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Functinal regulation of cardiac ATP-sensitive potassium channel by actin cytoekeleton

肌动蛋白细胞骨架对心脏 ATP 敏感钾通道的功能调节

基本信息

批准号：
08670774
负责人：
FURUKAWA Tetsushi
金额：
$ 1.66万
依托单位：
Tokyo Medical and Dental University, Medical Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Previously, we had reported that ATP-sensitive potassium channel (K_<ATP> channel)^- required intracellular ATP to maintain its activity and that this effect of ATP is similar to ATP action on polymerization/depolymerization of actin cytoskeleton. Thus, we examined functional modulation of K_<ATP> channel by cytoskeleton.Actin disruptors diminished K_<ATP> channel activity, while actin stabilizer maintained channel activity. Disruptors or stabilizers of microtubules did not affect K_<ATP> channel activity. Direct application of polymerized form of actin (F-actin) recovered lost channel activity. PIP_2, which binds to all actin binding proteins and inhibits their actin severing activity, act for maintainance of channel activity and recevered prtially lost channel activity. These data indicate that K_<ATP> channel activity was functionally modulated by the status of actin cytoskeleton.Next, we sought using yeast two hybrid system protein that would interact with nucleotide binding domain (NBD) of sulfonylurea receptor (SUR), a target of intracellular regulation of K_<ATP> channel. An unknown protein was found to interact with NBD1 of cardiac type SUR,(SUR2a), whose transcript are present ubiquitously and most predominantly in the heart. Isolation of full-length of cDNA revealed that there were four isoforms due to alternative splicing. In each isoform, there was a region that had high sequence homology to cytochrome C.Thus, K_<ATP> channel might be regulated by intracellular oxidization reaction.Currently, we are on the way to examine functional role of interaction of NBD1 in SUR2a and the unknown protein that we found.

以前，我们曾报道ATP敏感性钾通道（K_<ATP>channel）^-需要细胞内ATP来维持其活性，ATP的这种作用类似于ATP对肌动蛋白细胞骨架聚合/解聚的作用。因此，我们研究了细胞骨架对K_通道的功能调节<ATP>，肌动蛋白干扰剂减弱了K_<ATP>通道的活性，而肌动蛋白稳定剂维持了通道的活性。微管的破坏剂或稳定剂不影响钾<ATP>通道活性。直接应用聚合形式的肌动蛋白（F-肌动蛋白）恢复失去的通道活性。PIP_2与所有的肌动蛋白结合蛋白结合并抑制它们的肌动蛋白切割活性，其作用是抑制通道活性并恢复部分丧失的通道活性。这些结果表明，K_<ATP>通道的活性在功能上受肌动蛋白细胞骨架状态的调节。接下来，我们利用酵母双杂交系统蛋白，寻找与细胞内调节K_通道的靶点磺酰脲受体（SUR）的核苷酸结合域（NBD）相互作用<ATP>的蛋白。一种未知的蛋白质被发现与心脏型SUR的NBD 1（SUR 2a）相互作用，其转录物普遍存在于心脏中并且最主要存在于心脏中。全长cDNA的分离显示，有四个异构体由于选择性剪接。在每一个亚型中，都有一个区域与细胞色素C具有高度的序列同源性，因此，K通道<ATP>可能受细胞内氧化反应的调节。目前，我们正在研究NBD 1在SUR 2a中与我们发现的未知蛋白相互作用的功能作用。