Neurosteroid modulation of GABAa receptors

y-amino butyric acid type A (GABA(A)) receptors are the principal inhibitory neurotransmitter receptors of the brain. They play a significant role in nervous system disorders such as epilepsy and anxiety. Some drugs used in the treatment of such disorders target the GABA(A) receptors. Neurosteroids are one such class of drugs, though their exact mechanism of action on GABA(A) receptors is unknown. This thesis is a study of the mechanism of action of the neurosteroids on the Caenorhabditis elegans (C elegans) GABA(A) receptor. The C. elegans GABA(A) receptor provided us with a useful model to study neurosteroid interaction for two reasons. First, the different forms of the C. elegans GABA(A) receptor show differential sensitivity to neurosteroids, which allowed important receptor residues to be identified. Second, the C. elegans GABA(A) receptors are homomeric and hence simpler to study than the heterotrimeric mammalian GABA(A) receptors. The preliminary work had led to the identification of several residues that control the sensitivity of the neurosteroid pregnenolone sulfate (PS). The first objective of this thesis was to test whether mutations affecting PS sensitivity also affected the actions of other neurosteroids on the C. elegans GABA(A) receptors, to assess whether different neurosteroids use a shared site and similar mechanisms of action. The second objective was to identify residues in the C. elegans GABA(A) receptor important for the neurosteroid pregnanolone by creating additional mutant receptors using site directed mutagenesis. The final objective was to generate expression vectors for the mammalian GABA(A) receptors in order to eventually test whether neurosteroid mechanisms are conserved between C. elegans and mammals. In summary, the sites of action of different neurosteroids were found to be overlapping but not identical. Important amino acid residues of the C. elegans GABA(A) receptor were identified which mediate pregnanolone modulation of the GABA(A)receptor function. These results, along with the construction of mammalian GABA(A) receptor expression vectors, have set the stage for identifying residues important for neurosteroid modulation of the human GABA(A) receptors.