Dephosphorylation agents depress gap junctional communication between rat cardiac cells without modifying the connexin43 phosphorylation degree

The functional state of gap junctional channels and the phosphorylation sta tus of Connexine43 (Cx43), the major gap junctional protein in rat heart, w ere evaluated in primary cultures of neonatal rat cardiomyocytes. H7, able to inhibit a range of serine/threonine protein kinases, progressively reduc ed gap junctional conductance to approximately 13 % of its initial value wi thin 10 min except when protein phosphatase inhibitors were also present. T he dephosphorylating agent 2,3-Butanedione monoxime (BDM) produced both a q uick and reversible interruption of cell-to-cell communication as well as a parallel slow inhibition of junctional currents. The introduction of a non -hydrolysable ATP analogue (ATP gammaS) in the cytosol delayed the second c omponent, suggesting that it was the consequence of protein dephosphorylati on. Western blot analysis reveals 2 forms of Cx43 with different electrophoreti c mobilities which correspond to its known phosphorylated and dephosphoryla ted forms. After exposure of the cells to H7 (1 mmol/l, 1h) or BDM (15 mmol /l, 15 min), no modification in the level of Cx43 phosphorylation was obser ved. The lack of direct correlation between the inhibition of cell-to-cell communication and changes in the phosphorylation status of Cx43 suggest tha t the functional state of junctional channels might rather be determined by regulatory proteins associated to Cx43.