As an inhibitor of Ca$^2+$ release through ryanodine receptor
(RYR) channels, the skeletal muscle relaxant dantrolene has proven
to be both a valuable experimental probe of intracellular Ca$^2+$
signaling and a lifesaving treatment for the pharmacogenetic disorder
malignant hyperthermia. However, the molecular basis and specificity
of the actions of dantrolene on RYR channels have remained in question.
Here we utilize (3)Hryanodine 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 microm) was associated
with a 3-fold increase in the K(d) of (3)Hryanodine binding to
sarcoplasmic reticulum (SR) vesicles such that dantrolene effectively
reversed the 3-fold decrease in the K(d) for (3)Hryanodine binding
resulting from the malignant hyperthermia RYR1 Arg(615) --> Cys mutation.
Dantrolene inhibition of the RYR1 was dependent on the presence of
the adenine nucleotide and calmodulin and reflected a selective decrease
in the apparent 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 expressed in HEK-293 cells. By
comparison, the RYR3 isoform expressed in HEK-293 cells was significantly
inhibited 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.