EXPRESSION OF A CONNEXIN-43 BETA-GALACTOSIDASE FUSION PROTEIN INHIBITS GAP JUNCTIONAL COMMUNICATION IN NIH3T3 CELLS

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap jun ctions are comprised of a hexameric array of connexin polypeptides, th e expression of a mutant connexin polypeptide may exert a dominant neg ative effect on gap junctional communication. To examine this possibil ity, 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 in to NIH3T3 cells, a cell line which is well coupled via gap junctions a nd expresses high levels of Cx43. Transfectant clones were shown to ex press the fusion protein by northern and western analysis. X-Gal stain ing further revealed that all of the fusion protein containing cells a lso expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is i mmunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is simila r to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of c ell-cell contact. Examination of gap junctional communication (GJC) wi th dye injection studies further showed that dye coupling was inhibite d in the fusion protein expressing cells, with the largest reduction i n coupling found in a clone exhibiting little Cx43 localization at reg ions of cell-cell contact. When the fusion protein expression vector w as transfected into the communication poor C6 cell line, abundant fusi on protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheles s, dye coupling was inhibited in these C6 cells. Based on these observ ations, we propose that the fusion protein may inhibit GJC by sequeste ring the Cx43 protein intracellularly. Overall, these results demonstr ate that the Cx43/beta-gal fusion protein can exert a dominant negativ e 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.