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

本课题的目的是克隆大鼠肠系膜动脉表达的内皮衍生超极化因子(EDHF)的靶离子通道，并对该通道进行功能分析。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A7r5中提取总RNA。用钙激活钾离子通道(SKI、SK2、SK3、IK、BK)的特异性引物进行聚合酶链式反应分析。RT-PCR分析结果表明，SKI、SK2、SK3、IK和BK在大鼠脑表达，SK3和IK在大鼠心脏表达，SK3、IK和BK在大鼠肠系膜动脉表达，IK在A7r5表达。然后，用RT-PCR方法和cDNA1文库筛选的方法克隆了这些钾通道。采用大鼠心脏cDNA文库筛选的方法，从大鼠心脏中克隆了SK3基因。用RT-PCR方法从大鼠脑组织中克隆了SK3基因。用RT-PCR方法从大鼠脑组织中克隆了BK。Ik现已从A7r5的cDNA中克隆出来。序列分析表明，克隆的SK3与已报道的大鼠心脏SK3序列基本一致，但克隆的BK在BK通道的C末端插入了9个碱基对。这些通道在哺乳动物细胞中表达，并进行功能分析。我还对通道的显性负性通道进行了突变，并检测了突变通道对大鼠肠系膜动脉EDHF功能的影响。这些通道对大鼠肠系膜动脉的表达较为困难。我还用共聚焦显微镜观察了这些通道在大鼠肠系膜动脉中的定位。SK3看起来像是在内皮细胞中表达。EDHF的靶离子通道还需要进一步的实验研究。

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.