| Co-Investigator(Kenkyū-buntansha) |
YAMADA Mitsuhiko Faculty of Medicine, Osaka University, Assistant Professor, 医学部, 助手 (10263237)
TAKUMI Toru Faculty of Medicine, Osaka University, Assistant Professor, 医学部, 助手 (00222092)
HORI Masatsugu Faculty of Medicine, Osaka University, Professor, 医学部, 教授 (20124779)
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| Research Abstract |
ATP-sensitive potassium channels (K_<ATP>), which represent a family of K^+ channels inhibited by intracellular ATP,have been found in various tissues including heart, pancreatic beta-cells, and smooth muscle. K_<ATP> in different tissues exibit considerable variation in responce to K^+ channel openers (KCO). In the present study we investigated the molecular difference of K_<ATP> channels and establilsh a in vitro screening system to categorize KCOs and also for developping new KCOs. K_<ATP> consists of two different subunits, K^+ channel subunit and sulfonylurea receptor (SUR) subunit. We have cloned BIR/Kir6.2 and uK_<ATP>/Kir6.1, which are K^+ channel subunits. Then we isolated SUR2A and SUR2B cDNAa for SUR subunits. SUR2B was considered as a splice variant of SUR2A and had a very similar sequence to that of SUR1 in its carboxy terminal 42 amino acid residues. We expressed these cDNAs in HEK293T cells and assayd the cells with patch clamp method. When SUR2A and BIR were transfected in HEK cells, very similar channels to those in cardiac muscle were constructed. On the other hand, a combination of SUR2B and uK_<ATP> represents a K_<ATP>, whose properties were almost same as those of vascular smooth muscle K_<ATP>. Pinacidil could activate both SUR2A/Bir and SUR2B/uK_<ATP> channels. Small amount of diazoxide was able to open the SUR2B/uK_<ATP> channels but not those of SUR2A/Bir. Thus, using cloned K_<ATP> channel subunits and HEK293T cells, we reconstituted K_<ATP> channels of cardiac and vascular smooth muscle and evaluated the effects of KCOs on these channels. This in vitro system will be used to categorize KCOs and also develop new drugs.
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