Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle.
The mechanism of Ca2+ release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhanced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca2+ loading rate of longitudinal cisternae. Direct binding studies with [3H]ryanodine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae (approximately 100 nM range) with approximately 4 to 20 pmol bound per mg of protein. Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.
S
S
Fleischer
E M
EM
Ogunbunmi
M C
MC
Dixon
E A
EA
Fleer
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D007473:Ion Channels / Q000032:analysis / Q000187:drug effects*; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects; D011817:Rabbits; D012430:Ruthenium Red / Q000037:antagonists & inhibitors; D012433:Ryanodine / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000032:analysis / Q000648:ultrastructure*
D000470:Alkaloids; D007473:Ion Channels; D012430:Ruthenium Red; D012433:Ryanodine; D002118:Calcium