Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.
The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.
Jacquemond
V
V
Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore 21201.
Schneider
M F
MF
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D004594:Electrophysiology; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D008954:Models, Biological; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000494:pharmacology; D010320:Parvalbumins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011894:Rana pipiens; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
D009282:Naphthalenesulfonates; D010320:Parvalbumins; C016238:antipyrylazo III; D008274:Magnesium; D002118:Calcium