The major class of inhibitory neurotransmitter receptors, the GABA-A receptors, are modulated by a range of depressant drugs (eg: benzodiazepines, ethanol) and their dysfunction has been associated with several neurological disorders. However, in the absence of genomic clones, it has been difficult to establish any link between inappropriate receptor expression and the implicated disease states. In addition, little is known of how expression of subunit genes is regulated in a coordinated and localized manner. Studies on the structure, nucleotide sequence and chromosomal localization of the genes encoding the human beta-1, beta-3 and gamma-2 subunits have been completed. The cloned gene fragments have revealed polymorphisms that can be used to screen for any linkage between the subunit genes and known genetic disorders. The transcriptional start sites of the Hb3 gene have been identified by RNase protection assays. The Hb3 transcripts start from multiple sites, 110-150 bp upstream of the initiation ATG. Upstream regions of the Hb3 gene exhibit strong promotor activity in several cell lines. These cell lines include examples that normally express a beta-3 transcript (eg: PC12) as well as those in which beta-3 mRNA is absent (eg: HeLa). Through an analysis of several deleted constructs, it has been possible to identify a minimal promotor element of 140 bp. In order to identify sites at which transcription factors bind, the promotor region of the Hb3 gene has been characterized further by DNase footprinting. Nuclear extracts, prepared from HeLa or PC 12 cells, each protected a short region of 23 bases on both strands of the promotor sequence. The 23 bp segment that is protected has no clear homology with the binding sites of known transcription factors. At present, the precise function of this binding site is under investigation. Several immortalized cell lines that express GABA receptor subunits have been identified. Ongoing studies are aimed at identifying factors that regulate subunit expression in these model systems.
