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

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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.