IMMUNOCHEMICAL CHARACTERIZATION OF THE GAP JUNCTION PROTEIN CONNEXIN45 IN MOUSE KIDNEY AND TRANSFECTED HUMAN HELA-CELLS

Antibodies to the gap junction protein con nexin45 (Cx45) were obtaine d by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As s hown by immunoblotting and immunofluorescence, the affinity-purified a ntibodies recognized Cx45 protein in transfected human HeLa cells as w ell as in the kidney-derived human and hamster cell lines 293 and BHK2 1, respectively. In Cx45-transfected HeLa cells, this protein is phosp horylated as demonstrated by immunoprecipitation after metabolic label ing. The phosphate label could be removed by treatment with alkaline p hosphatase. A weak phosphorylation of Cx45 protein was also detected i n the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenos ine- or guanosine monophosphate (cAMP, cGMP) did not alter the level o f Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK2 1 cells. The addition of the tumor-promoting agent phorbol 12-myristat e 13-acetate (TPA) led to an increased P-32 phosphate incorporation in to the Cx45 protein in transfected cells. The Cx45 protein was found i n homogenates of embryonic brain, kidney, and skin, as well as of adul t lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and -26 were coexpressed in pr oximal tubules. No connexin43 protein was detected in renal tubules an d glomeruli at this stage of development. Our results suggest that cel ls in proximal and distal tubules are interconnected by gap junction c hannels made of different connexin proteins. The Cx45 antibodies chara cterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.