Abstracts

Rationale: Heterozygous loss-of-function mutations of the GABA_A receptor ?1 subunit (GABRA1) are associated with two human autosomal dominant idiopathic generalized epilepsy syndromes and heterozygous GABRA1 knockout (KO) causes epileptiform complexes in genetically modified mice. Here we determined the effects of heterozygous knockout on total and surface GABA_A receptor expression in the cortex and thalamus, two brain regions implicated in idiopathic generalized seizures.Methods: We dissected brains from wild type and heterozygous GABRA1 knockout mice of ages P33-37, the time point in development at which heterozygous mice develop epileptiform abnormalities. We cut live 400 m sagittal brain sections and incubated them with a membrane impermeable reagent that covalently attaches a biotin moiety to lysines of cell surface proteins. We then dissected the cortex and thalamus from each section, purified the cell surface proteins using immobilized neutravidin, and quantified total and cell surface GABA_A receptor subunit expression on Western blots.Results: In the cortex, heterozygous GABRA1 KO reduced surface ?1 subunit expression by a smaller amount (18 6%) than total expression (37 5%); there was no difference in reduction of surface and total ?1 subunit expression in the thalamus (29 8%). Although heterozygous GABRA1 KO reduced surface ?1 subunit expression, it did not reduce surface ?2 or ?3 subunit expression in either the cortex or thalamus, a finding that suggested that heterozygous neurons compensate for the loss of ?1 subunit by increasing expression of other ? subunit isoforms. We then found that heterozygous KO increased surface expression of ?3 subunit by 74% in the cortex and the thalamus.Conclusions: These data demonstrated that neurons compensate for the heterozygous loss of ?1 subunit expression by increasing the relative surface expression of the ?1 subunit and by increasing ?3 subunit expression. These findings suggest that the epilepsy phenotype is caused by altered surface GABA_A receptor composition rather than a reduction in GABA_A receptor expression.