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

有多种类型的细胞内钙离子(Ca^&lt；2+&gt；)信号作为内部第二信使调节许多不同的细胞过程。我们试图建立一种实验程序来同时分析钙信号与细胞内钙离子释放通道(Ryanodine Receptor：RyR)之间的关系，包括一些RyR1突变体，如恶性高热(MH)1。我们利用每隔1500 b.2引入的独特的限制性内切酶切点将RyR(RyR1)cDNA1分成11个片段。将重组的RyR1与绿色荧光蛋白(GFP)在RyR1的发散区(D2)融合表达，并在活细胞中可见。我们构建并表达了系列缺失的RyR1克隆来分析RyR1的结构-功能关系，发现3226-3724氨基酸的内部区域对于钙释放通道的功能形成并不是关键的。提示RyR1中可能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RyR1突变，并通过RyR1基因的定点突变实验证实了RyR1基因中的一个单核苷酸多态(SNPs)可以增加药物敏感性。

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.