Dantrolene Inhibition of Ryanodine Receptor Ca2+Release Channels

Abstract

As an inhibitor of Ca 2؉ release through ryanodine receptor (RYR) channels, the skeletal muscle relaxant dantrolene has proven to be both a valuable experimen-tal probe of intracellular Ca 2؉ signaling and a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia. However, the molecular basis and speci-ficity of the actions of dantrolene on RYR channels have remained in question. Here we utilize [ 3 H]ryanodine binding to further investigate the actions of dantrolene on the three mammalian RYR isoforms. The inhibition of the pig skeletal muscle RYR1 by dantrolene (10 ␮M) was associated with a 3-fold increase in the K d of [ 3 H]ryano-dine binding to sarcoplasmic reticulum (SR) vesicles such that dantrolene effectively reversed the 3-fold de-crease in the K d for [ 3 H]ryanodine binding resulting from the malignant hyperthermia RYR1 Arg 615 3 Cys mutation. Dantrolene inhibition of the RYR1 was de-pendent on the presence of the adenine nucleotide and calmodulin and reflected a selective decrease in the ap-parent affinity of RYR1 activation sites for Ca 2؉ relative to Mg 2؉ . In contrast to the RYR1 isoform, the cardiac RYR2 isoform was unaffected by dantrolene, both in native cardiac SR vesicles and when heterologously ex-pressed in HEK-293 cells. By comparison, the RYR3 iso-form expressed in HEK-293 cells was significantly inhib-ited by dantrolene, and the extent of RYR3 inhibition was similar to that displayed by the RYR1 in native SR vesicles. Our results thus indicate that both the RYR1 and the RYR3, but not the RYR2, may be targets for dantrolene inhibition in vivo. Ryanodine receptors (RYRs) 1 are intracellular Ca 2ϩ chan-nels specialized for the rapid and massive release of Ca 2ϩ , as is necessary for excitation-contraction (EC) coupling in striated