The Gating Behavior of a Channel for Ca2+-Induced Ca2+ Release in Fragmented Sarcoplasmic Reticulum1

Fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal muscle was passively loaded with 45 Ca 2+ . Its Ca 2+ -induced Ca 2+ release was measured in the presence of 0.1 M KCl and 5 mM MgCl 2 at 0°C by Millipore filtration. The following results were obtained. 1. The amounts of Ca 2+ -induced Ca 2+ release from heavy SR, light SR, and unfractionated SR were 80, 20, and 60% of the amounts of preloaded Ca 2+ respectively. Therefore, the experiments were carried out with unfractionated FSR. 2. The Ca 2+ -induced Ca 2+ release from FSR was inhibited by procaine, but unaffected by caffeine and trifluoperazine. The rate of Ca 2+ release decreased markedly with decreasing pH. 3. Various adenine nucleotides (ATP, AMPPNP, ADP, AMP) accelerated the Ca 2+ 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 Ca 2+ , the final amount of the Ca 2+ release was unaffected. The rate of Ca 2+ 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 2 mM. 5. In the presence of AMP, the amount of Ca 2+ released increased, while the relative rate was unaffected by increasing the external Ca 2+ concentration. The final amount released increased from 0 to 60% of the amount of preloaded Ca 2+ by increasing the free Ca 2+ concentration from 0.06 to 0.24 μM. The effect of external Ca 2+ on the release was reversible. 6. The ratio between the amount of preloaded Ca 2+ and that of Ca 2+ release was independent of the Ca 2+ concentration used for preloading. Furthermore, the dependence of the final amount of Ca 2+ -induced Ca 2+ release on external Ca 2+ 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 Ca 2+ 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 1, and that the gate opens in an all-or-none fashion when the external Ca 2+ concentration exceeds the threshold. The amount of Ca 2+ release per unit time (the Ca 2+ passes through the open gate of the active channel) is proportional to the concentration of internal Ca 2+ .