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 skel etal-muscle sarcoplasmic reticulum (SR), the former by an unknown mech anism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR, Dantrolene is used to treat malign ant hyperthermia (MH), a genetic predisposition to excessive intracell ular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH r esults from a point mutation in the SR RyR that alters the open probab ility of the channel, and is reflected in altered [H-3]ryanodine bindi ng parameters. Specific binding sites for [H-3]dantrolene and [H-3]rya nodine co-distribute on SR that has been isolated by discontinuous suc rose gradient centrifugation, If the two drug-binding sites are functi onally Linked, [H-3]dantrolene binding might be affected both by pharm acological and by genetic modulators of the functional state of the Ry R. Accordingly, we compared the characteristics of [H-3]dantrolene bin ding to porcine malignant-hyperthermia-susceptible and normal-skeletal -muscle SR, and examined the effects of RyR modulators on [H-3]dantrol ene binding to these membranes. Additionally, the feasibility of separ ating the SR binding sites for [H-3]dantrolene and [H-3]ryanodine was investigated. No significant differences in [H-3]dantrolene binding ch aracteristics 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 th e RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, an d neither dantrolene nor azumolene have any effect on [H-3]ryanodine b inding. Additionally, distinct peaks of [H-3]dantrolene and [H-3]ryano dine binding are detected in SR membranes fractionated by linear sucro se centrifugation, although no differences in protein patterns are det ected by SDS/PAGE or Western-blot analysis. We suggest that the bindin g sites for these two drugs are pharmacologically distinct, and may ex ist on separate molecules.