SULFONYLUREA RECEPTORS AND KATP CHANNELS

磺酰脲受体和 KATP 通道

• 批准号: 2518310
• 负责人: Joseph Bryan
• 金额: $ 22.36万
• 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/2518310
• 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.

该提案的长期目标是了解结构 以及连接细胞代谢与膜的离子通道的功能 电活动。 我们正在表征胰腺 α/β 细胞 K+ 通道的电活动受 ATP 调节 磺酰脲类药物。 该通道，I/ATP，被认为是高 α/β细胞中发现的亲和性磺酰脲受体或紧密结合 与该受体有关。 K/ATP 设置静息膜电位 在β细胞中，是葡萄糖诱导的关键离子通道 去极化导致胰岛素释放。 我们已经开发并 描述了一种碘化磺酰脲，称为碘甘脲，它 特异性光标记高亲和力 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细胞以及几种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 系（alphaTC-6 细胞）。 碘甘脲具有完全的生物活性， 结合的 K/D 相当于诱导胰岛素的 ED50 HIT 细胞中的分泌并抑制 K/ATP。 药理作用 光标记反应的特性是那些预期的高 分离的 HIT 细胞膜中定义的亲和力受体。 我们有 分离140 kDa光标记受体并获得肽序列。 针对该肽序列产生的抗体与 光标记蛋白质并由适当的合成物竞争 肽。 简并寡核苷酸/PCR策略已被用于 克隆编码受体 N 末端的 cDNA 片段。 主要 修订后的应用程序的目标是： 一个。 克隆 β 细胞受体的 cDNA 并对其进行测序。 b. 在非洲爪蟾卵母细胞和 COS 细胞中表达该 cDNA，以便 确定受体是否具有通道活性，如果有， 描述这项活动的特征。 c. 使用受体克隆来表征受体，并且，在 如果受体没有 K+ 通道活性，则隔离通道 亚单位。 d. 使用受体克隆来分离该家族的其他成员 蛋白质。