Structure and function of receptor-operated Ca^<2+> channels and development of specific blockers of the channels

本研究的目的是分离内皮素A型受体(ET_AR)激活的受体操纵型钙通道的编码基因。我们发现，传统的双电极电压钳方法不能用于筛选Ca^&lt；2+&gt；内流，因为受体的单独表达引起了大的内向电流，这是由于从细胞内储存中动员的Ca^&lt；2+&gt；引起的Ca^&lt；2+&gt；激活的氯离子电流。因此，为了区分通过细胞膜通道流入的钙和从储存库中动员的钙离子，我们决定在显微注射CRNA的卵母细胞中摄取来自不同组织的ET_AR和mRNA。为了提高方法的灵敏度，我们开发了一种用~(14)&gt；C-菊粉鉴定受损卵母细胞的方法，并优化了注射CRNA的ET_AR和mRNA的量。即使用改进的方法，我们也不能检测到受体激活的钙离子内流的阳性部分。因此，我们决定使用酵母-双杂交系统来分离与ET_AR直接相互作用的分子。利用这种方法，我们已经成功地分离了26个克隆，并正在分析它们在ET_AR介导的细胞内钙信号转导中的功能。

The purpose of the present study is to isolate cDNAs encoding for receptor-operated Ca^<2+> channels activated by endothelin type A receptor (ET_AR). We found that the conventional voltage-clamp method with two electrodes cannot be used for screening Ca^<2+> influx, because expression of the receptors alone induced a large inward current resulting from Ca^<2+>-activated Cl- currents due to Ca^<2+> mobilized from the intracellular stores. Therefore, to differentiate Ca^<2+> influx through channels across the cell membrane from Ca^<2+> mobilized from the stores, we decided to use ^<45>Ca^<2+> uptake into the oocytes which were microinjected with cRNA for ET_AR and mRNA prepared from various tissues. To increase the sensitivity of the method, we developed a method to identify the injured oocytes using ^<14>C-inulin and also optimized the amount of injected cRNA for ET_AR and mRNA. Even with the improved method, we could not detect a positive fraction showing receptor-activated Ca^<2+> influx. Therefore, we decided to use a yeast-two hybrid system to isolate molecules interacting directly with ET_AR. Using this method, we have succeeded in isolating 26 clones and are now analyzing their functional roles in ET_AR-mediated Ca^<2+> signaling in the cells.