Characterization of calmodulin-dependent and cyclic-AMP-dependent protein kinase stimulation of cardiac sarcoplasmic reticulum calcium transport.

Abstract

The mechanism by which calmodulin stimulates Ca2+ transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum (SR) was investigated. Under incubation conditions in which the majority of the phosphoprotein formed was Ca2+-dependent and no phospholamban phosphorylation was observed (10 degrees C, 15-sec incubations in the presence of 2 microM ATP), calmodulin was found to have no effect on the steady-state level of the acylphosphate phosphorylation site of Ca2+-ATPase. A significant stimulation of Mg2+, Ca2+-ATPase activity by calmodulin and a 3-fold increase in the turnover of the Ca2+ pump were, however, observed. As the ATP concentration in the incubation media was elevated (20 and 200 microM ATP), a significant degree of phosphoprotein formed was observed to be cyclic AMP (cAMP)-dependent. The degree of Ca2+-dependent phosphorylation remained constant. Under these conditions, calmodulin had no effect on the degree of phosphoprotein formed. However, when the experiments were conducted at 30 degrees C for 5 min in the presence of 500 microM ATP, a significant amount of the phosphoprotein formed was calcium-calmodulin-dependent and was additive to phosphoprotein formation observed in the presence of cAMP-dependent protein kinase. The ratio of calcium-calmodulin-dependent to cAMP-dependent phosphorylation was 1:1. K+ (110 mM) decreased the levels of phosphorylation observed in the presence of calcium and calmodulin, but had less of an effect on the levels observed in the presence of cAMP-dependent protein kinase. Autoradiographic analysis of SR membranes labeled with [32P]-ATP revealed two protein bands (24,500 and 40,000 daltons) phosphorylated in the presence of added calcium and calmodulin that were not observed in the absence of either of these additions to the reaction media. These results suggest that calmodulin stimulates Ca2+ transport by a direct effect on the Mg2+, Ca2+-ATPase. An indirect effect on Ca2+ transport via a calcium-calmodulin-dependent protein kinase, though, cannot be ruled out.