SULFONYLUREA RECEPTORS AND KATP CHANNELS

本提案的长期目标是了解 以及连接细胞代谢和细胞膜的离子通道的功能 电活动。 我们正在研究胰腺α/β细胞 K+通道，其电活动受ATP调节， 磺酰脲类。 这个通道，I/ATP，被认为是高的 在α/β细胞中发现的亲和性磺酰脲受体，或与 与这个受体有关。 K/ATP设置静息膜电位 在β细胞中，是葡萄糖诱导的 去极化导致胰岛素释放。 我们已经开发并 其特征在于碘化磺酰脲，称为碘格列本脲， 特异性光标记高亲和力的140 kDa磺酰脲受体 在来自包括神经元的各种细胞类型的分离膜中， 垂体细胞系、胰腺α-和β-细胞以及几种α- 和β细胞模型，即，仓鼠胰岛素分泌瘤（HIT 细胞）、大鼠胰岛素瘤（RIN细胞）和小鼠胰高血糖素分泌细胞 alphaTC-6细胞株。 碘格列本脲具有完全的生物活性， 结合的K/D相当于诱导胰岛素的ED 50 在HIT细胞中分泌并抑制K/ATP。 药理 光标记反应的性质是那些预期的高 在分离的HIT细胞膜中定义的亲和受体。 我们有 分离140 kDa的光标记受体并获得肽序列。 针对该肽序列产生的抗体与该肽序列交叉反应。 光标记的蛋白质，并通过适当的合成竞争 缩氨酸 简并寡核苷酸/PCR策略已用于 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： a. 克隆β细胞受体的cDNA并测序。 B. 在非洲爪蟾卵母细胞和COS细胞中表达该cDNA， 确定受体是否具有通道活性，如果是， 描述这一活动。 C. 使用受体克隆来表征受体， 如果受体不具有K+通道活性，则分离通道 亚单位。 D. 为了利用受体克隆来分离这个家族的其他成员， proteins.

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.