EXPRESSION OF MULTIPLE CONNEXINS IN CULTURED NEONATAL RAT VENTRICULARMYOCYTES

Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological prop erties and have different distributions in cardiac tissues with dispar ate conduction properties. We characterized the expression, phosphoryl ation, turnover, and subcellular distribution of these connexins in pr imary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and C x40 mRNA were specifically detected in RNA blots. Immunofluorescent st aining with antibodies specific for Cx43 and Cx45 revealed punctate la beling at appositional membranes, but no immunoreactive Cx40 was detec ted. Double-label immunofluorescence confocal microscopy of cultured m yocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were b oth identified by immunoprecipitation from [S-35]methionine-labeled cu ltures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipi tated from cultures metabolically labeled with [P-32]orthophosphate. P hosphoamino acid analysis demonstrated that Cx45 was modified on serin e residues, and Cx43 was phosphorylated on serine and threonine residu es. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results impl icate multiple mechanisms of gap junction regulation that may differ f or different connexins.