Phenotypic and genotypic analysis of rats with cerebellar GABAA receptors composed from mutant and wild-type alpha 6 subunits.

Abstract

The alcohol-sensitive (ANT) rat line, developed for high behavioral sensitivity to ethanol, also exhibits enhanced sensitivity to benzodiazepines, such as diazepam. The rat line carries a point mutation in the cerebellum-specific gamma-aminobutyric acid type A (GABAA) receptor subunit alpha 6, making their diazepam-insensitive (DIS) receptors sensitive to diazepam. We now report that phenotypes of individual ANT and alcohol-insensitive rats, classified on diazepam sensitivity of cerebellar [3H]Ro 15-4513 binding, correlated well with homozygous wild-type, homozygous mutant, and heterozygous genotypes, although some heterozygotes were biased toward the parental phenotypes. GABA down-modulated DIS [3H]Ro 15-4513 binding in mutant homozygotes but tended to up-modulate it in heterozygotes and wild-type homozygotes. Slopes for GABA inhibition of cerebellar t-butyl- bicyclophosphoro[35S]thionate binding were larger in mutant than in wild-type homozygotes, with heterozygotes being intermediate. Diazepam displacement of [3H]Ro 15-4513 binding in heterozygotes revealed three components, with their affinities indistinguishable from those in combined wild-type and mutant homozygotes. This lack of interaction in DIS binding between wild-type and mutant alpha 6 subunits was substantiated by experiments on recombinant receptors. The data suggest that the alpha 6 subunit-containing GABAA receptors in the heterozygotes are formed from individual mutant and wild-type subunits with their relative expression differing from animal to animal.