权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular Mechanisms of Cardiac Gap Junction

心脏间隙连接的分子机制

基本信息

批准号：
10557069
负责人：
TADA Michihiko
金额：
$ 8.64万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557069/
关键词：
Gap junction Connexin Cardiac myocytes Cai. aging system patch-clamp methods ZO-1 Gap junctional Protein パッチクランプ法 gap junctional protein connexin-43 部位特異的変異導入法パッチ・クランプシステム

项目摘要

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.

在可兴奋细胞中，细胞内Ca通过ryanodine受体从细胞内Ca储存结构（肌浆网）释放。为了确定这种释放的钙是否通过间隙连接传播到邻近细胞，从而构成一个长距离的信号网络，我们开发了一个细胞系统，其中表达连接蛋白43和兰尼定受体的细胞被仅表达连接蛋白43的细胞包围。使用Fura-2/AM的Ca成像系统观察Ca向邻近细胞的传播。接下来，我们评估了间隙连接通讯中连接蛋白43细胞外环中半胱氨酸残基的功能作用。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的细胞外二硫键对于这一过程至关重要。缝隙连接蛋白connexin 43定位于心肌细胞的闰盘，在心肌细胞收缩的同步化中起着关键作用。在转染的HEK 293细胞中使用共表达的表位标记的连接蛋白43和ZO-1的免疫共沉淀实验表明ZO-1连接连接蛋白43。使用缺失的ZO-1和缺失的connexin 43融合蛋白进行的亲和结合试验表明，connexin 43的C-末端区域与ZO-1的N-末端区域结合。ZO-1的N-末端结构域在表达连接蛋白43的细胞中的过表达导致连接蛋白43从细胞-细胞界面到细胞质结构的重新分布，与电耦合的损失一致。因此，我们的结论是连接蛋白43与ZO-1的连接可能有助于定位在闰盘的连接蛋白43，从而产生心肌细胞的功能性缝隙连接。