Molecular Mechanisms of Cardiac Gap Junction

在可兴奋的细胞中，细胞内的钙通过兰尼定受体从细胞内的钙储存结构-肌浆网释放出来。为了确定这种释放的钙是否通过缝隙连接传播到邻近细胞，从而构成一个远程信号网络，我们开发了一个细胞系统，在这个细胞系统中，同时表达连接蛋白43和兰诺定受体的细胞被只表达连接蛋白43的细胞包围。用Fura-2/AM钙离子成像系统观察钙离子向邻近细胞的扩散。接下来，我们评估了连接蛋白43细胞外环中半胱氨酸残基在缝隙连接通讯中的功能作用。半胱氨酸残基的突变表明形成了两对分子内二硫键。因此，连接蛋白43的胞外二硫键在这一过程中起着至关重要的作用。间隙连接蛋白Cx43定位于心肌细胞的间盘，在心肌细胞收缩的同步化过程中起着关键作用。共表达的表位标记的连接蛋白43和ZO-1在HEK293细胞中的免疫共沉淀实验表明，ZO-1连接连接蛋白43。用缺失的ZO-1和缺失的Cx43融合蛋白进行亲和结合分析表明，Cx43的C端区域与ZO-1的N端区域结合。在连接蛋白43表达的细胞中，ZO-1的N端结构域的过表达导致连接蛋白43从细胞-细胞界面重新分布到细胞质结构，与失去电耦合相一致。因此，我们得出结论，连接蛋白43与ZO-1的连接可能有助于将连接蛋白43定位于间盘，从而在心肌细胞中产生功能性缝隙连接。

In excitable cells, intracellular Ca is released via the ryanodine receptor from the intracellular Ca storing structure, the sarcoplasmic reticulum. To determine whether this released Ca propagates through gap junctions to neighboring cells and thereby constitutes a long-range signaling network, we developed a cell system in which cells expressing both connexin43 and ryanodine receptor are surrounded by cells expressing only connexin43. Propagation of Ca to neighboring cells was observed with a Ca imaging system using fura-2/AM. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin43 in gap junctional communication. Mutations of cysteine residues showed that two pairs of intramolecular disulfide bonds are formed. Thus, the extracellular disulfide bonds of connexin43 are crucial for this process. Gap junctional protein connexin43, localized to the intercalated discs of cardiac myocytes, plays a critical role in the synchronization of their contractions. Co-immunoprecipitation experiments using co-expressed epitope-tagged connexin43 and ZO-1 in transfected HEK293 cells indicated that ZO-1 links connexin43. Affinity binding assay using deleted ZO-1 and deleted connexin43 fusion proteins showed that the C-terminal region of connexin43 binds to the N-terminal region of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin43-expressing cells resulted in the redistribution of connexin43 from the cell-cell interfaces to the cytoplasmic structures, in accordance with the loss of electrical coupling. We therefore conclude that the linkage of connexin43 with ZO-1 may serve to localize the connexin43 at the intercalated discs, thereby generating the functional gap junction in cardiac myoctyes.