CALCIUM POOL SIZE MODULATES THE SENSITIVITY OF THE RYANODINE RECEPTOR-CHANNEL AND CALCIUM-DEPENDENT ATPASE OF HEAVY SARCOPLASMIC-RETICULUM TO EXTRAVESICULAR FREE CALCIUM-CONCENTRATION

We have examined calcium cycling and associated ATP consumption by iso lated heavy sarcoplasmic reticulum (HSR) vesicles incubated in conditi ons believed to exist in resting muscle. Our goals were to estimate th e 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 [C-14]-sucrose and electro n microscopy. HSR actively exchanged Ca2+ with the medium through a pa rtially open ryanodine-binding channel (RyR), as evidenced by the rapi d attainment of a steady-state gradient between HSR and medium, which was promptly increased by the closure of the channel with ruthenium re d (RR) or collapsed by its opening with caffeine. The ATP dependency w as evidenced by the sustained ATP consumption after the steady state w as attained and by the abrogation of the gradient following inhibition of the pump with thapsigargin (Tg) or the omission of ATP. When HSR v esicles were incubated in a comparatively large pool of calcium (appro ximate to 1 mu mol/mg HSR protein), ATP consumption was 1-1.5 mu mol x [min x mg protein](-1) at 0.1 mu M free Ca2+. Under such conditions, the main regulator of the sarcoplasmic Ca2+-dependent ATPase (SERCA) w as extravesicular-free Ca2+ concentration, with a four- to fivefold in crease between 0.1 and 2 mu M Ca2+, whereas RyR channel activity and t he replenishment of the HSR vesicles had only a modest effect on ATP c onsumption. When calcium pool size was reduced to 0.1 mu mol/mg HSR pr otein, a steady state was established at a lower level of HSR calcium. In spite of a slightly lower free extravesicular Ca2+ at equilibrium (approximate to 0.07 mu M following an initial concentration of 0.1 mu M), both ATP consumption and the open probability of the RyR channel were increased by a factor of three to five. Compared to the large cal cium pool, the sensitivity of both RyR channel and SERCA to extravesic ular free Ca2+ concentration as well as to caffeine and RR was markedl y enhanced. Conclusions: 1) In conditions present in resting muscle, H SR calcium is in dynamic equilibrium with the medium through a partial ly open RyR channel, which requires continuous ATP hydrolysis. 2) The availability of calcium is a major determinant of the sensitivity of b oth RyR channel and SERCA to free extravesicular Ca2+ and possibly oth er stimuli. 3) These observations are consistent with the concept that calcium cycling in resting muscle may account for a significant fract ion of muscle energy demands and further suggest that restricting calc ium availability may enhance the energetic demands of this process. (C ) 1998 Wiley-Liss. Inc.