Analysis on opening modes of ryanodine receptore-Ca^<2+> release channel

兰尼碱受体e-Ca^<2>释放通道的开放方式分析

• 批准号: 14570084
• 负责人: IKEMOTO Takaaki
• 金额: $ 2.05万
• 依托单位: Saitama Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570084/
• 关键词: Calcium ion; Ryanodine receptor; Skeletal muscle; Sarcoplasmic reticulum; Ca^<2+> -induced Ca^<2+> release; Dantrolene; Antihyperlipidemic agent; rhabdomyolysis; HMG-CoA reductase; Ca^<2+>によCa^<2+>放出

We analyzed the effect of HMG-CoA reductase inhibitors on Ca^<2+> release from the sarcoplasmic reticulum (SR) using chemically skinned skeletal muscle fibers. Cerivastatin (Cer, > 20μM) released Ca^<2+> from the SR, while pravastatin showed only a little effect. The rates of Ca^<2+> release were increased by Cer at all Ca^<2+> concentrations tested. Cer-induced Ca^<2+> release in the presence of Ca^<2+> was affected by adenosine monophosphate, Mg^<2+> and procaine in essentially the same way as for caffeine-induced Ca^<2+> release. The Ca^<2+>-uptake capacity of the SR was reduced after co-treatment with ryanodine and Cer at pCa6.0 to a much grater extent than with ryanodine alone. Thus, Cer-induced Ca^<2+> release in the presence of Ca^<2+> must be a result of the activation of the Ca^<2+>-induced Ca^<2+> release (CICR) mechanism of the ryanodine receptor. However, even under the condition that CICR was maximally inhibited by Mg^<2+> and procaine, or in the practical absence of Ca^<2+>, Cer still caused Ca^<2+> releases. These results indicate that Cer causes Ca^<2+> release also by activating some other mechanism(s) in addition to the activation of CICR. Either or both of these effects might be related to its adverse effect, rhabdomyolysisA novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.

我们使用化学去皮的骨骼肌纤维分析了HMG-CoA还原酶抑制剂对肌浆网（SR）Ca^2+释放的影响。西立伐他汀（Cer，> 20μM）可释放SR中的Ca^<2+>，而普伐他汀的作用很小。在所有的Ca^<2 +>浓度下，Cer都能增加Ca^<2 +>的释放速率。在Ca^<2 +>存在的情况下，铈诱导的Ca^<2 +>释放受到腺苷酸、Mg^<2+>和普鲁卡因的影响，其方式与咖啡因诱导的Ca^<2+>释放基本相同。在pCa 6.0时，与单独使用ryanodine相比，ryanodine和Cer共同处理后SR的Ca^<2+>-摄取能力降低的程度更大。因此，在Ca^2+存在的情况下，铈诱导的Ca^2+释放一定是兰尼碱受体的Ca^2+诱导的Ca ^2+释放（CICR）机制激活的结果。然而，即使在Mg^<2+>和普鲁卡因最大程度地抑制CICR的情况下，或者在实际上没有Ca^<2+>的情况下，Cer仍然引起Ca^<2+>的释放。这些结果表明，除了激活CICR外，Cer还通过激活某些其他机制引起Ca^2+释放。这些作用中的一种或两种可能与其副作用横纹肌溶解有关。开发了一种用于无放射性同位素的光亲和标记的新方法，其中通过激活光反应性基团，例如芳香族叠氮基或3-三氟甲基-3H-二氮杂环丙烯-3-基，并且通过Staudinger-Bertozzi反应将可检测标记锚定在能经受光解的烷基叠氮基部分上来实现连接产物的鉴定。该方法的化学基础是使用具有双官能团的模型化合物在光照射下在反应中间体的捕集剂的存在下确认的。该方法的实用性已通过特异性标记人HMG-CoA还原酶的催化部分得到证实。

项目成果

Hosoya et al.: "Novel bifunctional probe for radioisotope-free photoaffinity labeling : compact structure comprised of photospecic ligand ligation and detectable tag anchoring units."Organic & Biomolecular Chemistry. 2. 637-641 (2004)

Hosoya 等人：“用于无放射性同位素光亲和标记的新型双功能探针：由光特异性配体连接和可检测标签锚定单元组成的紧凑结构。”有机

Hosoya et al.: "[^<125>I]-N-[(3-Azido-5-iodo)benzyl]dantrolene and [^<125>I]-N-{[3-iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl}dantrolene : Photoaffinity Probes Specific for the Physiological Ca^<2+> Release from Sarcoplasmic Reticulum of Skeletal

Hosoya 等人：“[^<125>I]-N-[(3-Azido-5-iodo)benzyl]dantrolene 和 [^<125>I]-N-{[3-iodo-5-(3

Inoue et al.: "Ca^<2+>-releasing effect of cerivastatin on the sarcoplasmic reticulum of mouse and ratskeletal muscle fibers."Journal of Pharmacological Science. 93. 279-288 (2003)

Inoue等人：“西立伐他汀对小鼠和大鼠骨骼肌纤维肌浆网的Ca 2+ 释放作用。”药理学杂志。

Hosoya et al.: "Novel bifunctional probe for radioisotope-free photoaffinity labeling : compact structure comprised of photospecic ligand ligation and detectable tag anchoring units."Organic & Biomolecular Chemistry.. 2. 637-641 (2004)

Hosoya 等人：“用于无放射性同位素光亲和标记的新型双功能探针：由光特异性配体连接和可检测标签锚定单元组成的紧凑结构。”有机

Hosaka et al.: "α1-Syntrophin-deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration."Journal of Cell Biology. 158. 1097-1107 (2002)

Hosaka 等人：“α1-肌营养蛋白缺陷的骨骼肌在再生过程中表现出肥大和神经肌肉接头的异常形成。”细胞生物学杂志 158. 1097-1107 (2002)