Sullivan R, Lo C W
Biology Department, Goddard Laboratory, University of Pennsylvania, Philadelphia 19104-6017, USA.
J Cell Biol. 1995 Jul;130(2):419-29. doi: 10.1083/jcb.130.2.419.
Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.
间隙连接含有介导离子、代谢产物和细胞信号分子在细胞间移动的膜通道。由于间隙连接由连接蛋白多肽的六聚体阵列组成，突变连接蛋白多肽的表达可能对间隙连接通讯产生显性负效应。为了检验这种可能性，我们构建了一个连接蛋白43（Cx43）/β-半乳糖苷酶（β-gal）表达载体，其中细菌β-gal蛋白与Cx43的羧基末端读框融合。该载体被转染到NIH3T3细胞中，这是一种通过间隙连接紧密连接且表达高水平Cx43的细胞系。通过Northern和Western分析表明转染克隆表达融合蛋白。X-Gal染色进一步显示，所有含有融合蛋白的细胞也表达β-gal酶活性。用β-gal和Cx43抗体进行双重免疫染色表明，融合蛋白免疫定位于细胞质的核周区域以及细胞-细胞接触区域的点状斑点处。这种模式与亲代3T3细胞中Cx43的模式相似，只是在表达融合蛋白的细胞中，细胞-细胞接触区域的Cx43表达减少。通过染料注射研究检测间隙连接通讯（GJC）进一步表明，在表达融合蛋白的细胞中染料偶联受到抑制，在细胞-细胞接触区域几乎没有Cx43定位的克隆中偶联减少最为明显。当融合蛋白表达载体转染到通讯能力差的C6细胞系中时，观察到大量融合蛋白表达，但与转染的NIH3T3细胞不同，在细胞表面未检测到融合蛋白。然而，这些C6细胞中的染料偶联受到抑制。基于这些观察结果，我们提出融合蛋白可能通过在细胞内隔离Cx43蛋白来抑制GJC。总体而言，这些结果表明Cx43/β-gal融合蛋白可在两种不同细胞类型中对GJC产生显性负效应，并表明它可能是探究间隙连接生物学功能的一种有用方法。
