SCREENING FOR THE FUNCTIONAL DOMAINS IN THE RYANODINE RECEPTOR

1.通过引入独特的限制性内切酶位点（5’-XbaI、677SalI、2349Bsu36I、3798BstBI、5355MluI、6822SpeI、8476BsiWI、9804AvrII、11290NdeI、12675NheI、14427ClaI和edge- hindiii -3’end），将兔RyR1受体（RyR1） cDNA分成11个片段（约15000bp）。我们在CHO细胞中表达了由部分缺失克隆（16个克隆）编码的重组RyR1突变体，或通过与绿色荧光蛋白（GFP）融合的分裂磁带重组的部分克隆（12个克隆）。转染1、5、6、7和11的CHO细胞的GFP荧光信号在细胞质中除细胞核外呈点状结构，即使在质膜通透化后仍能保留，与全长表达RyR1的细胞相同。这些结果表明，RyR1分子有几个区域（氨基端、中央端和羧基端）作为细胞内钙储存区，负责向内质网传递保留/恢复信号。这些内质网滞留区域与恶性高热患者聚集的RyR1基因突变相对应，也提示这些区域对钙释放通道具有重要作用

1.We divided the ryanodine receptor (RyR1) cDNA (about 15000bp) from a rabbit into 11 cassettes by introducing unique restriction endonuclease sites (5'-XbaI, 677SalI, 2349Bsu36I, 3798BstBI, 5355MluI, 6822SpeI, 8476BsiWI, 9804AvrII, 11290NdeI, 12675NheI, 14427ClaI, and edge-HindIII-3'end).2.We expressed the recombinant RyR1 mutants encoded by partly deleted clones (16 clones) or by partial clones (12 clones) reconstructed by the divided cassettes fused with a green fluorescent protein (GFP) in CHO cells.3.The GFP fluorescent signals from the CHO cells transfected with the cassettes 1,5,6,7 or 11 were observed as the dot-like structures in the cytoplasm except for the nucleus and could be remained even after the plasma membranes permeabilized as seen in those cells having the full-length of RyR1 expressed.4.These results reveals that there are several regions (the amino terminal, the central and the carboxyl terminal regions) of the RyR1 molecule which are responsible for the retention / retrieval signals to the endoplasmic reticulum as the intracellular calcium stores of the cells. These regions for the ER retention are corresponded to the genetic mutations of RyR1 clustered in the malignant hyperthermia patients also suggesting that these regions have the important roles for the calcium release channel