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.
