The cerebellar GABA AR α6-R100Q polymorphism alters ligand binding in outbred Sprague–Dawley rats in a similar manner as in selectively bred AT and ANT rats

Abstract

The alcohol-tolerant AT and alcohol-nontolerant ANT rat lines have been selectively bred for innate sensitivity to ethanol-induced motor impairment. The cerebellar GABA A receptor (GABA AR) α6 subunit alleles α6-100R and α6-100Q are segregated in the AT and ANT rats, respectively. This α6 polymorphism might explain various differences in pharmacological properties and density of GABA ARs between the rat lines. In the present study, we have used nonselected outbred Sprague–Dawley rats homozygous for the α6-100RR (RR) and α6-100QQ (QQ) genotypes to show that these RR and QQ rats display similar differences between genotypes as AT and ANT rat lines. The genotypes differed in their affinity for [ 3H]Ro 15-4513 and classic benzodiazepines (BZs) to cerebellar “diazepam-insensitive” (DZ-IS) binding sites, in density of cerebellar [ 3H]muscimol binding and in the antagonizing effect of furosemide on GABA-induced inhibition of [ 3H]EBOB binding. The results suggest the involvement of α6-R100Q polymorphism in these line differences and in the differences previously found between AT and ANT rats. In addition, the α6-R100Q polymorphism induces striking differences in [ 3H]Ro 15-4513 binding kinetics to recombinant α6β3γ2s receptors and cerebellar DZ-IS sites. Association of [ 3H]Ro 15-4513 binding was ∼10-fold faster and dissociation was ∼3–4-fold faster in DZ-IS α6βγ2 receptors containing the α6-100Q allele, with a resulting change of ∼2.5-fold in equilibrium dissociation constant ( K D). The results indicate that in addition to the central role of the homologous α6-100R/Q (α1-101H) residue in BZ binding and efficacy, this critical BZ binding site residue has a major impact on BZ binding kinetics.