GABA A RECEPTOR SUBUNIT EXPRESSION IN CEREBELLAR NEURONS

这项提案将检查GABA/A受体亚单位的组成和 在活体和培养的小脑神经元中组装。GABA/A 受体是一种多亚单位、配体门控的离子通道，它介导 主要抑制性神经递质γ-氨基丁酸的作用 在中枢神经系统中。最近的研究表明， 受体由几个亚基组成，其中大部分由 基因家族。每个亚单位基因都表现出不同的 小脑的细胞和时间表达模式。而当 在某些小脑中只表达一种特定亚基的亚基。 群体中，在其他类型的细胞中存在多种亚基异构体。 这些结果，再加上电生理的发现，提高了 小脑神经元表达多种受体亚型的可能性 具有不同的亚基组成和功能特性。然而， 天然受体的亚基组成及其过程 受体是如何组装的，目前尚不清楚。 阐明GABA/A受体亚单位的组成和组装 为了识别出小脑神经元，将进行一些研究。 第一，受体亚单位在小脑细胞中的表达 培养将使用亚基特异性抗血清进行检查。中的更改 出生后发育过程中的亚单位水平将由Western 印迹分析。此外，颗粒和浦肯野的亚基分布 神经元将通过免疫组织化学进行检查。这些研究将 开始确定已识别细胞中GABA/A受体亚单位的组成 受体的群体和发育变化特征 表情。第二，小脑GABA/A的亚基组成 受体将用免疫沉淀法与受体一起测定 亚基特异性抗血清。这些研究将阐明哪些亚基 来自不同的类别，以及来自单个类别的亚基亚型是 共同组装成一个受体复合体。最后，GABA/A的组装 培养的小脑颗粒神经元的受体将用 亚基特异性的代谢标记和免疫沉淀 抗血清。这些研究将确定受体亚单位是否表现出 可比较的生物合成速率，并将检查亚基的过程 组装成寡聚受体复合体。总而言之，这些研究 将产生关于GABA/A受体亚单位的重要新信息 已鉴定的小脑细胞群体的组成和组装。 因为不同的亚基赋予不同的生理特性 受体，亚基组成的知识对于一个 中枢神经系统对GABA/A受体功能的认识 系统。

This proposal will examine GABA/A receptor subunit composition and assembly in cerebellar neurons in vivo and in culture. the GABA/A receptor is a multisubunit, ligand-gated ion channel that mediates the actions of gamma-aminobutyric acid, the major inhibitory neurotransmitter in the central nervous system. Recent studies have shown that the receptor is composed of several subunits, most of which are encoded by families of genes. Each of the subunit genes exhibits a distinct cellular and temporal pattern of expression in the cerebellum. While only one isoform of a specific subunit is expressed in some cerebellar populations, multiple subunit isoforms are present in other cell types. These results, coupled with electrophysiological findings, raise the possibility that cerebellar neurons express multiple receptor subtypes having diverse subunit compositions and functional properties. However, the subunit composition of native receptors and the process by which receptors are assembled remain unknown. To elucidate GABA/A receptor subunit composition and assembly in identified cerebellar neurons, a number of studies will be performed. First, receptor subunit expression in cerebellar cells in vivo and in culture will be examined using subunit-specific antisera. changes in subunit levels during postnatal development will be assessed by Western blot analysis. In addition, subunit distribution in granule and Purkinje neurons will be examined by immunohistochemistry. These studies will begin to define GABA/A receptor subunit composition in identified cell populations and characterize developmental changes in receptor expression. Second, the subunit composition of cerebellar GABA/A receptors will be determined using immunoprecipitation with receptor subunit-specific antisera. These studies will elucidate which subunits from different classes and which subunit isoforms from a single class are coassembled into a receptor complex. Finally, the assembly of GABA/A receptors in cultured cerebellar granule neurons will be examined using metabolic labeling followed by immunoprecipitation with subunit-specific antisera. These studies will determine whether receptor subunits exhibit comparable rates of biosynthesis and will examine the process of subunit assembly into oligomeric receptor complexes. Together, these studies will yield important new information concerning GABA/A receptor subunit composition and assembly in identified cerebellar cell populations. Since the different subunits confer distinct physiological properties to the receptor, knowledge of subunit composition is essential for an understanding of GABA/A receptor function throughout the central nervous system.