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

The alcohol-tolerant AT and -non-tolerant ANT rat lines have been selectively bred for innate sensitivity to ethanol-induced motor impairment. The cerebellar GABAA receptor 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 GABAA receptors between the rat lines. In the present study we have used non-selected 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 15–513 and classical benzodiazepines to cerebellar “diazepam-insensitive” 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 as well as cerebellar diazepam-insensitive sites. Association of [3H]Ro 15-4513 binding was ~10 fold faster and dissociation was ~3–4 fold faster in diazepam-insensitive α6βγ2 receptors containing the α6-100Q allele, with a resulting change of ~2.5-fold in equilibrium dissociation constant (KD). The results indicate that in addition to the central role of the homologous α6-100R/Q (α1-101H) residue in benzodiazepine binding and efficacy, this critical benzodiazepine binding site residue has a major impact on benzodiazepine binding kinetics.