Agonist-driven conformational changes in nAChRs

激动剂驱动的 nAChR 构象变化

• 批准号: 7215273
• 负责人: ROBERT L ROSENBERG
• 金额: $ 27.69万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7215273
• 关键词: Address; Agonist; Alzheimer' s Disease; Amino Acids; Area; Binding; Binding Proteins; Cholinergic Receptors; Cleaved cell; Cysteine; Data; Disulfides; Electrostatics; Epilepsy; Extracellular Domain; Goals; Homology Modeling; Lateral; Ligand Binding; Ligand Binding Domain; Ligands; Link; Measures; Methods; Modeling; Modification; Molecular; Molecular Conformation; Movement; Mutation; Neurons; Nicotine Dependence; Nicotinic Receptors; Personal Satisfaction; Play; Positioning Attribute; Property; Rate; Reagent; Receptor Activation; Relative (related person); Research; Rest; Role; Rotation; Structure; Sulfhydryl Compounds; Testing; Vestibule; Water; Xenopus oocyte; base; crosslink; cysteinylcysteine; extracellular; follow-up; mutant; nervous system disorder; neurotransmitter release; programs; receptor; research study

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms of agonist binding and agonist-dependent conformational changes during activation of neuronal nicotinic acetylcholine receptors (nAChRs) is essential for our understanding nicotine addiction, certain forms of epilepsy, Alzheimer's disease, and other neurological disorders. The research in this proposal focuses on alpha7 neuronal nAChRs because they are important in modulating neurotransmitter release. Specific residues that are important for ligand binding have been identified and characterized, but the conformational changes that couple agonist binding to channel gating are unclear. We use a homology model of the alpha7 receptors based on the crystal structure of the ACh Binding Protein to identify amino acids that may play important roles in agonist- or antagonist-dependent conformational changes. The specific goals of this research are to: (1) test the hypothesis that agonist binding causes a contraction or partial collapse of the ligand-binding pocket; (2) test the hypothesis that the beta9-beta10 hairpin structure, connecting the ligand-binding pocket to the transmembrane pore domain, undergoes conformational changes following the binding of agonists; and (3) determine whether lateral movement of the beta9-beta10 hairpin and/or rotational movements of subunits participate in agonist-driven activation. Several complementary experimental approaches will be employed. We will express alpha7 receptors in Xenopus oocytes and use electrophysiological approaches to probe functional properties. We will use the substituted cysteine accessibility method to identify residues that change accessibility during ligand binding. In addition, we will introduce electrostatic constraints and form disulfides or cysteine cross-links in (and near) the beta9-beta10 hairpin to limit movement and alter receptor activation.

描述（由申请人提供）：了解神经元烟碱乙酰胆碱受体（nAChR）激活过程中激动剂结合和激动剂依赖性构象变化的分子机制对于我们了解尼古丁成瘾、某些形式的癫痫、阿尔茨海默病和其他神经系统疾病至关重要。本提案中的研究重点是 α7 神经元 nAChR，因为它们对于调节神经递质释放很重要。对配体结合重要的特定残基已被鉴定和表征，但将激动剂结合与通道门控耦合的构象变化尚不清楚。我们使用基于乙酰胆碱结合蛋白晶体结构的 α7 受体同源模型来识别可能在激动剂或拮抗剂依赖性构象变化中发挥重要作用的氨基酸。本研究的具体目标是：（1）检验激动剂结合导致配体结合袋收缩或部分塌陷的假设； (2) 检验连接配体结合袋和跨膜孔结构域的β9-β10发夹结构在激动剂结合后发生构象变化的假设； (3)确定β9-β10发夹的横向运动和/或亚基的旋转运动是否参与激动剂驱动的激活。将采用几种互补的实验方法。我们将在非洲爪蟾卵母细胞中表达 α7 受体，并使用电生理学方法来探测功能特性。我们将使用取代的半胱氨酸可及性方法来识别在配体结合过程中改变可及性的残基。此外，我们将引入静电约束并在 beta9-beta10 发夹中（及其附近）形成二硫化物或半胱氨酸交联，以限制运动并改变受体激活。