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 (Vj-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 Vj-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 Vj-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 Vj-sensing and gating. We hypothesize that the residues in the NT domain of Cx45 are important for its Vj-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 Vj-gating properties in model cells. Our results revealed an important role of these two residues on fast Vj-gating kinetics and formation of morphological and functional GJ channels. In contrast, no Vj-gating change was observed on a GFP tagged Cx45 at its carboxyl terminus.

中文翻译：

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氨基末端结构域在跨结电压依赖性Cx45间隙连接的门控动力学中起重要作用。

由Cx45形成的间隙连接（GJ）通道存在于心脏的动作电位传播最慢的节点细胞中。节点细胞中缓慢传播速度（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，较小的细胞大小和较低的Cx45 GJ耦合电导。与其他心脏GJ相比，Cx45 GJ不仅具有最低的单位通道电导率，而且具有跨结电压依赖性门控（Vj-gating）的最高程度和最快的动力学以及缓慢的恢复。这些独特的门控特性可能会使Cx45 GJ更容易因动态解耦而降低到更低的耦合水平，尤其是在延长连接延迟和/或提高心率时。尚未研究确定Cx45（连接蛋白属于γ基）GJ的Vj门控性质的分子机制。以前对属于α或β基团的氨基末端（NT）域嵌合体或其他点连接蛋白的点变体的功能研究表明，它们的NT域在确定其Vj门控性质中起着重要作用。同源连接蛋白GJ的晶体和低温电子显微镜结构表明，NT域位于GJ孔中，该位置可能在Vj感应和门控中起作用。我们假设Cx45的NT域中的残基对其Vj门控特性很重要。人Cx45 NT结构域与α和β组连接蛋白的蛋白质序列比对显示，Cx45中的第二个和第八个残基与大多数这些连接蛋白不同。我们在这两个残基上生成了总共14个变体，并研究了它们在模型细胞中形成功能性GJ的能力及其Vj门控性质。我们的结果揭示了这两个残基在快速Vj门控动力学以及形态和功能GJ通道形成中的重要作用。相比之下，在GFP标记的Cx45的羧基末端未观察到Vj门控变化。