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首页> 外文学位 >Insights into the regulation of the ryanodine receptor: Differential effects of calcium(2+), magnesium(2+) and pharmacological agents on ATP binding.
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Insights into the regulation of the ryanodine receptor: Differential effects of calcium(2+), magnesium(2+) and pharmacological agents on ATP binding.

机译：深入了解ryanodine受体的调节：钙（2+），镁（2+）和药理剂对ATP结合的差异作用。

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摘要

The ryanodine receptors (RyRs) are proteins involved in the release of calcium ions from the sarcoplasmic reticulum. In this study, we have investigated the effects of main cellular effectors of RyR1, Ca 2+ and Mg2+ as well as pharmacologically important agents on the binding characteristics of a third cellular effector of the channel, ATP. Employing an innovative approach using ESR spectroscopy and an ATP analog that carries an ESR-active reporter group, spin-labeled ATP, we directly observed for the first time a total of eight ATP binding sites present on the tetrameric RyR1. We showed that the number of ATP binding sites accessible to the ATP analog and the respective dissociation constants directly depended on the presence and concentrations of Ca2+ and Mg 2+ ions. The RyR1 inhibitions induced by the presence of Ca 2+ or Mg2+ ions resulted in the binding of respectively four and eight ATP analog to the channel, pointing to the existence of two different inhibition mechanisms. Our results also implied that the accessibility of 8 sites seems to correspond to an open channel, while only 4 sites seemed to indicate a stably closed channel. ATP binding modulation to RyR1 therefore appears to play a central role in stabilizing the different conformations adopted by the channel during opening and closing events.; Ryanodine, caffeine, dantrolene and tetracaine are exogenous compounds known or suggested to affect the activity of RyR1. By using ESR spectroscopy, we investigated the ATP binding characteristics to the channel in the presence of these pharmacological agents. We demonstrated that different classes of exogenous effectors act differently on RyR1 by interfering with the negative feedback action of calcium ions on the channel with or without decreasing the ATP binding affinity. We also showed that dantrolene, the unique compound used to treat patients with maligniant hyperthermia, may be able to restore the missing negative feedback action of calcium ions on RyR1. ATP binding modulation to RyR1 appears to be crucial and in correlation with the modified activity of the channel induced by pharmacological agents. We demonstrated that ATP binding studies are a major tool to determine the mode of action of pharmacological agents on RyR1.; We attempted to localize the ATP binding sites of RyR1 by photoaffinity labeling using 2-N3-ATP. Our efforts were not successful, potentially due a low photoaffinity labeling efficiency and a low yield of recovery of the labeled peptide. Therefore the precise location of these ATP binding sites on RyR1 is still to be discovered.; Attempt to clone the cDNA of the human RyR1 into a high expression yeast strain were unfortunately also unsuccessful, most of the problems being due to the large size of the gene coding for the 550000 Da protein monomer of RyR1.
机译：ryanodine受体（RyRs）是参与从肌质网释放钙离子的蛋白质。在这项研究中，我们研究了RyR1，Ca 2+和Mg2 +的主要细胞效应子以及药理学上重要的药物对通道第三细胞效应子ATP结合特性的影响。我们采用ESR光谱技术和带有ESR活性报告基团的ATP类似物（自旋标记的ATP）采用创新方法，首次直接观察到四聚体RyR1上共有八个ATP结合位点。我们表明，ATP类似物可及的ATP结合位点的数量和各自的解离常数直接取决于Ca2 +和Mg 2+离子的存在和浓度。由Ca 2+或Mg2 +离子的存在引起的RyR1抑制导致分别有四个和八个ATP类似物与通道结合，这表明存在两种不同的抑制机制。我们的结果还暗示，8个站点的可访问性似乎对应于一个开放频道，而只有4个站点似乎表示一个稳定的封闭频道。因此，对RyR1的ATP结合调节似乎在稳定通道在打开和关闭过程中所采用的不同构象方面起着核心作用。 Ryanodine，咖啡因，dantrolene和丁卡因是已知或建议影响RyR1活性的外源性化合物。通过使用ESR光谱，我们研究了在这些药理剂存在下ATP与通道的结合特征。我们证明了不同类别的外源性效应子通过干扰钙离子在通道上的负反馈作用（或不降低ATP结合亲和力）而对RyR1的作用不同。我们还显示，丹特林是用于治疗恶性高热患者的独特化合物，它可能能够恢复钙离子对RyR1缺失的负反馈作用。 ATP对RyR1的结合调节似乎至关重要，并且与药理药物诱导的通道修饰活性相关。我们证明了ATP结合研究是确定药理剂对RyR1作用方式的主要工具。我们尝试使用2-N3-ATP通过光亲和标记来定位RyR1的ATP结合位点。我们的努力未成功，可能是由于光亲和性标记效率低以及标记肽的回收率低。因此，仍需要发现这些ATP结合位点在RyR1上的精确位置。不幸的是，尝试将人RyR1的cDNA克隆到高表达酵母菌株中也是失败的，大多数问题是由于编码RyR1的550000 Da蛋白单体的基因的大小很大。

著录项

  • 作者

    Viana Dias, Jose Miguel.;

  • 作者单位

    Southern Methodist University.;

  • 授予单位 Southern Methodist University.;
  • 学科 Biology Molecular.; Biology Cell.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 142 p.
  • 总页数 142
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;细胞生物学;生物化学;
  • 关键词

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