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Cloning and functional analysis of the target ion channel of endothelium-derived hyperpolarizing factor

内皮源性超极化因子靶离子通道的克隆及功能分析

基本信息

批准号：
12670076
负责人：
FUKAO Mitsuhiro
金额：
$ 2.18万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670076/
关键词：
EDHF endothelium vascular smooth muscle EDRF 血管内皮血管平滑筋イオンチャネル

项目摘要

The aim of this project was to clone the target ion channel of endothelium-derived hyperpolarizing factor (EDHF) expressed in rat mesenteric artery and to do the functional analysis of the channel. First, total RNA was extracted from rat brain, lung, heart, aorta, mesenteric artery and rat aortic smooth muscle cell line A7r5. RNA was reverse transcribed to cDNA and PCR analysis using specific primers for Ca^<2+>-activated potassium ion channels (SKI, SK2, SK3, IK, BK) were performed. The results of RT-PCR analysis exhibited that SKI, SK2, SK3, IK and BK was expressed in rat brain, SK3 and IK in rat heart, SK3, IK and BK in rat mesenteric artery and IK in A7r5. Next, these potassium channels were cloned with RT-PCR method and cDNA 1 ibrary screening. SK3 cDNA was cloned from rat heart using cDNA library screening of rat heart. SK3 was also cloned from rat brain using RT-PCR method. BK was cloned from rat brain using RT-PCR. IK was now cloning from cDNA of A7r5. Sequencing analysis showed that cloned SK3 was almost the same to the SK3 reported previously in rat heart, however cloned BK had 9 base pair insertion in the C-terminal side of BK channel. These channels were expressed in mammalian cel ls and functional analysis was performed. I also mutated the channels, which act as dominant negative of the channels, and examined the effect of mutated channels on EDHF function in rat mesenteric artery. The expression of these channels to rat mesenteric artery was difficult. I also examined the localization of these channels in rat mesenteric artery with con focal microscopy. SK3 looks like expressed in endothelial cells. Further experiment should be needed to clarify the target ion channel of EDHF.

本课题的目的是克隆大鼠肠系膜动脉内皮源性超极化因子（EDHF）靶离子通道，并对其进行功能分析。首先，从大鼠脑、肺、心脏、主动脉、肠系膜动脉和大鼠主动脉平滑肌细胞系A7 r5中提取总RNA。将RNA逆转录为cDNA，并使用Ca^<2+>激活的钾离子通道（SKI、SK 2、SK 3、IK、BK）的特异性引物进行PCR分析。RT-PCR分析结果显示，SK 1、SK2、SK 3、IK和BK在大鼠脑中表达，SK 3和IK在大鼠心脏中表达，SK 3、IK和BK在大鼠肠系膜动脉中表达，IK在A7 r5中表达。然后用RT-PCR和cDNA文库筛选的方法克隆了这些钾离子通道。利用大鼠心脏cDNA文库筛选技术，从大鼠心脏中克隆到SK 3 cDNA。用RT-PCR方法从大鼠脑组织中克隆了SK 3基因。采用RT-PCR方法从大鼠脑组织中克隆了BK基因。目前已从A7 r5的cDNA中克隆了IK。测序分析表明，克隆的SK 3与先前报道的大鼠心脏SK 3几乎相同，但克隆的BK在BK通道的C端侧有9个碱基对的插入。这些通道在哺乳动物细胞中表达并进行功能分析。我还突变了通道，这是通道的显性阴性，并检查了突变通道对大鼠肠系膜动脉EDHF功能的影响。这些通道在大鼠肠系膜动脉表达困难。我还研究了这些通道在大鼠肠系膜动脉的定位与共聚焦显微镜。SK 3似乎在内皮细胞中表达。EDHF的靶离子通道尚需进一步的实验研究。