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Reaction mechanism of sarcoplasmic reticulum calcium release channels

肌浆网钙释放通道的反应机制

基本信息

批准号：
02670417
负责人：
SHIGEKAWA Munekazu
金额：
$ 1.47万
依托单位：
National Cardiovascluar Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670417/
关键词：
Cardicac sarcoplasmic reticulum Ca channel cDNA cloning Protein Kinase Phosphorylation Ryanodine Calmodulin CHO cells

项目摘要

1)The sequence of 4968(or 4976 with an insertion)amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by-cloning and sequencing the CDNA. This protein is homologous in antino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with MRNA derived from the cardiac ryanodine receptor CDNA exhibit Ca^<2+>-dependent Cl^- current in response caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor MRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac MRNA. Ibis results, in conjunction with cloning and analysis of partial CDNA sequence, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.2)The exogenous addition of tie catalytic subunit of cAMP-depeiident protein kinase(PK … More A), cOMP-dependent protein kinase(PKG), or calmodulin(CaM)induced rapid, phosphorylation of the ryanodine receptor(Ca^<2+> release channel)in canine cardiac microsomes treated with lmM[gamma- ^<32>P]ATP. Added pro(ein kinase C(PKC)also phosphorylated the cardiac ryanodiiie receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependen(phosphorylation of the ryanodine receptor was comparable to the maximum level of[ ^3H]ryanodine binding in cardiac microsomes. wherets the level of CaM-dependent phosphorylation was about 4 times greater. Phospliorylation by PKA. PKG, and PKC increased[ ^3H]rryanodine binding in cardiac microsomes by 22<plus-minus>5, 17<plus-minus>4, and 15<plus-minus>9 %(average <plus-minus>SD, n=4-5), respectively. In contrast, incubation of microsomes with 5 mu M CaM alone and 5 mu M CaM plus 1 mM ATP decreased[ ^3H]ryanodine binding by 38<plus-minus>14 and 53<plus-minus>15 %(average <plus-minus>SD. n=6). respectively. Pliosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA. FIKO. and PKC predominantly phosphorylate serine residue(s)in the same phospliopeptide(peptide I). whereas the endogetious CaM-kinase phosphorylates serine residue(s)in a different phosphopeptide(peptide 4). Photoaffinity labeling or microsomes with photoreactive ^<125>I-labeled CaM revealed that CaM bound to a high niolecular weight protein, which was immunoprecipitated by a monoclogial antibody against the cardiac ryanodine receptor. These results suggest that protein kittase-det, eiidciit pliospliorylatioii and CaM play important regulatory roles iii tlie function of the cardiac sarcoplasniic reticulum Ca^<2+> release channel.3)We constructed an expression plasmid(pMAMCRR51)that carries the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the mouse mammary tumor virus promotor and E. coli xanthine-guanine phosphoribosyltransferase(gpt)as a selectable marker. Chinese hamster ovary(CHO)cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca^<2+> transients were. selected. Immunoprecipitation with a monocional antibody against the canine cardiac ryatiodine receptor revealed that the cell clones thus selected exhibited Ca^<2+>-dependent[ ^33H]ryanodine binding activity, which was stimulated by 10 mM ATP or 1 M KCI. The apparent dissociation constatit(Kd)for [ ^3H]ryanodine was 5.2 nM. which was similar to the Kd observed with cardiac microsomes. Immunoprecipitation also demonstrated that the cell clones produced a protein indistinguishable in Mr from the ryatiodine receptor in canine cardiac microsomes. The amount of ryanodine receptor expressed in CHO cells increased significantly after dexamethasone induction. We examined the effect of Ca^<2+> and caffeine on Ca^<2+> release from intracellular Ca^<2+> stores using saponin-skinned CHO cells. Addition of micromolar levels of Ca^<2+> evoked rapid Ca^<2+> release from skinned CHO cells expressing the cardiac ryanodine receptor. In the presence of 0.1 muM Ca^<2+>, however, some Ca^<2+> release observed with the skinned CHO cells, but addition of 5 mM caffeitie stimulated Ca^<2+> release. The amounl of Ca^<2+> release induced by addition of micromolar levels of Ca^<2+> or 5 muM caffeine was similar to that observed after addition of ionomycin. These results clearly demonstrate that the cardiac ryanoditie receptor expressed in CHO cells functions as a Ca^<2+>-induced Ca^<2+> release channel. Less

1)通过对兔心肌肌浆网ryanodine受体cDNA的克隆和序列测定，推导出ryanodine受体的4968（或4976个插入）氨基酸序列。这种蛋白质在抗氨基酸序列上是同源的，并且与骨骼肌兰尼碱受体共享特征性结构特征。将来自心脏兰尼碱受体cDNA的mRNA注入非洲爪蟾卵母细胞，卵母细胞对咖啡因的反应表现出Ca^2+依赖性Cl^-电流，这表明功能性钙释放通道的形成。RNA印迹杂交分析与探针特异性的心脏兰尼碱受体mRNA显示，胃和大脑包含一个可杂交的RNA种类的大小类似的心脏mRNA。结合部分cDNA序列的克隆和分析，结果表明脑内含有心肌型兰尼碱受体mRNA。2）外源性添加cAMP依赖性蛋白激酶（PK）催化亚基， ...更多信息 A）、cOMP依赖性蛋白激酶（PKG）或钙调素（CaM）诱导了用1 mM [γ-P]ATP处理的犬心脏微粒体中兰尼碱受体（Ca^2+释放通道）的快速磷酸化<32>。加入蛋白激酶C（PKC）也使心脏兰尼酸受体磷酸化，但速率相对较慢。在心肌微粒体中，观察到的ryanodine受体的PKA、PKG或PKC依赖性磷酸化水平与[ ^3H]ryanodine结合的最大水平相当。其中CaM依赖性磷酸化水平高约4倍。PKA磷酸化。PKG和PKC分别使心肌微粒体中[ ^3H]兰尼碱结合增加<plus-minus>22.5%、<plus-minus>17.4%和<plus-minus>15.9%（平均<plus-minus>SD，n=4-5）。与此相反，将微粒体与5 μ M CaM单独孵育，以及与5 μ M CaM加1 mM ATP孵育，[ ^3H]ryanodine的结合分别降低了38 <plus-minus>14%和53 <plus-minus>15%（平均<plus-minus>SD。n=6）。分别多肽图谱和磷酸化氨基酸分析提供了证据表明PKA。菲科而PKC主要磷酸化同一多肽（肽I）中的丝氨酸残基。而内源性CaM激酶磷酸化不同磷酸肽（肽4）中的丝氨酸残基。光亲和标记或微粒体与光反应性^<125>I-标记的CaM显示，CaM结合到高分子量的蛋白质，这是免疫沉淀的单克隆抗体对心脏ryanodine受体。3）构建了兔心肌兰尼碱受体cDNA全序列的表达质粒pMAMCRR 51，将其与小鼠乳腺肿瘤病毒启动子和大肠杆菌E.大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖转移酶（GPT）作为选择标记。用pMAMCRR 51转染中国仓鼠卵巢（CHO）细胞，麦考酚酸抗性细胞显示咖啡因诱导的细胞内Ca^2+瞬变。选定.用抗犬心肌ryanodine受体的单克隆抗体进行免疫沉淀，结果显示，这样选择的细胞克隆显示出Ca^2+依赖的[^33 H]ryanodine结合活性，这种活性可被10 mM ATP或1 M KCl刺激。[ ^3H]ryanodine的表观解离常数（Kd）为5.2 nM。这与用心脏微粒体观察到的Kd相似。免疫沉淀也表明，细胞克隆产生的蛋白质难以区分从犬心脏微粒体ryatiodine受体的先生。地塞米松诱导后CHO细胞Ryanodine受体表达量显著增加。我们用带皂皮的CHO细胞检测了Ca^2+和咖啡因对细胞内Ca ^2+库释放Ca^2+的影响。加入微摩尔水平的Ca^2+可诱导表达心肌兰尼碱受体的去皮CHO细胞快速释放Ca^2+。然而，在0.1 μ M Ca^2+存在的情况下，在去皮的CHO细胞中观察到一些Ca^2+释放，但加入5 mM咖啡因刺激了Ca^2+释放。加入微摩尔浓度的Ca ^<2+>或5 μ M咖啡因诱导的Ca ^<2+>释放速率与加入离子霉素后观察到的相似。这些结果清楚地表明，在CHO细胞中表达的心脏ryanoditie受体起着Ca^2+诱导的Ca^2+释放通道的作用。少