Structure and function of ryanodine receptors

用体外诱变和基因敲除小鼠等分子生物学和遗传学方法研究了兰尼定受体亚型(即RyR-L、2和3)的结构与功能的关系。我们在这项研究项目中的主要发现如下。RyR-2缺失突变小鼠的产生及特性研究。由于RyR-2在胚胎心脏中的功能尚不清楚，我们产生了缺失RyR-2的突变小鼠。突变小鼠的结果显示RyR-2对胚胎心肌细胞的收缩不是必需的功能，然而，出现心脏骤停的RyR-2缺陷小鼠在胚胎第10天左右死亡。我们的结果表明，RyR-2通过调节胚胎心肌细胞的腔静息钙水平来维持细胞内钙的储存是必不可少的。兰尼定受体一级结构上的分子内功能区。我们利用RyRs缺失的肌肉，将骨骼肌兴奋-收缩偶联的关键区域定位到RyR-1分子上的D2区域。通过cDNA表达和单通道检测相结合，我们在RyR-1分子上定位了钙释放通道形成结构域和负责钙通道失活与钙结合的部分(命名为D3区)。定位于骨骼肌三联体连接的新蛋白的鉴定。我们通过分离特异性的单抗和克隆cDNA，从骨骼肌的三联体连接中鉴定出mitsugum29(MG29)和MG23为新的跨膜蛋白。MG29是突触素家族的一个新成员，提示突触前区和三联体连接具有共同的进化相关成分。

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.