NEURONAL NICOTINIC RECEPTORS--EXPRESSION AND REGULATION

描述：(改编自申请者摘要)神经元烟碱 乙酰胆碱受体(AChRs)的研究将使用稳定转染法 表达表位标记鸡神经元型AChR基因的Qt-6细胞系 在可诱导启动子下构建。A7 AChR将给予特殊照顾 强调是因为它们的不同寻常的性质(它们起着同聚体的作用， 有很高的钙通透性，位于突触外 睫状神经节)。还需要研究的是a3、b4、a5+的组合 B2亚基(因为这些类型的AChR在突触后发现于 睫状神经节)。细胞系将被用来分析亚基 相互作用、组装路径和功能特性。表面 AChRs将使用毒素和表位标签的单抗进行定量。 功能检测采用全细胞膜片钳记录。装配 中间体将通过脉冲标记和免疫来鉴定 降水。亚基组成将通过以下方式分析 亚基和亚基单抗的免疫沉淀和免疫印迹分析 标签。细胞内的分布将通过共聚焦进行评估 显微镜。表达A7的神经细胞系也将用于 比较一下。 这些研究应该揭示细胞和分子的重要方面 神经型尼古丁受体的生物学，并与 尼古丁成瘾中的尼古丁AChRs。

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.