EXPRESSION OF MULTIPLE CONNEXINS IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES
EXPRESSION OF MULTIPLE CONNEXINS IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES
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DOI：
10.1161/01.res.76.3.381
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发表时间：
1995-03-01
影响因子：
20.1
通讯作者：
BEYER, EC
中科院分区：
文献类型：
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作者：
DARROW, BJ;LAING, JG;BEYER, EC
在哺乳动物心肌细胞中发现了三种缝隙连接蛋白：连接蛋白43(Cx43)、连接蛋白45(Cx45)和连接蛋白40(Cx40)。这些蛋白质形成具有不同电生理特性的通道，并且在具有不同传导特性的心脏组织中具有不同的分布。我们研究了这些连接蛋白在原代培养的新生大鼠心肌细胞中的表达、磷酸化、周转和亚细胞分布。Cx43、Cx45和Cx40的mRNA在RNA印迹中特异检测到。抗Cx43和Cx45抗体的免疫荧光染色显示对位膜有斑点标记，但未检测到Cx40免疫反应。培养心肌细胞的双标记免疫荧光共聚焦显微镜显示Cx43和Cx45共定位。免疫沉淀法从[S-35]蛋氨酸标记的培养物中鉴定出Cx43和Cx45，但抗Cx40抗体不沉淀任何放射性标记的蛋白。Cx45和Cx43的磷酸化形式都是从用[P-32]正磷酸代谢标记的培养物中免疫沉淀的。磷酸氨基酸分析表明，Cx45在丝氨酸残基上被修饰，而Cx43在丝氨酸和苏氨酸残基上被磷酸化。脉冲追逐标记实验表明，Cx43和Cx45的半衰期分别为1.9和2.9小时。因此，Cx43和Cx45的翻转速度都相对较快，这表明心肌缝隙连接具有动态重塑的潜力。这些结果暗示了缝隙连接调节的多种机制，不同的连接蛋白可能会有所不同。
Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [S-35]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [P-32]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.