Molecular cloning of ATP-sensitive K^+ channels and development of the method evaluating K^+ channel openers.

ATP 敏感 K^ 通道的分子克隆和评估 K^ 通道开放剂的方法的开发。

基本信息

批准号：
06557044
负责人：
KURACHI Yoshihisa
金额：
$ 9.86万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557044/
关键词：
ATP-sensitive potassium channels heart smooth muscle cloning sulfonylurea receptor ATP K^+ channel opener culture cell スルフオニル尿素受容体カリウムチャネル ATP感受性チャネルスルフォニルウレア cDNA パッチクランプ K_<ATP>チャネル NDP G蛋白

项目摘要

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.

在包括心脏，胰腺β细胞和平滑肌在内的各种组织中发现了代表由细胞内ATP抑制的K^+通道家族的ATP敏感钾通道（K_ <ATP>）。 K_ <ATP>在不同组织中，对K^+通道开瓶（KCO）的反应差异很大。在本研究中，我们研究了K_ <ATP>通道的分子差异和建立体外筛选系统，以分类KCOS以及开发新的KCO。 K_ <ATP>由两个不同的亚基组成，分别是K^+通道亚基和磺酰脲受体（SUR）亚基。我们已经克隆了BIR/KIR6.2和UK_____ <ATP>/kir6.1，它是K^+通道亚基。然后，我们将SUR2A和SUR2B cDNAA分离为SUR亚基。 SUR2B被认为是SUR2A的剪接变体，并且在其42个氨基酸残基中具有与SUR1的序列非常相似。我们在HEK293T细胞中表达了这些cDNA，并用斑块夹法分析了细胞。当在HEK细胞中转染SUR2A和BIR时，构建了与心肌中非常相似的通道。另一方面，SUR2B和UK___________________________________________________________________________________________________________________________________________________ <ATP>几乎与血管平滑肌K_ <ATP>相同。 Pinacidil could activate both SUR2A/Bir and SUR2B/uK_<ATP> channels.少量的二氮氧化物能够打开SUR2B/UK____________________/bir的通道。因此，使用克隆的K_ <ATP>通道亚基和HEK293T细胞，我们重组了心脏和血管平滑肌的K_ <ATP>通道，并评估了KCOS对这些通道的影响。该体外系统将用于对KCO进行分类并开发新药。