INCOMPATIBILITY OF CONNEXIN-40 AND CONNEXIN-43 HEMICHANNELS IN GAP-JUNCTIONS BETWEEN MAMMALIAN-CELLS IS DETERMINED BY INTRACELLULAR DOMAINS

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functiona l heterotypic gap junction channels. This property may contribute to t he preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium , expressing Cx43. When mouse Cx40 and Cx43 were individually expresse d in cocultured human HeLa cells, no punctate immunofluorescent signal s were detected on apposed plasma membranes between different transfec tants, using antibodies specific for each connexin, suggesting that Cx 40 and Cx43 hemichannels do not dock to each other. We wanted to ident ify domains in these connexin proteins which are responsible for the i ncompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We f ound that exchange of both extracellular loops (E1 and E2) in this sys tem (Cx4043E1,2) was required for formation of homotypic and heteroty pic conductive channels, although the electrical properties differed f rom those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channel s. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced int o Cx40 (Cx4043E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx 43 contributed to selectivity. A third construct, in which only the C- terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx4043C3, s howed neither homotypic nor heterotypic coupling with Cx40 and Cx43 tr ansfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.