title: Calcium pool size modulates the sensitivity of the ryanodine receptor channel and calcium-dependent ATPase of heavy sarcoplasmic reticulum to extravesicular free calcium concentration.
abstract: We have examined calcium cycling and associated ATP consumption by isolated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditions believed to exist in resting muscle. Our goals were to estimate the magnitude of calcium cycling under those conditions and identify the main mechanisms involved in its regulation. The integrity of the HSR vesicles was documented by the retention of [14C]-sucrose and electron microscopy. HSR actively exchanged Ca2+ with the medium through a partially open ryanodine-binding channel (RyR), as evidenced by the rapid attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium red (RR) or collapsed by its opening with caffeine. The ATP dependency was evidenced by the sustained ATP consumption after the steady state was attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR vesicles were incubated in a comparatively large pool of calcium (approximately 1 micromol/mg HSR protein), ATP consumption was 1-1.5 micromol x [min x mg protein](-1) at 0.1 microM free Ca2+. Under such conditions, the main regulator of the sarcoplasmic Ca2+-dependent ATPase (SERCA) was extravesicular-free Ca2+ concentration, with a four- to fivefold increase between 0.1 and 2 microM Ca2+, whereas RyR channel activity and the replenishment of the HSR vesicles had only a modest effect on ATP consumption. When calcium pool size was reduced to 0.1 micromol/mg HSR protein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca2+ at equilibrium (approximately 0.07 microM following an initial concentration of 0.1 microM), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large calcium pool, the sensitivity of both RyR channel and SERCA to extravesicular free Ca2+ concentration as well as to caffeine and RR was markedly enhanced.
  
  
  CONCLUSIONS
  1) In conditions present in resting muscle, HSR calcium is in dynamic equilibrium with the medium through a partially open RyR channel, which requires continuous ATP hydrolysis. 2) The availability of calcium is a major determinant of the sensitivity of both RyR channel and SERCA to free extravesicular Ca2+ and possibly other stimuli. 3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fraction of muscle energy demands and further suggest that restricting calcium availability may enhance the energetic demands of this process.
authors.affiliation: Division of Endocrinology and Metabolism, Jewish General Hospital, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Marie
authors.forename: J E
authors.initials: JE
authors.lastname: Silva
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D007476:Ionophores; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D012433:Ryanodine; D000001:Calcimycin; D002110:Caffeine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium