究表明，它们的 NT 结构域在决定其 V-门控特性方面发挥着重要作用。同源连接蛋白 GJ 的晶体和冷冻电子显微镜结构表明，NT 结构域排列在 GJ 孔中，该位置可能在 V 感应和门控中发挥作用。我们假设 Cx45 的 NT 结构域中的残基对于其 V-门控特性很重要。与人 Cx45 NT 结构域的蛋白质序列比对与α和β组的连接蛋白显示，Cx45 的第二和第八个残基与这些连接蛋白中的大多数不同。我们在这两个残基上总共生成了 14 个变体，并研究了它们形成功能性 GJ 的能力及其在模型细胞中的 V-门控特性。我们的结果揭示了这两个残基在快速 V-门控动力学和形态和功能 GJ 通道形成中的重要作用。相比之下，在 Cx45 的羧基末端标记 GFP 时没有观察到 V-门控变化。
