The Skeletal Muscle Ryanodine Receptor Identified as a Molecular Target of [3H]Azidodantrolene by Photoaffinity Labeling
| Autor: | Jerome Parness, Kalanethee Paul-Pletzer, Leslie S. Jimenez, Hiromi Morimoto, Sanjay S. Palnitkar |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Swine
Immunoprecipitation Photoaffinity Labels Ligands Tritium Biochemistry Dantrolene Adenosine Triphosphate medicine Animals Muscle Skeletal Photoaffinity labeling Calpain Chemistry Ryanodine receptor Hydrolysis Myocardium Endoplasmic reticulum Malignant hyperthermia Skeletal muscle Ryanodine Receptor Calcium Release Channel medicine.disease Precipitin Tests Molecular biology Peptide Fragments Molecular Weight Dithiothreitol Sarcoplasmic Reticulum medicine.anatomical_structure Mechanism of action Reducing Agents Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Cattle Rabbits medicine.symptom medicine.drug |
| Zdroj: | Biochemistry. 40:531-542 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi001502s |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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