Molecular cloning of GABA(A) receptor subunits from seizure prone (DBA/2J) and resistant (C57BL/6J) inbred mice

Abstract

Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter in brain. The structure of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor is of particular interest because it contains several binding sites for clinically significant drugs such as benzodiazepines (BZ), barbiturates and alcohol. In addition, there is evidence which suggests the involvement of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor in several neurological afflictions. Molecular cloning of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor subunits is the first step towards further understanding of this receptor through a molecular biological approach. The object of my thesis research is the use of molecular biological techniques to study the structure of subunits of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor at the cDNA and mRNA levels, extending this study to an animal model of epilepsy, in an attempt to establish the contribution of changes in the structure of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor to seizure susceptibility. Molecular cloning techniques have allowed determination of the nucleic acid and amino acid sequences of six subunits of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptors {dollar}(\alpha\sb{lcub}1-3{rcub},{dollar} {dollar}\gamma\sb{lcub}1-2{rcub}{dollar} and {dollar}\delta){dollar} from inbred DBA/2J and C57BL/6J mice. These subunits from mice have a high degree of sequence identity with those from rat, cattle and humans. GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor subunit proteins thus have structures which appear to be well conserved in mammals; presumably this reflects their key functional role. No amino acid sequence differences have been found between the two strains for these six subunits. Changes in regulation of gene expression of the receptor subunits could result in subunit under- or over-expression followed by dysfunction of the receptor. Unfortunately, our Northern blot data from the cerebellum, cortex and rest of the brain in both DBA/2 and C57BL/6 mouse brains did not indicate any major differences in these six subunits' expression. Our negative data should prove useful to others who are considering this obvious avenue of research to account for differential susceptibility to audiogenic seizures in DBA/2J and C57BL/6J mice. The discovery of {dollar}\gamma\sb{lcub}\rm 2L{rcub}{dollar} and {dollar}\gamma\sb{lcub}\rm 2S{rcub}{dollar} subunits supplies evidence that the generation of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor heterogeneity occurs not only by the combinatorial association of subunit types and subunit isoforms, each of which are encoded by distinct genes, but also by alternative splicing of subunit gene transcripts. Our limited data about regional distribution and developmental profile of the subunit messenger mRNAs give another indication of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor subtype diversity. The correlations existing among subunits in their regional distribution and developmental patterns may offer some hints as to the subunit combinations occurring in different native receptors and their possible functions in the brain.