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Structure and function of ryanodine receptors

兰尼定受体的结构和功能

基本信息

批准号：
09470022
负责人：
TAKESHIMA Hiroshi
金额：
$ 8.38万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The structure-function relationship of ryanodine receptor subtypes (namely, RyR-l, 2 and 3) have been investigated by means of molecular biological and genetic methords, for example in vitro mutagenesis and generation of knockout mice. Our major findings in this research project are listed below.1. Generation and characterization of mutant mice lacking RyR-2. Because function of RyR-2 was unknown in embryonic heart, we have generated mutant mice lacking RyR-2. The results in the mutant mice showed non-essential function of RyR-2 for contraction of embryonic cardiac muscle cells, however, the RyR-2-deficient mice exhibiting cardiac arrest die around embryonic day 10. Our results obtained indicate that RyR-2 is essential for the maintenance of intracellular Ca^<2+> stores by regulating luminal resting Ca^<2+> levels in embryonic cardiac muscle cells.2. Intramolecular functional regions on the primary structure of the ryanodine receptor. We have mapped a region critical for skeletal muscle excitation-contraction coupling to the called D2 region on the RyR-1 molecule in our cDNA expression system using RyRs-deficient muscle. By combination of cDNA expression and single channel measurement, we have located the Ca^<2+> release channel-forming domain and a portion (designated as D3 region) responsible for Ca^<2+> binding for channel inactivation on the RyR-1 molecule.3. Identification of novel proteins localized in skeletal muscle triad junction. We have identified mitsugumin29 (MG29) and MG23 as novel transmembrane proteins from skeletal muscle the triad junction by isolating specific monoclonal antibodies and cloning cDNAs. MG29 was shown to be a novel member of the synaptophysin family, suggesting that the presynaptic region and triad junction share evolutionally related components.

兰尼碱受体亚型(即RyR-1、2和3)的结构-功能关系已经通过分子生物学和遗传学方法进行了研究，例如体外诱变和产生敲除小鼠。我们在这个研究项目中的主要发现如下： 1．缺乏 RyR-2 的突变小鼠的产生和表征。由于 RyR-2 在胚胎心脏中的功能尚不清楚，因此我们产生了缺乏 RyR-2 的突变小鼠。突变小鼠的结果显示RyR-2对于胚胎心肌细胞的收缩不是必需的功能，然而，RyR-2缺陷的小鼠表现出心脏骤停在胚胎第10天左右死亡。我们获得的结果表明RyR-2通过调节胚胎心肌细胞中的管腔静息Ca^2+水平对于维持细胞内Ca^2+储存是必需的。2．兰尼定受体一级结构上的分子内功能区。我们使用 RyRs 缺陷肌肉，将骨骼肌兴奋-收缩耦合的关键区域映射到我们的 cDNA 表达系统中 RyR-1 分子上的 D2 区域。通过结合cDNA表达和单通道测量，我们定位了RyR-1分子上的Ca^2+释放通道形成结构域和负责Ca^2+结合导致通道失活的部分(称为D3区)。3．鉴定位于骨骼肌三联体连接处的新型蛋白质。我们通过分离特异性单克隆抗体和克隆 cDNA，将 mitsugumin29 (MG29) 和 MG23 鉴定为来自骨骼肌三联体连接处的新型跨膜蛋白。 MG29被证明是突触素家族的新成员，表明突触前区域和三联体连接共享进化相关的成分。