Kinetic Studies of Calcium-Induced Calcium Release in Cardiac Sarcoplasmic Reticulum Vesicles

摘要

Fast Ca²⁺ release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca²⁺. Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca²⁺] and [Mg²⁺]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa ≤5. These results suggest that channels respond differently when exposed to sudden [Ca²⁺] changes than when exposed to Ca²⁺ for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg²⁺ produced a marked inhibition of release kinetics at pCa 6 (K0.5 = 63 μM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg²⁺] tested and increased at pCa 6 the K0.5 for Mg²⁺ inhibition, from 63 μM to 136 μM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca²⁺ and Mg²⁺ followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.