Molecular Mechanisms of Cardiac Gap Junction

在可兴奋的细胞中，细胞内钙通过ryanodine受体从细胞内钙储存结构肌浆网释放。为了确定这种释放的钙离子是否通过间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connexin43和ryanodine受体的细胞被只表达connexin43的细胞包围。用fura-2/AM的Ca成像系统观察Ca向邻近细胞的传播。接下来，我们评估了半胱氨酸残基在connexin43细胞外环中在间隙连接通信中的功能作用。半胱氨酸残基的突变表明形成了两对分子内二硫键。因此，connecin43的细胞外二硫键对这一过程至关重要。间隙连接蛋白connexin43定位于心肌细胞间插盘，在心肌细胞收缩同步中起关键作用。在转染的HEK293细胞中使用表位标记的connexin43和ZO-1共表达的共免疫沉淀实验表明，ZO-1与connexin43连接。用缺失的ZO-1和缺失的connexin43融合蛋白进行亲和结合实验表明，connexin43的c端区域与ZO-1的n端区域结合。在connexin43表达的细胞中，ZO-1的n端结构域过表达导致connexin43从细胞-细胞界面重新分布到细胞质结构，符合电偶联的丧失。因此，我们得出结论，connexin43与ZO-1的连锁可能有助于将connexin43定位于间盘，从而在心肌中产生功能性间隙连接。

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.