Pharmacological distinction between dantrolene and ryanodine binding sites: Evidence from normal and malignant hyperthermia-susceptible porcine skeletal muscle

Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR, Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [H-3]ryanodine binding parameters. Specific binding sites for [H-3]dantrolene and [H-3]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation, If the two drug-binding sites are functionally Linked, [H-3]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [H-3]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [H-3]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [H-3]dantrolene and [H-3]ryanodine was investigated. No significant differences in [H-3]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the B-max ratio for [H-3]dantrolene/[H-3]ryanodine was 1.4(+/-0.1): 1 in both muscle types. [H-3]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [H-3]ryanodine binding. Additionally, distinct peaks of [H-3]dantrolene and [H-3]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.