A Screening System for Development of Novel Insulin Secretagogues

开发新型胰岛素促分泌素对糖尿病的治疗具有重要意义。磺酰脲类药物广泛用于治疗2型糖尿病。磺酰脲类药物通过抑制胰腺β细胞中ATP敏感性K^+通道来刺激胰岛素分泌。我们最近发现，β细胞钾<ATP>通道由内向整流钾通道亚基Kir6.2和磺酰脲类受体亚基SURI组成，SURI对磺酰脲类药物格列本脲具有高亲和力。我们还克隆了一种名为SUR 2A的SUR 1亚型，它对格列本脲的亲和力较低。为了建立新型磺酰脲及其衍生物有效性的筛选系统，我们确定了1）β细胞K_通道的亚基化学计量<ATP>，2）SUR 1中的磺酰脲结合位点; 3）我们产生了Kir6.2缺陷型小鼠。利用SUR 1和Kir6.2的融合蛋白，我们发现当<ATP>SUR 1亚基和Kir6.2亚基分别与K_通道的活性相关时， ...更多信息 nit以1：1的摩尔比共表达。由于内向整流钾通道被认为是同源或异源四聚体，这表明β细胞钾<ATP>通道是由四个Kir6.2亚基和四个SUR 1亚基组成的异源八聚体。制备了SUR 1和SUR 2A之间的各种嵌合体。我们检测了^3H标记的格列本脲与转染了这些嵌合体的COS-1细胞的结合，以及格列本脲对<36>转染了每种嵌合体和Kir6.2的COS-1细胞的^ Rb外排的影响。我们发现格列本脲的高亲和力结合位点位于SUR1.3的第15和第16跨膜段之间。我们<ATP>通过对Kir6.2进行遗传破坏来产生K_通道缺陷小鼠，Kir6.2形成了通道的K^+离子选择性孔。纯合子小鼠（Kir6.2^&lt;-/-&gt;）缺乏K<ATP>通道活性。在K_<ATP>通道缺陷小鼠（Kir6.2^&lt;-/-&gt;）中，通过胰岛的灌流和分批孵育来评估，没有发现对葡萄糖或磺酰脲甲苯磺丁脲的响应的显著胰岛素分泌。我们的研究将为胰岛素分泌新药的开发提供有用的信息。少

Development of novel insulin secretagogues is important for the treatment of diabetes mellitus. Sulfonylureas are widely used in the treatment of type 2 diabetes. Sulfonylureas stimulate insulin secretion by inhibiting ATP-sensitive K^+ channels in pancreatic beta-cells. We have recently shown that the beta-cell K_<ATP> channel comprises the inward rectfier K^+ channel subunit Kir6.2 and the sulfonylurea receptor subunit SURI which shows high affinity for the sulfonylurea glibenclamide. We also cloned an isoform of SUR1, called SUR2A which shows low affinity for glibenclamide. To develop screening system of the effectiveness of novel sufonylureas and their derivatives, we have determined 1) the subunit stoichiometry of the beta-cell K_<ATP> channel, 2) sulfonylurea binding sites in the SUR1 ; and 3) we generated Kir6.2 deficient mice.1). By using fusion proteins of SUR1 and Kir6.2, we found that the activity of K_<ATP> channels is optimized when the the SUR1 subunit and the Kir6.2 subu … More nit are coexpressed with a molar ratio of 1 : 1. Since inward rectifier K^+ channels are thought to function as homo- or hetero-tetramers, this suggests that the beta-cell K_<ATP> channel functions as a hetero- octamer composed of four Kir6.2 subunits and four SUR1 subunits.2). Various chimeras between SUR1 and SUR2A were prepared. We examined ^3H labeled glibenclamide binding to COS-1 cells transfected with these chimeras and also the effect of glibenclamide on ^<36>Rb efflux from COS-1 cells transfected with each chimera and Kir6.2. We found that high affinity binding site for glibenclamide locates between 15 th and 16th transmembrane segement of SUR1.3). We generated K_<ATP> channel-deficient mice by genetic disruption of Kir6.2, which forms the K^+ ion-selective pore of the channel. The homozygous mice (Kir6.2^<-/->) lack K_<ATP> channel activity. No significant insulin secretion in response to either glucose or the sulfonylurea tolbutamide was found in K_<ATP> channel-deficient mice (Kir6.2^<-/->), as assessed by perifusion and batch incubation of pancreatic islets. Our studies should provide useful information for the development of new drugs for insulin secretion. Less