Probing a putative dantrolene-binding site on the cardiac ryanodine receptor
Probing a putative dantrolene-binding site on the cardiac ryanodine receptor
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DOI：
10.1042/bj20041336
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发表时间：
2005-05-01
影响因子：
4.1
通讯作者：
Parness, J
中科院分区：
文献类型：
--
作者：
Paul-Pletzer, K;Yamamoto, T;Parness, J
丹曲林是骨骼肌肌浆网细胞内钙释放的抑制剂。用[H-3]叠氮杂三烯和人工合成的结构域肽进行的直接光亲和标记实验表明，该药物针对骨骼肌RyR亚型RyR1(Ryanodine Receptor 1)的590-609个氨基酸[称为DP1(结构域肽1)]。尽管在心脏亚型RyR2(残基601-620)中存在相同的序列，但丹曲林对RyR2的特异性标记尚未得到证实，尽管一些功能研究表明丹曲林对心脏功能有保护作用。在这里，我们测试RyR2中是否存在丹曲烯活性结构域，如果存在，是否可以调制该结构域。我们现身。来自RyR1(DP1-2S；残基590-628)和RyR2(DP1-2c；残基601-639)的延长的DP1序列可以被[H-3]叠氮齿三烯光标记。在Western印迹和免疫沉淀实验中，以DP1区为表位的抗RyR1单抗只能识别RyR1而不能识别RyR2，但同时识别DP1-2c和DP1-2s。这表明，尽管RyR2序列在体外具有与丹曲林结合的内在能力，但这个位点在天然通道蛋白中可能很难获得。为了检验是否有可能调节这个位点，我们测量了[H-3]丹曲林与心脏SR的结合作为游离钙的函数。我们发现>；=10 mM的EGTA使[H-3]丹曲林与RyR2的结合增加了近2倍。这些数据表明，RyR2上的丹曲烯结合位点是构象敏感的。该部位可能成为对细胞内钙释放功能障碍敏感的心血管疾病的潜在治疗靶点。
Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [H-3]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show. that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photo-labelled by [H-3]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [H-3]dantrolene to cardiac SR as a function of free Ca2+. We found that >= 10 mM EGTA increased [H-3]dantrolene binding to RyR2 by similar to 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.