Regulation of skeletal muscle sarcolemmal ATP-dependent calcium transport by calmodulin and cAMP-dependent protein kinase

Abstract

Skeletal muscle sarcolemma preparations, predominantly in the form of inside-out vesicles, were obtained from porcine muscle by a LiBr-extraction procedure. In the presence of ATP, these preparations were able to accumulate 94 nmol Ca/mg protein after 20 min at 37 °C. Sarcolemmal calcium uptake was completely blocked by the calcium ionophore, A23187, but was unaffected by monovalent cation ionophores. Calcium uptake was markedly inhibited by vanadate, with an approximate K i of 0.5 μ m. Oxalate (5 m m) had little effect on the initial phase of calcium uptake, while inorganic phosphate, at concentrations up to 50 m m, had a significant stimulatory effect on sarcolemmal calcium uptake. In contrast to ATP, acetylphosphate had minimal ability and p-nitrophenylphosphate had no ability to support calcium uptake. The maximal initial velocity of skeletal muscle sarcolemmal calcium uptake was 10.0 nmol Ca mg −1 min −1 at 37 °C, with a K 1 2 for Ca 2+ of 0.88 μ m. Addition of either 1 μ m calmodulin, or 5 μ m cAMP and 0.1 mg/ml cAMP-dependent protein kinase, increased the V max to 12.5 and 12.8 nmol Ca mg −1 min −1, respectively, and decreased the K 1 2 for Ca 2+ to 0.67 and 0.70 μ m, respectively; simultaneous addition of calmodulin and cAMP-dependent protein kinase increased the V max to 15.2 nmol Ca mg −1 min −1 and further lowered the K 1 2 to 0.51 μ m. When concentrations of NaCl from 10 to 60 m m were added to vesicles that had been loaded with calcium in the presence of ATP, an immediate release of calcium occurred. This process had an approximate K 1 2 for sodium of 10–20 m m and an approximate maximal rate of 50 nmol Ca mg −1 min −1. We conclude that skeletal muscle sarcolemma contains a cAMP-dependent protein kinase-and calmodulin-stimulatable ATP-dependent calcium transport, as well as a sodium: calcium exchange activity.