Structure and function of mitochondrial ATP-sensitive K^+ channel.

线粒体 ATP 敏感 K^ 通道的结构和功能。

基本信息

批准号：
11670080
负责人：
GONOI Tohru
金额：
$ 2.43万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670080/
关键词：
ATP-sensitive K^+ Channel Mitochondria Kir6.1 Kir6.2 SUR1 SUR2 Jurkat cell cAMP-dependent protein kinase ATP感受性K^+チャネル代謝パッチクランプ法 Kir6.2

项目摘要

ATP-sensitive K^+ (K_<ATP>) channels open and close depending on ATP concentration in the cytoplasm, and control excitability of the cells. We have previously shown that K_<ATP> channel of pancreatic β-cell comprises SUR1, a member of ABC protein, and Kir6.2, a member of inward rectifier K channels. In heart and skeletal muscles, and blood vessels, K_<ATP> channels of similar properties comprise SUR2A/B and Kir6.x, regulating muscle contraction during hypoxia or ischemia.Although similar K_<ATP> channel is expressed in mitochondria, and regulating energy metabolism, its constituting molecules are unknown.Using the patch-clamp technique, we recorded ion channel activity from the mitochondrial inner membrane of Jurkat cells. K^+ ion selective channels with conductance of 55 pS were observed, of which activity being suppressed by 2 mM ATP.These properties and burst kinetics are similar to the channel comprising Kir6.1 molecule. In other to study if Kir6.1 molecules are induced during hypo … More xia, A7r5 cells, derived from blood vessel were cultured in medium containing Co^<2+> ions, mimicking hypoxic conditions. Western blotting using anti Kir6.1 antibody revealed that Kir6.1 protein became expressed in 3-4 days after an addition of the ions. However, no activity of channels comprising activity Kir6.1 was observed in the plasma membrane of heart of blood vessel muscles. Following these results, a possibility that Kir6.1 molecule induced in ischemia composes mitochondrial K_<ATP> channel is currently investigated.In other series of experiments effects of protein kinase A mediated phosphorylation of K_<ATP> channels comprising SUR1 and Kir6.2 were studied. We found both subunits are phosphorylated and channel properties are modulated in a different manner.Finally, effects of anti-diabetic reagents, troglitazone, pioglitazone, and KAD-1229, were studied on reconstituted K_<ATP> channels. The studies are helpful in understanding structure-function relationship of K_<ATP> channels in the plasma membrane as well as mitochondria. Less

ATP 敏感的 K^+ (K_<ATP>) 通道根据细胞质中的 ATP 浓度打开和关闭，并控制细胞的兴奋性我们之前已经证明胰腺 β 细胞的 K_<ATP> 通道包含 SUR1（一种）。 ABC 蛋白的成员，Kir6.2 是内向整流 K 通道的成员，在心脏和骨骼肌以及血管中，K_<ATP> 通道具有相似的特性，包括 SUR2A/B 和Kir6.x，在缺氧或缺血时调节肌肉收缩。虽然相似的K_<ATP>通道在线粒体中表达，并调节能量代谢，但其组成分子未知。利用膜片钳技术，我们记录了线粒体中离子通道的活性观察到 Jurkat 细胞内膜的 K^+ 离子选择性通道，电导率为 55 pS，其活性被 2 mM ATP 抑制。这些特性和突发动力学与该通道相似。为了研究 Kir6.1 分子是否在缺氧期间被诱导，将来自血管的 A7r5 细胞培养在含有 Co^2+ 离子的培养基中，使用蛋白质印迹法模拟缺氧条件。抗Kir6.1抗体显示，在添加离子后3-4天，Kir6.1蛋白表达，然而，在血管肌肉的心脏膜质中没有观察到包含活性Kir6.1的通道的活性。根据这些结果，目前正在研究缺血诱导的 Kir6.1 分子组成线粒体 K_<ATP> 通道的可能性。在其他系列实验中，研究了蛋白激酶 A 介导的包含 SUR1 和 Kir6.2 的 K_<ATP> 通道磷酸化的影响我们研究发现两个亚基都被磷酸化，并且通道特性以不同的方式调节。最后，抗糖尿病药物曲格列酮的作用，吡格列酮和 KAD-1229 对重建的 K_<ATP> 通道进行了研究，这些研究有助于理解质膜和线粒体中 K_<ATP> 通道的结构与功能关系。