IMMUNOCHEMICAL AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF MURINE CONNEXIN40 AND CONNEXIN43 IN MOUSE-TISSUES AND TRANSFECTED HUMAN-CELLS

Human HeLa or SkHep1 cells, defective in intercellular communication t hrough gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells mere restored in gap junctional coupling as shown by 100-fold increased electrical conduct ance. When studied by the double whole-cell patch-clamp technique, Cx4 0 HeLa transfectants exhibited single channel conductances of gamma = 121 +/- 7 pS and gamma = 153 +/- 5 pS. They were voltage gated with an equivalent gating charge of z = 4.0 +/- 0.5 for a voltage of half max imal inactivation U-0 = 44 +/- 7 mV. The corresponding values of conne xin43 (Cx43) HeLa transfectants are: gamma = 60 +/- 4 pS and gamma = 4 0 +/- 2 pS as well as z = 3.7 +/- 0.8 and U-0 = 73 +/- 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx40- than in Cx43-transfectants though their total junctional conductance was si milar or even higher than for Cx43-transfectants. In order to characte rize cell and tissue distribution as well as phosphorylation of connex in40 and -43 proteins, antibodies to C terminal oligopeptides of these proteins were prepared and used for immunoblotting, immunoprecipitati on, and immunofluorescence analysis of transfected cells where they ex hibited the punctate pattern characteristic of gap junctions on contac ting membranes. Phosphorylation of connexin40 was shown by immunopreci pitation from P-32-labeled, transfected SkHep1 cells. Analyses of prot ein distribution in tissues revealed that the amount of connexin40 det ected in heart was higher than in lung which is the inverse of the lev el of connexin40 mRNA in these tissues, suggesting posttranscriptional control of expression. Connexin40 protein in adult mouse heart and sk in is about 20-fold more abundant than in the corresponding embryonic tissue. Connexin43 in adult mouse heart appears to be more highly phos phorylated than in embryonic heart or in transfected human cells.