Essential Role of Calmodulin in RyR Inhibition by Dantrolene.
Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.).
Ye Win
YW
Oo
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.).
Nieves
N
Gomez-Hurtado
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.).
Kafa
K
Walweel
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.).
Dirk F
DF
van Helden
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.).
Mohammad S
MS
Imtiaz
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.).
Bjorn C
BC
Knollmann
School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Callaghan, New South Wales, Australia (Y.W.O., K.W., D.F.H., M.S.I., D.R.L.); and Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, Tennessee (N.G.-H., B.C.K.) Derek.Laver@newcastle.edu.au.
Derek R
DR
Laver
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003620:Dantrolene / Q000008:administration & dosage*; D008305:Malignant Hyperthermia / Q000188:drug therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D009465:Neuromuscular Agents / Q000008:administration & dosage*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012756:Sheep
D002147:Calmodulin; D009465:Neuromuscular Agents; D019837:Ryanodine Receptor Calcium Release Channel; D003620:Dantrolene; D002118:Calcium