γ-aminobutyric acid is the major inhibitory neurotransmitter in the adult mammalian central nervous system (CNS) and may also play a neurotrophic role during CNS development. Diversification of GABA<sub>A</sub> receptor mediated responses are in part a result ofvariation in subunit composition in the receptor complex. This variation arises both from the number of different subtypes of GABA<sub>A</sub> receptor subunits (α1-6, β1-4, γ1-3, δ1, ρ1-3, ε, ρ), as well as from post-transcriptional processes such as RNA splicing. In this thesis, I have investigated the developmental onset of GABA<sub>A</sub> receptor gene expression and the distribution and temporal expression of GABA<sub>A</sub> receptor subunit mRNAs and 12 splice variants within the developing and adult murine CNS. Preliminary studies using S 1 nuclease protection analysis demonstrated that α1, β3 and γ2 were the predominant subtypes of GABA<sub>A</sub> receptor subunits expressed at embryonic day 14 and in the adult murine CNS. In situ hybridisation analysis demonstrated overlapping but distinct spatial and temporal patterns of GABA<sub>A</sub> subunit mRNA expression during postnatal development and in the adult murine CNS. Analysis of γ2 mRNA splice variants demonstrated that the γ2S transcript is the predominant γ2 mRNA expressed during latter stages of embryo genesis, while the γ2L transcript is the predominant γ2 isoform present inthe adult CNS. Since there is a 29 to 47 percent amino acid identity among the various GABA<sub>A</sub> receptor subunits, I have also demonstrated through site-directed mutagenesis studies, that changes in a conserved amino acid in the cysteine loop of the bovine a 1 GABA<sub>A</sub> receptor subunit resulted in a loss of agonist and antagonist binding (DI49N), while a change in a conserved amino acid in the M1 transmembrane domain of the bovine α1 GABA<sub>A</sub> receptor subunit resulted in loss of agonist binding and reduction in the B<sub>max</sub> and K<sub>d</sub> for antagonist binding (P243A). 'These results are in contrast to the effect of identical mutations in the bovine β1 subunit and suggest that if the pentameric GABA<sub>A</sub> receptor assembly is composed of (α1)2(β1)1(γ2)2, then changes in highly conserved amino acids in the α1 receptor subunit would have a greater distortion on the structure of the receptor complex.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:299023 |
| Date | January 1998 |
| Creators | Sutherland, Margaret Lloy |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://oro.open.ac.uk/54847/ |
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