Screening For Special Molecules Of Store-operated Calcium Channel And Exploring The Property Of The Target Molecules

Store-operated calcium channel (SOC) is a kind of calcium channels existing in many cell types. The basic operating mechanism of SOC is that when the intracellular calcium store depletes, the signal molecules, stromal interaction molecule 1 (STIM1), located on the membrane of the store, will aggregate and bind to the channel protein Orai1, then change the channel protein's structure and open the channel. SOC can regulate many physiological processes, such as regulating transcriptions of some genes, controlling the activity of some enzymes and modulating exocytosis. Meanwhile, SOC can be related to some diseases like severe combind immunodeficiency. However, up to present, there are not enough antagonists and agonists of SOC, and also almost all of the known antagonists and agonists are lack of specificity, which impede our profound understanding of SOC.Our research aims at getting some specific antagonists or agonists of SOC which will make people know SOC better. Using Drosophila S2 cells and the technique of intracellular Ca2+concentration measurement, the basal cellular assay model for screening for the antagonist and agonist of SOC was established. After examing the dependability and stability of the basal cell model, we screened the compounds that were extracted for Chinese herbal medicine. Finally we got an effective compound corosolic acid, which could block store-operated calcium entry (SOCE). The inhibiting effect of corosolic acid was conformed by many experiments. We further explored the inhibiting mechanism of corosolic acid. 1.The establishment of cellular model of screening for antagonist and agonist of SOCUsing Drosophila S2 cells as the cellular model to screen for the antagonist and agonist of SOC. After exploring, the optimized growing environment for Drosophila S2 cells is that 25℃without CO2. Using the known SOC agonist thapsigargin (TG), intracellular calcium indicator Fluo-4 AM, we established the basal cellular assay model for screening antagonist and agonist of SOC. Then using three known Ca2+ channel inhibitors Gd3+,SKF-96365 and 2APB, we examined the dependability and stability of the basal model. And the results show that the basal model is dependable and stable.2. The screening of compounds extracted from Chinese herbal medicine and the exploring of the property of target compoundUsing the established model, we screened some compounds extracted from Chinese herbal medicine and finally got an effective compound corosolic acid which exhibited inhibiting effect. And many other experiments were carried on to confirm corosolic acid can block SOC. Then we explored corosolic acid's inhibiting mechanism and found that corosolic acid could block calcium release from the store. Therefore, we infer that corosolic acid probably take the inhibiting effect through blocking calcium release.