CALCIUM-DEPENDENT BLOCK OF RYANODINE RECEPTOR-CHANNEL OF SWINE SKELETAL-MUSCLE BY DIRECT BINDING OF CALMODULIN

The interaction of the Ca2+ binding protein calmodulin (CaM) with the ryanodine receptor of the sarcoplasmic reticulum (SR) of pig skeletal muscle was investigated by [H-3]-ryanodine binding, planar bilayer recordings, and rapid filtration of Ca-45(2+)-loaded SR. Inhibition of [H-3]-ryanodine binding by CaM was phosphorylation-independent, had an IC50 of approximately 0.1 mu M and was optimal at 10 mu M Ca2+. CaM also inhibited [H-3]-ryanodine binding to CHAPS-solubilized and purified ryanodine receptors, suggesting a direct CaM-ryanodine receptor interaction. In single channel recordings, CaM blocked Ca2+ release channels in a Ca2+-dependent manner by decreasing the number of open events per unit time without affecting the mean open time or unitary channel conductance. Rapid filtration of Ca-45(2+) passively loaded into SR vesicles showed that CaM blocked Ca2+ release within milliseconds of exposure of SR to a Ca2+ release medium containing 10 mu M CaM. In controls, an increase in extravesicular Ca2+ from 7 nM to 10 mu M resulted in a release of 47+/-10% of the Ca-45(2+) in 20 ms. CaM reduced the release to 23+/-12% in the same period. These results are compatible with a direct mechanism of Ca2+ release channel blockade by CaM and suggest that CaM could play a significant role in the inactivation of SR Ca2+ release during excitation-contraction coupling.