Natural mutation of GABAA receptor alpha 6 subunit alters benzodiazepine affinity but not allosteric GABA effects.

The binding of the imidazobenzodiazepine, [3H]Ro 15-4513, to cerebellar granule cell-specific GABAA/benzodiazepine receptors is typically insensitive to benzodiazepine receptor agonists such as diazepam. A mutation in the alpha 6 subunit, causing replacement of the arginine at the 100 position by glutamine (Q100), has recently been found in an alcohol- and benzodiazepine-sensitive rat line. The mutant alpha 6(Q100)beta 2 gamma 2 recombinant receptors are sensitive to diazepam. The binding of [3H]Ro 15-4513 to cerebellar diazepam-insensitive receptors is enhanced by GABA, whereas binding to diazepam-sensitive receptors is inhibited. Recombinant receptors consisting of beta 2 and gamma 2 subunits together with the wildtype alpha 6 or mutant alpha 6(Q100) subunit showed positive modulation of [3H]Ro 15-4513 binding by GABA, whereas alpha 1 beta 2 gamma 2 receptors showed negative modulation. The picrotoxin-sensitive binding of a convulsant, t-butylbicyclophosphoro[35S]thionate ([35S]TBPS), was inhibited in the alpha 6 beta 2 gamma 2 and alpha 6(Q100) beta 2 gamma 2 receptors by GABA at concentrations less than one-tenth of those required in the alpha 1 beta 2 gamma 2 receptors. GABA effects on [35S]TBPS binding were only slightly affected by diazepam in the alpha 6(Q100) beta 2 gamma 2 receptors, while profound effects were seen in the alpha 1 beta 2 gamma 2 receptors in the presence of diazepam. The results with the mutant receptor suggest that the alpha 1 and alpha 6 subunits are responsible for differential allosteric actions by GABA on other binding sites, independently of the structures defining the benzodiazepine binding pharmacology.