STRUCTURES OF ACETYLCHOLINE RECEPTORS

乙酰胆碱受体的结构

• 批准号: 3393532
• 负责人: Arthur Karlin
• 金额: $ 30.4万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3393532
• 关键词: Raman spectrometry; X ray crystallography; acetylcholine; affinity chromatography; autoradiography; binding proteins; cations; chemical binding; crosslink; disulfide bond; electron microscopy; fluorescence; immunochemistry; local anesthetics; membrane channels; membrane permeability; membrane proteins; membrane reconstitution /synthesis; membrane structure; neurochemistry; neuropharmacology; neurophysiology; nicotinic receptors; protein sequence; protein structure; radiotracer; reagent /indicator; stoichiometry; synapses; ultracentrifugation

The acetylcholine (ACH) receptor of electric tissue and skeletal muscle transduces the binding of ACH into a change in the permeability fo the post-synaptic membrane to cations. The receptor has the polypeptide chain composition alpha2/Beta/gamma/delta. The overall objectives of the project are to determine the three dimensional structure of the receptor in the membrane and the location within this structure of the functional sites, such as the ACH binding sites and the cation- conducting channel. We will identify the amino acid residues contributing to the ACH binding sites and to the noncompetitive inhibitor (NCI) sites. We will crosslink a Cys 192 and Cys 193, which we have shown are in the ACH binding site, to other residues in the site. We will also directly affinity label other residues in this site. We will probe the NCI sites using a rapid- mixing, rapid-photolabeling technique, which allows us to label the receptor in its transient channel-open and channel-closed states. With this techniques, we will probe the mechanism of block by local anestheties and other NCIs and will test the hypothesis that the NCI sites are within the channel. The amino acid residues that react with labels directed either to the ACH sites or to the NCI sites will be located in the sequences of the chains. We will label susceptible residues in membrane-bound receptor with membrane-impermeant reagents. We will locate these residues in the sequences of the chains in order to determine the folding pattern of the chains in the membrane. We propose to determine the three dimensional structure of the receptor by x-ray crystallography. The location of the functional sites of the receptor in a high resolution structure will provide a basis for our understanding of the function of the receptor in molecular terms.

电组织和骨骼的乙酰胆碱（ACH）受体 肌肉转导ACH的结合到变化 阳离子后膜的渗透性。 这 受体具有多肽链组成 alpha2/beta/gamma/delta。 项目的总体目标 确定受体的三维结构 在膜和该结构内的位置 功能位点，例如ACH结合位点和阳离子 - 导电渠道。 我们将识别氨基酸残基 有助于ACH结合位点和非竞争力 抑制剂（NCI）位点。 我们将交叉链接A CYS 192和CYS 193， 我们已经显示的是ACH结合位点，与其他 站点中的残留物。 我们还将直接相关标签其他 该站点中的残留物。 我们将使用快速 - 混合，快速标签技术，这使我们能够标记 其瞬态通道和通道关闭的受体 国家。 通过这种技术，我们将探究 局部麻醉和其他NCI封锁，将测试 假设NCI位点在通道内。 氨基 与针对ACH的标签反应的酸残基 站点或NCI站点将位于 链。 我们将在膜结合中标记易感残留 具有膜覆盖试剂的受体。 我们将找到 这些残留在链的序列中，以便 确定膜中链的折叠模式。 我们 提议确定 X射线晶体学的受体。 功能的位置 高分辨率结构中受体的部位将提供 我们理解受体在中的功能的基础 分子项。