SULFONYLUREA RECEPTORS AND KATP CHANNELS

磺酰脲受体和 KATP 通道

• 批准号: 2143700
• 负责人: Joseph Bryan
• 金额: $ 19.88万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-20 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143700
• 关键词: B lymphocyte; Xenopus; Xenopus oocyte; adenosine triphosphate; chemical binding; complementary DNA; drug receptors; glyburide; molecular cloning; molecular site; nucleic acid sequence; polymerase chain reaction; potassium channel; protein structure function; receptor binding; sulfonylurea; tissue /cell culture

The long-term objective of this proposal is to understand the structure and function of ion channels that link cellular metabolism with membrane electrical activity. We are characterizing a pancreatic alpha/Beta-cell K+ channel whose electrical activity is modulated by ATP and sulfonylureas. This channel, I/ATP, is thought either to be the high affinity sulfonylurea receptor found in alpha/Beta-cells or to be tightly associated with this receptor. K/ATP sets the resting membrane potential in Beta-cells and is a key ion channel in the glucose-induced depolarization that leads to insulin release. We have developed and characterized an iodinated sulfonylurea, termed iodoglyburide, which specifically photolabels a high affinity, 140 kDa sulfonylurea receptor in isolated membranes from various cell types including neurons, pituitary cell lines, pancreatic alpha-and Beta-cells and several alpha- and Beta-cell models, i.e., a hamster insulin secreting tumor (HIT cells), a rat insulinoma (RIN cells) and a mouse glucagon secreting cell line (alphaTC-6 cells). Iodoglyburide is fully biologically active, has a K/D for binding equivalent to the ED50 for induction of insulin secretion in HIT cells and inhibits K/ATP. The pharmacological properties of the photolabeling reaction are those expected for the high affinity receptor defined in isolated HIT cell membranes. We have isolated the 140 kDa photolabeled receptor and obtained peptide sequence. Antibodies produced against this peptide sequence crossreact with the photolabeled protein and are competed by the appropriate synthetic peptides. A degenerate oligonucleotide/PCR strategy has been used to clone a cDNA fragment coding the N-terminus of the receptor. The major goals of the revised application are: a. To clone and sequence a cDNA for the Beta-cell receptor. b. To express this cDNA in Xenopus oocytes and COS cells in order to ascertain whether the receptor has channel activity and, if so, characterize this activity. c. To use the receptor clone to characterize the receptor, and, in the event the receptor does not have K+ channel activity, to isolate channel subunits. d. To use the receptor clone to isolate other members of this family of proteins.

这项建议的长期目标是理解结构 以及连接细胞代谢和膜的离子通道的功能 电活动。我们正在研究一种胰岛α/β细胞 K+通道，其电活动由ATP和 磺脲类药物。这条通道，I/ATP，被认为是最高的 在α/β细胞中发现的亲和性磺脲受体或紧密结合 与这种受体有关。K/ATP决定静息膜电位 在β细胞中，是葡萄糖诱导的关键离子通道 导致胰岛素释放的去极化。我们已经开发出了 表征了一种名为碘格列本脲的碘化磺酰脲，它 光标记高亲和力的140 kDa磺酰脲受体 在包括神经元在内的各种细胞类型的分离膜中， 垂体细胞系，胰腺α-细胞和β-细胞以及几个α-细胞 和Beta细胞模型，即仓鼠胰岛素分泌肿瘤(HIT 细胞)、大鼠胰岛素瘤(RIN细胞)和小鼠胰高血糖素分泌细胞 细胞株(αTC-6细胞)。Iodoglyburide完全具有生物活性， 结合的K/D相当于诱导胰岛素的ED50 HIT细胞分泌并抑制K/ATP。药理学 光标记反应的性质是预期的高 亲和力受体在分离的HIT细胞膜中定义。我们有 分离得到140 kDa的光标受体，获得多肽序列。 针对该肽序列产生的抗体与 光标记蛋白并与适当的合成蛋白竞争 多肽。简并的寡核苷酸/聚合酶链式反应策略已被用于 克隆编码该受体N-端的基因片段。少校 修订后的申请的目标是： A.克隆β细胞受体基因并测序。 B.在非洲爪哇卵母细胞和COS细胞中表达该基因 确定受体是否具有通道活性，如果是， 描述这项活动的特征。 C.使用受体克隆来表征受体，并且在 受体不具有K+通道活性的事件，以隔离通道 亚单位。 D.使用受体克隆来分离这个家族的其他成员 蛋白质。