The skeletal muscle ryanodine receptor identified as a molecular target of [3H]azidodantrolene by photoaffinity labeling.
Dantrolene is a skeletal muscle relaxant which acts by inhibiting intracellular Ca(2+) release from sarcoplasmic reticulum (SR). It is used primarily in the treatment of malignant hyperthermia (MH), a pharmacogenetic sensitivity to volatile anesthetics resulting in massive intracellular Ca(2+) release. Determination of the site and mechanism of action of dantrolene should contribute to the understanding of the regulation of intracellular Ca(2+) release in skeletal muscle. Photoaffinity labeling of porcine SR with [(3)H]azidodantrolene, a photoactivatable analogue of dantrolene, has identified a 160 kDa SR protein with immunologic cross-reactivity to skeletal muscle ryanodine receptor (RyR) as a possible target [Palnitkar et al. (1999) J. Med. Chem. 42, 1872-1880]. Here we demonstrate specific, AMP-PCP-enhanced, [(3)H]azidodantrolene photolabeling of both the RyR monomer and a 160 or 172 kDa protein in porcine and rabbit SR, respectively. The 160/172 kDa protein is shown to be the NH(2)-terminus of the RyR cleaved from the monomer by an endogenous protease activity consistent with that of n-calpain. MALDI-mass spectrometric analysis of the porcine 160 kDa protein identifies it as the 1400 amino acid NH(2)-terminal fragment of the skeletal muscle RyR reportedly generated by n-calpain [Shevchenko et al. (1998) J. Membr. Biol. 161, 33-34]. Immunoprecipitation of solubilized, [(3)H]azidodantrolene-photolabeled SR protein reveals that the cleaved 160/172 kDa protein remains associated with the C-terminal, 410 kDa portion of the RyR. [(3)H]Dantrolene binding to both the intact and the n-calpain-cleaved channel RyR is similarly enhanced by AMP-PCP. n-Calpain cleavage of the RyR does not affect [(3)H]dantrolene binding in the presence of AMP-PCP, but depresses drug binding in the absence of nucleotide. These results demonstrate that the NH(2)-terminus of the RyR is a molecular target for dantrolene, and suggest a regulatory role for both n-calpain activity and ATP in the interaction of dantrolene with the RyR in vivo.
Paul-Pletzer
K
K
Department of Anesthesia, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Palnitkar
S S
SS
Jimenez
L S
LS
Morimoto
H
H
Parness
J
J
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000378:metabolism; D002417:Cattle; D003620:Dantrolene / Q000031:analogs & derivatives* / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006868:Hydrolysis; D008024:Ligands; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D011233:Precipitin Tests; D011817:Rabbits; D019163:Reducing Agents / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000302:isolation & purification / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014316:Tritium
D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D019163:Reducing Agents; D019837:Ryanodine Receptor Calcium Release Channel; C119472:azidodantrolene; D014316:Tritium; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D002154:Calpain; D003620:Dantrolene; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D004229:Dithiothreitol