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

受体操纵的 Ca^2 通道的结构和功能以及该通道的特异性阻断剂的开发

基本信息

批准号：
15390075
负责人：
MIWA Soichi
金额：
$ 8.38万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390075/
关键词：
Endothelin receptor Vascular smooth muscle cell Ca^<2+> channel Xenopus oocyte Ca^<2+> uptake yeast two-hybrid GST pull-down cDNA cloning cDNAクローニングエンドセリン収縮受容体作動性カルシウムチャネル

项目摘要

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.

本研究的目的是分离编码内皮素A型受体（ET_AR）激活的受体操纵性Ca^2+通道的cDNA。我们发现，传统的双电极电压钳方法不能用于筛选Ca^2+内流，因为单独表达受体会诱导一个由Ca ^2+激活的Cl-电流引起的大的内向电流，而这是由于Ca^2+从细胞内储存库中动员出来。因此，为了区分Ca^2+通过细胞膜通道流入和从储存中动员的Ca^2+，我们决定使用<45>微注射ET_AR cRNA和从各种组织制备的mRNA的卵母细胞中的Ca^2+摄取。为了提高该方法的灵敏度，我们开发了一种使用β-C-菊粉鉴定受损卵母细胞的方法<14>，并优化了ET_AR和mRNA的cRNA注射量。即使使用改进的方法，我们也不能检测到受体激活的Ca^2+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，我们成功地分离了26个克隆，现在正在分析它们在细胞中ET_AR介导的Ca^<2+>信号传导中的功能作用。