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Inhibitory effect of dantrolene on skeletal muscle RYR in the presence of calmodulin

Y.W. Oo, D.F. van Helden, M.S. Imtiaz and D.R. Laver, School of Biomedical Sciences and Pharmacy, University of Newcastle , University Drive, Callaghan, NSW 2308, Australia.

Dantrolene is a muscle relaxant that has been used clinically as the treatment for malignant hyperthermia (MH) response to volatile anaesthetics such as halothane. Our group recently reported that dantrolene inhibition of RyR1 and RyR2 required calmodulin (CaM) which is usually absent in single channel recording experiments because it readily dissociates from RyRs (Oo et al., 2015). However, nothing more is known about the properties of dantrolene inhibition of single RyR1 channels. Therefore, this study aimed to characterize dantrolene inhibition on RyR1 in the presence of exogenous CaM. CaM has a biphasic regulation on RyR1: activation at resting cytoplasmic Ca2+ (100 nmol/l) or inhibition at higher [Ca2+] (Tripathy et al., 1995). Therefore, we measured the effects of dantrolene on RyR1 differentially regulated by CaM at various cytosolic Ca2+ concentrations. Moreover, RyR1 can be both physiologically and pathologically activated by substances such as ATP, halothane and the interdomain domain destablising peptide, DP4, that mimics the effects of inherited mutations underlying MH susceptibility. Therefore, we tested if dantrolene can restore the channel activity affected by these activators.

RyR1 was isolated from rabbit skeletal muscle and incorporated into artificial lipid bilayers and their open probability Po was measured using single channel recording. Control RyR1 activity was measured for periods of 1 min, and then during 1 min exposure to dantrolene and then again after washout, all done in the presence of exogenous 100 nmol/l CaM.

Dantrolene caused inhibition of RyR1 and RyR2, but only when CaM was presence in the cytoplasmic bath. In the presence of cytoplasmic [Ca2+] (100 nmol/l) and 2 mmol/l ATP, dantrolene inhibited both RyR1 and RyR2 with identical hyperbolic dose-responses with IC50 of 0.16 ± 0.03 μmol/l and with a saturating Po of 52 ± 4 % of control. In the absence of dantrolene, RyR1 has a bell-shaped cytoplasmic Ca2+ activation curve with half activation at 1.75 ± 0.62 μmol/l and half inhibition at 0.21 ± 0.02 mmol/l and a peak Po = 0.94 ± 0.06. Dantrolene reduced Po in sub-activating and inhibiting [Ca2+] but failed to reduce peak Po. Dantrolene caused 50-60% reduction in Po of RyR1 activated by cytoplasmic Ca2+ alone (100 nmol/l) or in conjunction with halothane (5 mmol/l) or DP4 (10 μmol/l). To conclude, dantrolene inhibits RyR1 activity by: 1) decreasing RyR1 sensitivity to Ca2+ activation; 2) increasing sensitivity to Ca2+ inhibition; and 3) decreasing channel activation by halothane or by 4) reducing the effect of inter-domain disruption in the RyR1 protein.

Oo YW Gomez-Hurtado N, Walweel K, van Helden DF, Imtiaz MS, Knollmann BC, Laver DR. (2015). Essential role of calmodulin in RyR inhibition by dantrolene. Mol Pharmacol 88, 57-63.

Tripathy A, Xu L, Mann G. & Meissner G. (1995). Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor). Biophys J 69, 106-119.