Abstract
Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.
Highlights
In RyR1 that have been identified appear in only about 50% of affected families [6, 7]
Because Malignant hyperthermia (MH) syndromes are linked to mutations in the RyR1 channel in various vertebrates, as well as in humans [2, 4, 34, 35], and because dantrolene binds to a specific site on RyR1 [36], previous studies have focused on the role of dantrolene in modulating RyR1 channel activity
We demonstrate that azumolene disrupts the tight Ca2ϩ release-coupled graded activation of store-operated Ca2ϩ entry (SOCE) that is normally seen in adult mouse skeletal muscle fibers without substantially inhibiting sarcoplasmic reticulum (SR) Ca2ϩ release
Summary
Cell Culture—C1148 cells, a Chinese hamster ovary (CHO) cell line stably expressing RyR1, were maintained in Ham’s F-12. For SR Ca2ϩ content assessment, the fiber was mechanically skinned in the absence of Rhod-5 and Ca2ϩ and incubated with an intracellular-like solution (in mM, 140 potassium glutamate, 6.5 MgCl2, 6 creatine phosphatase, 0.5 CaCl2, 20 2-bromoethanesulfonate/BES-KOH) containing 0.2 mM EGTA and 20 M Rhod-5N AM (Invitrogen) for 1 h at room temperature to load the SR, followed by extensive washes and incubation for an additional 30 min to allow the complete deesterification of the dye. The fiber was exposed to an SR-depleting solution (in mM, 100 potassium glutamate, 16 sodium glutamate, 20 EGTA-KOH, 5 1,2-bis(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, 0.07 MgCl2, 0.25 ATP, 1 creatine phosphatase, 10 BES-KOH) with C/R (30 mM/5 M), in the presence of azumolene or Me2SO carrier for 1000 s These experiments were repeated six times, and mean values of 10 regions of interest per fiber were analyzed. A value of p Ͻ 0.05 was used as criterion for statistical significance
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