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Simultaneous measurement of ATP-sensitive K current and fluorescent imaging.

同时测量 ATP 敏感 K 电流和荧光成像。

基本信息

批准号：
14370012
负责人：
TAKANO Makoto
金额：
$ 9.09万
依托单位：
JICHI MEDICAL University (2004)Kyoto University (2002-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370012/
关键词：
Kir6.2 SUR2A SUR FRET ATP

项目摘要

The ATP-sensitive K^+ (K_<ATP>) channel is a heteromultimer composed of four inwardly rectifying K^+ channels (Kir6.2) and four sulfonylurea receptors (SUR). Intracellular ATP binds to Kir6.2 and inhibits K_<ATP> channel. Nucleotide diphosphates (NDPs) binds to SUR, thereby increases the open probability of, and decreases the ATP-sensitivity of K_<ATP> channel. SUR is a member of ATP-binding cassette (ABC) superfamily. possessing two nucleotide binding folds (NBF). Prokaryotic members of ABC superfamily such as HisP and RbsA are termed "half-size ABC proteins", and possess only one NBF, and form a functional dimer in the cell membrane. We splited SUR2A into two "half-size molecules" before and after NDB1, and found that they could reassemble with Kir6.2 to form a functional K_<ATP> channel. C-terminal half SUR (C640) conferred glibenclamide sensitivity to Kir delta C36, whereas C640 did not increase open probability of Kir6.2 delta C36. We also made fusion proteins of yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) with split SURs and Kir6.2, and tried to identify fluorescent resonance energy transfer (FRET) between CYP and YFP. However, pharmacological stimuli with K channel openers and glibenclamide failed to induce the changes of FRET ratio. We also found that fluorescent labeled ATP could successfully inhibited the fusion protein of GFP and Kir6.2delta C36. However, it was difficult to identify FRET between fluorescent labeled ATP and GFP.

ATP敏感性K^+（K_<ATP>）通道是由四个内向整流K^+通道（Kir6.2）和四个磺酰脲受体（SUR）组成的异源多聚体。细胞内ATP与Kir6.2结合，抑制K_<ATP>通道。核苷酸二磷酸（NDP）与SUR结合，增加了K通道的开放概率，降低了K通道的ATP敏感性<ATP>。SUR是ATP结合盒（ABC）超家族的成员。具有两个核苷酸结合折叠（NBF）。ABC超家族的前体成员如HisP和RbsA被称为“半大小ABC蛋白”，它们只有一个NBF，在细胞膜上形成功能性二聚体。我们将SUR 2A在NDB 1前后分裂成两个“半分子”，发现它们可以与Kir6.2重组形成功能性K_<ATP>通道。C-末端半SUR（C640）赋予格列本脲对Kir δ C36的敏感性，而C640并未增加Kir6.2 δ C36的开放概率。我们还制备了黄色荧光蛋白（YFP）或青色荧光蛋白（CFP）与分裂的SURs和Kir6.2的融合蛋白，并试图鉴定荧光素酶和YFP之间的荧光共振能量转移（FRET）。然而，药物刺激与K通道开放剂和格列本脲未能诱导FRET比率的变化。荧光标记的ATP能有效抑制GFP与Kir6.2 delta C36融合蛋白的表达。然而，荧光标记的ATP和GFP之间的FRET很难识别。