Use of antibodies in the analysis of connexin 43 turnover and phosphorylation

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characte rized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet ) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brai n. Relatively little of the 41-kDa protein was detectable in heart homogena tes. Cultured rat leptomeningeal cells expressed high levels of the gap jun ction protein Cx43 and were used to analyze its turnover and phosphorylatio n. Pulse-chase experiments in leptomeningeal cells with [S-35]methionine in dicated that the 41-kDa form of connexin 43 was the first immunoprecipitabl e translation product. Radiolabel subsequently appeared in the lower band o f the doublet at 43 kDa, followed by a shift into the higher band and turno ver of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [P-32] P-I, indicated that phosphorylation of connexin 43 was limited to the 43-kD a protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shif ted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on West ern blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for mo st, if not all, of Cx43 phosphorylation in this system. (C) 2000 Academic P ress.