The Skeletal Muscle Ryanodine Receptor Identified as a Molecular Target of [³H]Azidodantrolene by Photoaffinity Labeling

Abstract

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.

Dantrolene requires Mg 2+ to arrest malignant hyperthermia

“…Functional studies on muscle fibers suggested the RyR as the potential target for the action of dantrolene (25,26), and respective binding sites were mapped to the protein (27)(28)(29). However, no action of dantrolene was found in most studies on single RyRs incorporated into lipid bilayers (26,30,31).…”

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

“…[ 3 H]Azidodantrolene specifically photolabels the N-terminal 1400-amino acid fragment of RyR1 cleaved by an endogenous, SR membrane-bound calpain (33) and is localized within sequence Leu 590 -Cys 609 based on the following evidence. 1) Of several synthetic RyR1 domain peptides examined, [ 3 H]azidodantrolene specifically photolabels only peptides containing the Leu 590 -Cys 609 sequence (named DP1 for domain peptide-1), and 2) an anti-RyR1 monoclonal antibody recognizing DP1 inhibits [ 3 H]azidodantrolene photolabeling of RyR1 (34).…”

Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor

“…Recently, we have demonstrated that [ 3 H]azidodantrolene, a pharmacologically active, photoaffinity analog of dantrolene, specifically labels the N-terminal, 1400-amino acid residue fragment of RyR1 cleaved by n-calpain, a tissue-specific isoform of this Ca 2ϩ -and thiol-activated protease (13). Several studies have demonstrated that this portion of the RyR plays a significant role in the regulation of channel function (14 -18).…”

“…A monoclonal anti-RyR1 antibody raised against rabbit terminal cisternae (25) that recognizes both the intact rabbit RyR1 and the 172 kDa, n-calpain-cleaved, N-terminal fragment of this channel (13) (26), and the specific activity was determined to be 28 Ci/mmol.…”

“…Microsomal membranes from CHO cells were obtained by ultracentrifugation of cell lysates prepared after sonication of cultured cells, as described (30). Leupeptin was specifically omitted from buffers used in membrane preparation to specifically allow for n-calpain cleavage of RyR1 and subsequent identification of its 172-kDa, N-terminal fragment, if present (13,33 (13). For inhibition of photolabeling by mAb antiRyR1, mouse ascites were added to the binding buffer at dilutions of 1:50, 1:25, and 1:10 in the presence of protease inhibitors (see "Membrane Preparation" above) with the addition of 1 mM leupeptin.…”