Mechanism by which cyclic adenosine 3':5'-monophosphate-dependent protein kinase stimulates calcium transport in cardiac sarcoplasmic reticulum.

Abstract

How cyclic(c)AMP-dependent protein kinase (PK) stimulates the Ca pump of cardiac sarcoplasmic reticulum vesicles was studied. The Ca2+ dependence of Ca uptake rates by 30 .mu.g/ml canine cardiac sarcoplasmic reticulum was measured at 25.degree. C in 120 mM KCl, 40 mM histidine buffer (pH 6.8), 5 mM MgATP and an ATP-regenerating system (75 .mu.g/ml pyruvate kinase (PK) + 5 mM phosphoenolpyruvate) with 50 mM phosphate as Ca-precipitating anion. Preincubation with PK, 100 .mu.g/ml, plus 1 .mu.M cAMP for 10 min increased Ca uptake rates from 2- to 3-fold at Ca2+ concentrations 0.25-2.0 .mu.M. This stimulation was associated with a decrease in the Ca2+ concentrations needed to produce 50% maximal Ca uptake velocity from 2.38 .+-. 0.21 to 1.07 .+-. 0.10 .mu.M (n = 7, P < 0.001). The Ca2+ dependence of Ca uptake in nonphosphorylated cardiac sarcoplasmic reticulum vesicles exhibited positive cooperativity, but cooperativity was not evident in the corresponding preparations from fast rabbit skeletal muscle. The estimated Hill coefficient for control vesicles was 1.77 .+-. 0.15; this value decreased significantly to 1.24 .+-. 0.08 (P < 0.01) after preincubation with PK. After exposure to PK cardiac sarcoplasmic reticulum exhibited the low Ca2+ cooperativity seen with fast skeletal sarcoplasmic reticulum. Phosphorylation of cardiac sarcoplasmic reticulum by PK appears to increase the apparent Ca2+-sensitivity of the Ca pump while decreasing positive cooperativity between the 2 Ca2+-binding sites of the Ca pump.