Elucidation of the mechanisms underlying the regulation of ATP-sensitive K^+ channels by sulfonylureas

ATP敏感K^(K&lt；ATP&gt；)通道是连接细胞代谢状态和膜电位的关键分子。在胰岛β细胞中，KK_lt；ATP&gt；通道不仅是胰岛素分泌的葡萄糖感受器，而且也是胰岛素促分泌剂磺脲类药物的靶点，被广泛用作口服降糖药治疗糖尿病。在心肌细胞和神经元中，钾通道被认为参与了缺血时的细胞保护。我们已经证明，β细胞钾通道是磺脲受体Suri和新克隆的内向整流型钾通道成员Kir6.2的复合体，而心肌细胞钾通道是新克隆的磺脲受体SUR2和Kir6.2的复合体。本研究旨在阐明钾通道调节的分子机制。[3-细胞(SUR1/Kir6.2)和心脏(SUR2/Kir6.2)钾通道对磺脲格列本脲和K通道开放剂…的敏感性更多的二氮杂氮是不同的，由不同的SUR亚基决定。为了确定给予格列本脲和二氮卓敏感性的结构域，我们在SUR1和SUR2之间制备了一系列嵌合体，并研究了嵌合K_&lt；ATP&gt；通道的性质。所有嵌合的SURS在含有Kir6.2的COS-1细胞中表达时均能产生电流。接下来，我们研究了格列本脲和二氮嗪对嵌合电流的影响，以^&lt；86；rb^外排来衡量，并缩小了赋予这些试剂敏感性的区域。此外，我们构建了SUR1和SUR2之间的各种嵌合体，并通过测量@SH标记的格列本脲与COS 1细胞中表达的嵌合体(手稿在3@E1准备中)的结合活性，几乎确定了格列本脲响应区。此外，我们还研究了G蛋白对重组K&lt；ATP&gt；通道的影响。我们发现G蛋白α亚基直接调节K&lt；ATP&gt；通道的活性，并且这种调节在[3-cell(SUR1/Kir6.2)和心脏(SUR2/Kir6.2)K&lt；ATP&gt；通道之间是不同的，提示这种差异是由不同的SuR亚基决定的。较少

ATP-sensitive K^+(K_<ATP>) channels are key molecules which link the cell's metabolic status to its membrane potential. In pancreatic beta-cells, KK_<ATP> channel is not only a glucose sensor m insulin secretion but also a target for the insulin secretagogue, the sulfonylurea, widely used as oral hypoglycemic agents in the treatment of diabetes mellitus. In cardiac myocytes and neurons, K_<ATP> channels are supposed to be involved in cytoprotection in ischemia, We have shown that beta-cell K_<ATP> channel is a complex of the sulfonylurea receptor SURI and a newly cloned inward rectifier K_<ATP> channel member Kir6.2, and that cardiac myocyte K_<ATP> channel is a complex of the newly cloned sulfonylurea receptor SUR2 and the Kir6.2. Our purpose in this study is to clarify the molecular mechanisms underlying the regulation of K^+ channels.Sensitivities of the [3-cell (SUR1/Kir6.2) and cardiac (SUR2/Kir6.2) K_<ATP> channels to the sulfonylurea glibenclamide and the K_<ATP> channel opener … More diazoxide are different, and are determined by distinct SUR subunits. To determine the domains that confer glibenclamide and diazoxide sensitivities, we prepared a series of chimeras between SUR1 and SUR2, and studied the properties of the chimeric K_<ATP> channels. All chimeric SURs could generate currents when expressed in COS 1 cells with Kir6.2. Next, we have examined the effects of the glibenclamide and diazoxide on the chimeric currents measured as ^<86>Rb^+ efflux, and narrowed the region that confer the sensitivities to these reagents. Furthermore, we have constructed various chimeras between SUR1 and SUR2, and have almost determined the glibenclamide-responsive region by measuring the binding activities of @SH-labeled glibenclamide for the chimeras expressed in COS 1 cells (manuscript in 3@E1preparation).In addition, we have investigated the effects of G-protein on the reconstituted K_<ATP> channels. We have shown that G-protein alpha subunit directly regulates the K_<ATP> channel activity, and that the regulation is different between [3-cell (SUR1/Kir6.2) and cardiac (SUR2/Kir6.2) K_<ATP> channels, suggesting that the difference is determined by distinct SUR subunits. Less