Phenotypic and Genotypic Analysis of Rats with Cerebellar GABAA Receptors Composed from Mutant and Wild-Type α6 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 γ-aminobutyric acid type A (GABA_A) receptor subunit α6, 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-butylbicyclophosphoro³⁵S]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 α6 subunits was substantiated by experiments on recombinant receptors. The data suggest that the α6 subunit-containing GABA_A receptors in the heterozygotes are formed from individual mutant and wild-type subunits with their relative expression differing from animal to animal.