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 [3H]-ryanodine binding, planar bilayer recordings, and rapid filtration of 45Ca(2+)-loaded SR. Inhibition of [3H]-ryanodine binding by CAM was phosphorylation-independent, had an IC50 of approximately 0.1 microM and was optimal at 10 microM Ca(2+). CaM also inhibited [3H]-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 Ca(2+)-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 45Ca2+ passively loaded into SR vesicles showed that CaM blocked Ca2+ release within milliseconds of exposure of SR to a Ca2+ release medium containing 10 microM CaM. In controls, an increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 47 +/- 10% of the 45Ca2+ 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.
Fuentes
O
O
Department of Physiology, University of Wisconsin School of Medicine, Madison.
Valdivia
C
C
Vaughan
D
D
Coronado
R
R
Valdivia
H H
HH
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D066298:In Vitro Techniques; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002147:Calmodulin; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium