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.

[3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum.

Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understanding both the mechanism of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [3H]Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific activity for detection of photo-cross-linked proteins by fluorographic methods. This was accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was demonstrated to be >/=95% tritium-labeled at the imine position by NMR spectroscopy, and the specific radioactivity of [3H]azidodantrolene sodium was empirically determined by HPLC and liquid scintillation counting to be 24.4 Ci/mmol, approximately 85% of theoretical maximum. [3H]Azidodantrolene was found to be pharmacologically active in ligand-receptor binding studies with skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed by SDS-PAGE and tritium fluorography have identified a approximately 160-kDa specifically labeled protein as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor may be related to the skeletal muscle ryanodine receptor.

Pharmacological distinction between dantrolene and ryanodine binding sites: evidence from normal and malignant hyperthermia-susceptible porcine skeletal muscle.

Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR. Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [3H]ryanodine binding parameters. Specific binding sites for [3H]dantrolene and [3H]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation. If the two drug-binding sites are functionally linked, [3H]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [3H]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [3H]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [3H]dantrolene and [3H]ryanodine was investigated. No significant differences in [3H]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the Bmax ratio for [3H]dantrolene/[3H]ryanodine was 1.4(+/-0.1):1 in both muscle types. [3H]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [3H]ryanodine binding. Additionally, distinct peaks of [3H]dantrolene and [3H]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.

Identification of dantrolene binding sites in porcine skeletal muscle sarcoplasmic reticulum.

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.

Dantrolene and azumolene inhibit [3H]PN200-110 binding to porcine skeletal muscle dihydropyridine receptors.

We tested whether the hydantoin muscle relaxants dantrolene, azumolene, or aminodantrolene could alter the binding of [3H]PN200-110 to transverse tubule dihydropyridine receptors or the binding of [3H]ryanodine to junctional sarcoplasmic reticulum Ca2+ release channels. All three drugs inhibited [3H]PN200-110 binding with azumolene (IC50 approximately 20 microM) 3-5 times more potent than dantrolene or aminodantrolene. In contrast, 100 microM azumolene and dantrolene produced a small inhibition of [3H]ryanodine binding (less than 25%) while aminodantrolene was essentially inert. Hence there was a preferential interaction of hydantoins with dihydropyridine receptors instead of ryanodine receptors. Skeletal muscle dihydropyridine receptors may participate in the mechanism of action of dantrolene and azumolene.

Taxol binds differentially to flagellar outer doublets and their reassembled microtubules.

Taxol induces the in vitro assembly of calcium stable microtubules from flagellar tubulin solubilized from sea urchin (Strongylocentrotus purpuratus) sperm tail outer doublets by sonication. Assembly occurs in the presence or absence of exogenous GTP. The drug (10 microM) reduces the critical concentration of protein required for assembly to less than or equal to 0.04 mg/ml. 3H-Taxol binds specifically to both isolated flagellar outer doublets and to reassembled microtubules with calculated maximal binding ratios of 0.25 and 1.32 moles taxol/mole polymerized flagellar tubulin dimer, respectively. We suggest that the discrepancy in maximal binding ratios may result from the presence of an endogenous molecule(s) along the surface of outer doublet microtubules that restricts taxol binding to that structure.

Taxol binds to cellular microtubules.

Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. B. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [3H]taxol and the formation of microtubule bundles. [3H]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubules in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [3H]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaN3 prevents the characteristic taxol-induced bundle formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.

Taxol binds to polymerized tubulin in vitro.

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5'-guanylyl-alpha,beta-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.