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

本项目旨在克隆大鼠肠系膜动脉内皮衍生超极化因子（EDHF）表达的靶离子通道，并对其功能进行分析。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。将RNA逆转录为cDNA，利用Ca^<2+>-活化钾离子通道（SKI, SK2, SK3， IK， BK）特异引物进行PCR分析。RT-PCR分析结果显示，SKI、SK2、SK3、IK和BK在大鼠脑中表达，SK3和IK在大鼠心脏中表达，SK3、IK和BK在大鼠肠系膜动脉中表达，IK在A7r5中表达。接下来，利用RT-PCR方法和cDNA 1文库筛选对这些钾离子通道进行克隆。利用大鼠心脏cDNA文库筛选，从大鼠心脏中克隆SK3 cDNA。用RT-PCR方法从大鼠脑中克隆SK3。利用RT-PCR从大鼠脑中克隆BK。IK现在从A7r5的cDNA克隆。测序分析表明，克隆的SK3与先前报道的大鼠心脏SK3基本相同，但克隆的BK在BK通道的c端侧有9个碱基对插入。这些通道在哺乳动物细胞中表达，并进行了功能分析。我还突变了作为显性负通道的通道，并检测了突变通道对大鼠肠系膜动脉EDHF功能的影响。这些通道难以表达到大鼠肠系膜动脉。我也用共聚焦显微镜检查了这些通道在大鼠肠系膜动脉中的定位。SK3似乎在内皮细胞中表达。EDHF的靶离子通道需要进一步的实验来阐明。

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.