title: Identification of dantrolene binding sites in porcine skeletal muscle sarcoplasmic reticulum.
abstract: Dantrolene, an intracellularly acting skeletal muscle relaxant, inhibits Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling by an unknown mechanism. The drug is used to treat malignant hyperthermia, a genetic sensitivity to volatile anesthetics which results in the massive release of intracellular Ca2+ from affected skeletal muscle. We hypothesize that determination of the site of action of dantrolene will lead to further understanding of the regulation of sarcoplasmic reticulum calcium release. We report the identification of specific dantrolene binding sites in porcine skeletal muscle sarcoplasmic reticulum using a rapid filtration binding assay for [3H]dantrolene. The binding isotherm in the heavy sarcoplasmic reticulum fraction indicates a single binding site with a Kd of 277 +/- 25 nM and a Bmax of 13.1 +/- 1.5 pmol/mg of protein. Pharmacological specificity is characterized by inhibition of [3H]dantrolene binding with unlabeled dantrolene, or azumolene, a physiologically active congener, but not with aminodantrolene, which is physiologically inactive. Drug binding is maximal at pH 6.5-7.5, requires no Ca2+ or Mg2+, and is inhibited by salt concentrations above 100 mM. [3H]Dantrolene binding is greatest in the sarcoplasmic reticulum, which contains the ryanodine receptor, the primary calcium release channel. No binding is detected in the fractions enriched for sarcolemma or transverse tubules. We suggest that dantrolene inhibits calcium release from the sarcoplasmic reticulum by either direct or indirect interaction with the ryanodine receptor.
authors.affiliation: Department of Anesthesia, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick 08901, USA.
authors.forename: J
authors.initials: J
authors.lastname: Parness
authors.forename: S S
authors.initials: SS
authors.lastname: Palnitkar
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D003620:Dantrolene / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D009994:Osmolar Concentration; D010080:Oxazoles / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine
chemical_list: D015220:Calcium Channels; D007093:Imidazoles; D009124:Muscle Proteins; D010080:Oxazoles; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; C061387:azumolene; D003620:Dantrolene; D008274:Magnesium; C094969:aminodantrolene; D002118:Calcium