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

点击翻译按钮获取中文摘要

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.