NEURONAL NICOTINIC RECEPTORS--EXPRESSION AND REGULATION

描述：（改编自申请人的摘要） 乙酰胆碱受体（AChRs）将使用稳定转染的 QT-6鹌鹑细胞系表达表位标记的鸡神经元AChR基因 在诱导型启动子下的构建体。 a7 AChR将被给予特殊的 强调是因为它们不寻常的性质（它们起同聚物的作用， 具有高钙渗透性，位于突触外， 睫状神经节）。 还将研究a3、b4、a5 + b2亚基（因为这些类型的AChR在突触后发现， 睫状神经节）。 细胞系将用于分析亚基 相互作用、组装途径和功能特性。 表面 将使用毒素和针对表位标签的mAb定量AChR。 将通过全细胞膜片钳记录测定功能。 组件 将通过脉冲标记和免疫分析鉴定中间体。 降水 亚单位组成将通过 使用针对亚基的mAb进行免疫沉淀和免疫印迹分析， 标签. 将通过共聚焦显微镜评价细胞内的分布。 显微镜 表达α 7的神经元细胞系也将用于 对比 这些研究应该揭示细胞和分子的重要方面， 神经元烟碱受体的生物学，并与 尼古丁依赖中的烟碱乙酰胆碱受体。

DESCRIPTION: (Adapted from applicant's Abstract) Neuronal nicotinic acetylcholine receptors (AChRs) will be studied using stably transfected QT-6 quail cell lines to express epitope-tagged chick neuronal AChR gene constructs under inducible promoters. a7 AChRs will be given special emphasis because of their unusual properties (they function as homomers, have high calcium permeability and are located extrasynaptically in ciliary ganglia). Also to be studied are combinations of a3, b4, a5 + b2 subunits (because AChRs of these types are found postsynaptically in ciliary ganglia). The cell lines will be used to analyze subunit interactions, assembly pathways, and functional properties. Surface AChRs will be quantitated using toxins and mAbs to the epitope tags. Function will be assayed by whole cell patch clamp recording. Assembly intermediates will be identified by pulse labeling and immune precipitation. Subunit composition will be analyzed by immunoprecipitation and immunoblot analysis using mAbs to subunits and tags. Distribution within the cells will be evaluated by confocal microscopy. Neuronal cell lines which express a7 will also be used for comparison. These studies should reveal important aspects of the cell and molecular biology of neuronal nicotinic receptors and are relevant to the role of nicotinic AChRs in nicotine addiction.