The skeletal muscle ryanodine receptor identified as a molecular target of [3H]azidodantrolene by photoaffinity labeling.
The skeletal muscle ryanodine receptor identified as a molecular target of [3H]azidodantrolene by photoaffinity labeling.
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通过光亲和标记，骨骼肌兰尼碱受体被鉴定为[3H]叠氮丹曲林的分子靶标。
DOI：
10.1021/bi001502s
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Parness,J
中科院分区：
文献类型：
--
作者：
Paul-Pletzer,K;Palnitkar,SS;Jimenez,LS;Morimoto,H;Parness,J
丹曲林是一种骨骼肌松弛剂，通过抑制肌浆网（SR）的细胞内Ca 2+释放发挥作用。它主要用于治疗恶性高热（MH），这是一种对挥发性麻醉剂的药物遗传学敏感性，导致大量细胞内Ca 2+释放。丹曲林的作用部位和机制的确定应有助于了解骨骼肌细胞内钙释放的调节。用[3 H]叠氮丹曲林（一种可光活化的丹曲林类似物）对猪SR进行光亲和标记，已鉴定出一种160 kDa SR蛋白，该蛋白与骨骼肌兰尼碱受体（RyR）具有免疫交叉反应性，可能是靶点[Palnitkar et al.（1999）J. Med. Chem.42，1872 - 1880]。在这里，我们证明了具体的，AMP-PCP增强，[3 H]叠氮丹曲林光标记的RyR单体和160或172 kDa的蛋白质在猪和兔SR，分别。160/172 kDa的蛋白质被证明是从单体切割的RyR的NH 2-末端的内源性蛋白酶活性与n-钙蛋白酶一致。猪160 kDa蛋白质的MALDI-质谱分析鉴定其为骨骼肌RyR的1400个氨基酸的NH 2-末端片段，据报道由n-钙蛋白酶产生[Shevchenko等人（1998）J. Biol. 161，33 - 34]。溶解的，[3 H]叠氮丹曲林光标记的SR蛋白的免疫沉淀显示，切割的160/172 kDa蛋白仍然与C-末端，410 kDa的部分的RyR。AMP-PCP类似地增强了[3 H]丹曲林与完整和n-钙蛋白酶切割通道RyR的结合。在AMP-PCP存在下，RyR的n-钙蛋白酶裂解不影响[3 H]丹曲林结合，但在不存在核苷酸的情况下抑制药物结合。这些结果表明，NH 2-末端的RyR是丹曲林的分子靶点，并建议调节作用的n-calpain活性和ATP在体内的相互作用丹曲林与RyR。
Dantrolene is a skeletal muscle relaxant which acts by inhibiting intracellular Ca2+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 Ca2+release. Determination of the site and mechanism of action of dantrolene should contribute to the understanding of the regulation of intracellular Ca2+release in skeletal muscle. Photoaffinity labeling of porcine SR with [3H]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, [3H]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 NH2-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 NH2-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, [3H]azidodantrolene-photolabeled SR protein reveals that the cleaved 160/172 kDa protein remains associated with the C-terminal, 410 kDa portion of the RyR. [3H]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 [3H]dantrolene binding in the presence of AMP-PCP, but depresses drug binding in the absence of nucleotide. These results demonstrate that the NH2-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.