The gating behavior of a channel for Ca2+-induced Ca2+ release in fragmented sarcoplasmic reticulum.

Abstract

Fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal muscle was passively loaded with 45Ca2+. Its Ca2+-induced Ca2+ release was measured in the presence of 0.1 M KCl and 5 mM MgCl2 at 0 degrees 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 microM 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 2 mM. 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 microM. 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 Ca2+ release on external Ca2+ was unaffected by internal Ca2+.