CEMBRANOIDS--NEW INHIBITORS OF CHOLINERGIC RECEPTORS

烟碱型乙酰胆碱受体(ACHR)无疑将是 第一个离子通道，其结构和功能将被理解 在原子水平上。目前这种原型蛋白质的模型 优雅地解释一大堆数据，但它们是不完整的 而且经常是矛盾的。从研究中学到了很多东西 结合多个相互作用的非竞争性抑制物(NCIS) 位于受体蛋白界面的离子通道内的位置 与膜脂在仍未确定的部位。这个项目 将使用基于以下内容的策略来研究NCIS的高亲和力站点 最近的两个发现：1.一个不带电荷的疏水家族 竞争结合的非竞争性抑制物，西膜类化合物 使用通道阻滞剂苯环利定(PCP)。2.观察 PCP对来自细胞的AchR的亲和力高于 来自心肌细胞的。这种物种差异对于 西膜素醋酸酯(EUAC)。我们假设五氯苯酚与 对氨基酸(A.A.)在M2的第6和第10位 跨膜片段，以及PCP之间的亲和力差异 受体是由于它们的三个氨基酸取代 各就各位。我们进一步假设EUAC与A.A.有约束力。在M2 位置10和13，与PCP站点重叠，但显示 这两种受体之间的差异很小。以下是 实验被用来检验这些假设：一、结合 学习。放射性配基与富含AchR膜结合的测量 来自鱼雷的电风琴将被用来确定效力 20个西膜类化合物的排名及其结合部位与 抑制剂的。II.野生型的电生理研究 感受器。来自电子器官的Achr将在非洲爪哇表达 乳牛卵母细胞及其对乙酰胆碱诱导的抑制作用 将使用电压钳位技术来研究电流。三. 修饰受体的研究。混合ACHR，构建自 肌肉和电子器官受体的亚单位，将在 卵母细胞。这些杂交种对五氯苯酚的亲和力将被测试。 随后，第6、10、13和13位的单个氨基酸 其他M2片段将通过定点突变进行修改 以评估每种氨基酸对结合能的贡献 PCP和EUAC。

The nicotinic acetylcholine receptor (AchR) will undoubtedly be the first ion channel whose architecture and function will be understood at the atomic level. Present models of this prototypic protein explain elegantly a large body of data and yet, they are incomplete andoften contradictory. Much has been learned from studies of noncompetitive inhibitors (NCIs) which bind at several interacting sites inside theion channel, at the interface of receptor protein with membrane lipids of at still undetermined sites. This project will study the high-affinity site for NCIs using a strategy based on two recent discoveries: 1. A family of uncharged, hydrophobic noncompetitive inhibitors, the cembranoids, that compete for binding with the channel blocker phencyclidine (PCP). 2. The observation that PCP displays higher affinity for the AchR from electrocyte than that from myocyte. This species difference is much smaller for the cembranoid eupalmerin acetate (EUAC). We hypothesize that PCP binds to the amino acids (a.a.) at positions 6 and 10 of the M2 transmembrane segments, and that the PCP affinity difference between receptors is due to three amino acid substitutions at those positions. We further hypothesize that EUAC binds to a.a. at M2 positions 10 and 13, which overlap with the PCP site but display little difference between the two receptors. The following experiments are proposed to test these hypotheses: I. Binding studies. Measurements of radioligand binding to AchR-rich membranes from Torpedo electric organ will be used to determine the potency rank for 20 cembranoids and the relationship of their binding site to those of inhibitors. II. Electrophysiological studies on wild-type receptors. The AchR from electric organ will be expressed in Xenopus laevis oocytes and the inhibition by cembranoids of Ach-induced currents will be studied using a voltage-clamp technique. III. Studies on modified receptors. Hybrid AchR's, constructed from subunits of muscle and electric organ receptors, will be expressed in oocytes. The affinity of these hybrids for PCP will be tested. Subsequently, individual amino acids at positions 6, 10, 13, and others of M2 segments will be modified by site-directed mutagenesis to assess the contribution of each amino acid to the binding energies of PCP and EUAC.