CEMBRANOIDS--NEW INHIBITORS OF CHOLINERGIC RECEPTORS

烟碱乙酰胆碱受体（AchR）无疑将是 第一离子通道，其结构和功能将被理解 在原子水平上。 展示这种原型蛋白的模型 优雅地解释了大量的数据，但它们是不完整的 而且常常自相矛盾。 从对以下问题的研究中我们学到了很多东西： 非竞争性抑制剂（NCI），其结合在几个相互作用的 离子通道内的位点，在受体蛋白的界面 与膜脂的位置仍然未定。 这个项目 将使用基于以下的策略研究NCIs的高亲和力位点： 两个最新的发现：1。一个不带电荷的，疏水的 非竞争性抑制剂，西松烷类， 与通道阻滞剂苯环利定（PCP）。 2.观察 PCP对电细胞AchR的亲和力高于 这是来自myocyte。 这种物种差异对于 西松烷醇乙酸盐（EUAC）。 我们假设五氯酚 至氨基酸（a.a.）在M2的位置6和10， 跨膜片段，以及PCP之间的亲和力差异 受体是由于这些受体上的三个氨基酸取代造成的 岗位 我们进一步假设EUAC结合a.a.在M2 位置10和13，与PCP部位重叠，但显示 两种受体之间的差异很小。 以下 提出了实验来验证这些假设：I.结合 问题研究 放射性配体与富含AchR的膜结合的测量 将使用鱼雷电器官的电流来确定效力 20种西松烷类化合物的排序及其结合位点与 那些抑制剂。 二.野生型的电生理学研究 受体。 来自发电器官的AchR将在非洲爪蟾中表达 西松烷类化合物对乙酰胆碱诱导的卵母细胞凋亡的抑制作用 将使用电压钳技术研究电流。 三. 研究修饰受体。 混合AchR，由 肌肉和电器官受体的亚基，将在 卵母细胞 将测试这些杂交体对五氯苯酚的亲和力。 随后，在位置6、10、13和14处的单个氨基酸， 其余M2片段将通过定点突变进行修饰 以评估每个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.