Organ level Inhibition of Inter cellular communication by mutant connexin expression vector

缝隙连接是一种特殊的细胞-细胞连接，它形成细胞间通道，并介导低分子代谢物和离子的直接转移。通过缝隙连接的细胞间通讯被认为在控制细胞生长、分化和维持动态平衡方面发挥着重要作用。最近，有报道称，一些人类遗传性疾病，如Charcot-Marie-Tooth病、非综合征性感音神经性耳聋和皮肤病，都是由缝隙连接蛋白(Cxexin)基因点突变引起的。然而，这些疾病的病理过程由于缝隙连接的异常而被了解得很少。在本研究中，我们建立了一种在活细胞中可视化细胞功能的方法，同时实时识别连接蛋白的定位。含有绿色荧光蛋白(Gep)和野生型或显性负性突变体连接蛋白43(Cx43)的融合蛋白的表达载体为cons…更多地构建并转染到原代新生大鼠心肌细胞和通讯缺陷的HeLa细胞中。通过显微注射缝隙连接渗透性荧光染料(Alexa 568，M.W.730)。用荧光钙指示剂(Fura Red)结合共聚焦扫描显微镜监测心肌细胞内钙动力学。野生型Cx43-GFP在通讯缺陷的Hela细胞中进行了功能缝隙连接。相反，突变的Cx43-GFP以显性-负性方式抑制原代新生大鼠心肌细胞之间的染料偶联。突变的Cx43-GFP诱导搏动心肌细胞钙瞬变的去同步化频率显著高于野生型Cx43-GFP。这些结果表明，显性-负性Cx43可通过钙瞬变的去同步化引起心肌细胞间同步搏动的抑制，提示通过缝隙连接抑制细胞间通讯可能导致心律失常和心脏收缩障碍。CX-GFP表达载体为研究缝隙连接在体内的定位和功能提供了有用的系统。较少

Gap junctions are specialized cell-cell junctions that form intercellular channels and mediate the direct transfer of low molecular weight metabolites and ions. Intercellular communication via gap junction is believed to play important roles in the control of cell growth, differentiation and maintenance of homeostasis. Recently, it has been reported that several human hereditary diseases such as Charcot-Marie-Tooth disease, non-syndromic sensorineural deafness and skin diseases are caused by point mutations of gap junction protein (connexin) genes. However, the pathological processes of these diseases due to abnormalities in gap junctions are poorly understood.In the present study, we have established a method for visualization of cellular function in living cells, while identifying the localization of connexins in real-time. Expression vectors that contain fusion proteins of green fluorescent protein (GEP) and, either wild-type or a dominant-negative mutant connexin43 (Cx43) were cons … More tructed and transfected into primary neonatal rat cardiomyocytes and into communication-deficient HeLa cells. Intercellular communication was estimated by microinjection of gap junction-permeable fluorescent dye (Alexa 568,m.w. 730). Intracellular calcium dynamics in cardiomyocytes were monitored by a fluorescent calcium indicator (Fura Red) in combination with confocal scanning microscopy. Wild-type Cx43-GFP made functional gap junctions in otherwise communication-deficient Hela cells. In contrast, the mutated Cx43-GFP Inhibited dye coupling among primary neonatal rat cardiomyocytes in a dominant-negative manner. The mutated Cx43-GFP induced desynchronization of calcium transients among beating cardiomyocytes with a significantly higher frequency than in wild-type Cx43-GFP. These results indicate that dominant-negative Cx43 can induce inhibition of synchronous beating among cardiomyocytes through desynchronization of calcium transients, and suggest that inhibition of intercellular communication via gap junction might cause arrhythmia and contraction disturbance in the heart. Cx-GFP expression vectors provides a useful systems for studies of localization and function of gap junctions in vivo. Less