SPATIO-TEMPORAL ANALYSIS OF CA^<2+> RELEASE CHANNEL RELATED TO THE VARIOUS TYPE OF THE INTRACELLULAR CA^<2+> SIGNALS

细胞内钙离子（Ca^<2+>）信号作为第二信使参与多种细胞过程的调控。我们试图建立同时分析Ca^2+信号和细胞内Ca^2+诱导的Ca^2+释放（CICR）通道（Ryanodine receptor：RyR）之间关系的实验程序，包括RyR 1的一些突变体，如恶性高热（malignant hyperthermia，MH）1。利用间隔1500 bp引入的特异性限制性内切酶位点将RyR 1型cDNA（约15000 bp）分为11个盒.我们表达了融合绿色荧光蛋白（GFP）的重组RyR 1，并在活体细胞中观察到.我们构建并表达了RyR 1的串联缺失克隆，分析了RyR 1的结构与功能关系，发现RyR 1的3226 - 3724位氨基酸的内部区域不是Ca^2+释放通道功能形成的关键区域.提示RyR 1中存在多个位点作为滞留信号进入内质网.我们在MH患者中发现了新的RyR 1突变，并通过RyR 1 cDNA的定点突变实验证实了RyR 1基因中的单核苷酸多态性（SNP）之一，以增加药物敏感性。

There are various types of intracellular calcium ion (Ca^<2+>) signaling as an internal second messenger to regulate so many diverse cellular processes. We have tried to establish the experimental procedure for analyzing the relationships between the Ca^<2+> signaling and the intracellular Ca^<2+>-induced Ca^<2+> release (CICR) channel (ryanodine receptor : RyR) simultaneously, including some RyR1 mutants such as the malignant hyperthermia (MH)1. We divided the type 1 of RyR (RyR1) cDNA (about 15000bp) into 11 cassettes by utilization of unique restriction endonuclease sites introduced at intervals of 1500 bp.2. We expressed the recombinant RyR1 fused with a green fluorescent protein (GFP) in the divergent region (D2) of RyR1 to be visualized in living cells.3. We constructed and expressed serial deletion RyR1 clones to analyze the structure-function relationships of RyR1 and found that the internal region from the amino acids 3226 to 3724 was not a critical for the functional formation of the Ca^<2+> release channel.4. There suggested to be multiple sites in RyR1 as the retention signals into endoplasmic reticulum.5. We found novel RyR1 mutations in MH patients and confirm the one of the single nucleotide polymorphisms (SNPs) in the RyR1 gene to increase drug sensitivities by the site-directed mutagenesis experiments of the RyR1 cDNA.