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 Ki of 0.5 μm. Oxalate (5 mm) had little effect on the initial phase of calcium uptake, while inorganic phosphate, at concentrations up to 50 mm, 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 Ca2+ of 0.88 μm. Addition of either 1 μm calmodulin, or 5 μm cAMP and 0.1 mg/ml cAMP-dependent protein kinase, increased the Vmax to 12.5 and 12.8 nmol Ca mg-1 min-1, respectively, and decreased the K 1 2 for Ca2+ to 0.67 and 0.70 μm, respectively; simultaneous addition of calmodulin and cAMP-dependent protein kinase increased the Vmax to 15.2 nmol Ca mg-1 min-1 and further lowered the K 1 2 to 0.51 μm. When concentrations of NaCl from 10 to 60 mm 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 mm 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.
Bibliographical noteFunding Information:
1 This research was funded, in part, by grants from the Muscular Dystrophy Association, the Minnesota Swine Center, and NIH GM-31382. Preliminary reports of some of this material have already appeared [J. R. Mickelson, E. M. Gallant, and C. F. Louis (1984) Biophys. J. 45, 79a; J. R. Mickelson and C. F. Louis (1985) Biophys. J. 47,452a]. ‘Postdoctoral fellow of the Muscular Association. whom correspondence addressed.