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

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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.