Fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal muscle was passively loaded with ⁴⁵Ca²⁺. Its Ca²⁺-induced Ca²⁺ release was measured in the presence of 0.1M KCl and 5mM MgCl² at 0°C by Millipore filtration. The following results were obtained.
1. The amounts of Ca²⁺-induced Ca²⁺ release from heavy SR, light SR, and unfractionated SR were 80, 20, and 60% of the amounts of preloaded Ca²⁺, respectively. Therefore, the experiments were carried out with unfractionated FSR. 2. The Ca²⁺induced Ca²⁺ release from FSR was inhibited by procaine, but unaffected by caffeine and trifluoperazine. The rate of Ca²⁺ release decreased markedly with decreasing pH. 3. Various adenine nucleotides (ATP, AMPPNP, ADP, AMP) accelerated the Ca²⁺ release, and the accelerating effect was reversible. CTP had no effect on the release, but inhibited the accelerating effect of AMPPNP. 4. In the presence of 15 μM external free Ca²⁺, the final amount of the Ca²⁺ release was unaffected. The rate of Ca²⁺ release was markedly increased by AMP; the dependence of the rate on AMP concentration followed a Michaelis-Menten type equation with a Hill coefficient of 1 and an apparent affinity for AMP of about 2mM. 5. In the presence of AMP, the amount of Ca²⁺ released increased, while the relative rate was unaffected by increasing the external Ca²⁺ concentration. The final amount released increased from 0 to 60% of the amount of preloaded Ca²⁺ by increasing the free Ca²⁺ concentration from 0.06 to 0.24 μM. The effect of external Ca²⁺ on the release was reversible. 6. The ratio between the amount of preloaded Ca²⁺ and that of Ca²⁺ release was independent of the Ca²⁺ concentration used for preloading. Furthermore, the dependence of the final amount of Ca²⁺-induced Ca^2- release on external Ca²⁺ was unaffected by internal Ca ²⁺.
The results suggest that most of the FSR vesicles have no or only one channel for the Ca ²⁺-induced Ca²⁺ release, that the channel has active and inactive forms and the equilibrium between them shifts to the active form when adenine nucleotide binds to the channel with a Hill coefficient of I, and that the gate opens in an allor-none fashion when the external Ca²⁺ concentration exceeds the threshold. The amount of Ca²⁺ release per unit time (the Ca²⁺ passes through the open gate of the active channel) is proportional to the concentration of internal Ca²⁺.