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

We examined the mechanism by which cyclic AMP-dependent protein kinase (PK) stimulates the calcium pump of cardiac sarcoplasmic reticulum vesicles. The Ca dependence of calcium uptake rates by 30 /ig/ml canine cardiac sarcoplasmic reticulum was measured at 25 °C in 120 mM KCl, 40 mM histidine buffer (pH 6.8), 5 mM MgATP, and an ATP-regenerating system (75 fig/ml pyruvate kinase + 5 mM phosphoenolpyruvate) with 50 mM phosphate as calcium-precipitating anion. Preincubation with PK, 100 /ig/ml, plus 1 fiM cyclic AMP for 10 minutes increased calcium uptake rates from 2to 3-fold at Ca concentrations between 0.25 and 2.0 fiM. This stimulation was associated with a decrease in the Ca concentration needed to produce 50% maximal calcium uptake velocity from 2.38 ± 0.21 to 1.07 ± 0.10 /IM (n 7, P< 0.001). The Ca dependence of calcium uptake in nonphosphorylated cardiac sarcoplasmic reticulum vesicles exhibited positive cooperativity, whereas 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, therefore, cardiac sarcoplasmic reticulum exhibited the low Ca cooperativity seen with fast skeletal sarcoplasmic reticulum. Phosphorylation of cardiac sarcoplasmic reticulum by PK thus appears to increase the apparent Ca-sensitivity of the calcium pump while decreasing positive cooperativity between the two Ca-binding sites of the calcium pump. Ore Res 44: 384-391,1979