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 rec ordings, and rapid filtration of Ca-45(2+)-loaded SR. Inhibition of H -3-ryanodine binding by CaM was phosphorylation-independent, had an I C50 of approximately 0.1 mu M and was optimal at 10 mu M Ca2+. CaM als o inhibited H-3-ryanodine binding to CHAPS-solubilized and purified ryanodine receptors, suggesting a direct CaM-ryanodine receptor intera ction. In single channel recordings, CaM blocked Ca2+ release channels in a Ca2+-dependent manner by decreasing the number of open events pe r unit time without affecting the mean open time or unitary channel co nductance. Rapid filtration of Ca-45(2+) passively loaded into SR vesi cles showed that CaM blocked Ca2+ release within milliseconds of expos ure 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 relea se 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 Ca2release during excitation-contraction coupling.