Characterization of wild-type (R100R) and mutated (Q100Q) GABAA alpha 6 subunit in Sardinian alcohol non-preferring rats (sNP)

Sardinian alcohol non-preferring (sNP) rats, selected for their low ethanol preference and consumption, carry a point mutation (R100Q) in the gene coding for GABA(A) receptor alpha(6) subunit, which becomes more sensitive to diazepam-evoked GABA currents. We performed binding studies in the cerebellum of normal (RR) and mutated (QQ) sNP rats using 3H]Ro 15-4513, an inverse agonist for the benzodiazepine site which binds both diazepam insensitive and diazepam sensitive sites. Saturation curves performed on cerebellar membrane from genotyped rats indicated an higher affinity of 3H]Ro 15-4513 for GABA(A) receptors in QQ with respect to RR rats (K(d) values 4.0+/-0.67 and 6.24+/-0.95 nM, respectively), with similar B(max) values (3.5+/-0.25 and 3.9+/-0.39 pmol/mg protein, respectively). Diazepam displacement curves showed a two component model for both genotypes, with similar K(i1) values for QQ and RR (3.6+/-0.62 and 4.9+/-0.33 nM, respectively). In QQ rats diazepam is able to completely displace 3H]Ro 15-4513 (K(i2)=1.48+/-0.27 microM), while in RR rats the diazepam sensitive sites are still present (K(i2)>10 microM). The basal mRNA and protein expression level of the alpha(6) subunit were similar in RR and QQ rats. The electrophysiological profile of oocytes of Xenopus laevis injected with cerebellar synaptosomes showed that ethanol positively modulated GABA-evoked currents significantly more in QQ than in RR rats. These data contribute to the characterization of the function of GABA(A) alpha(6) subunit and its involvement in determining alcohol related behavior.