Alterations of the benzodiazepine site of rat α6β2γ2‐GABAA receptor by replacement of several divergent amino‐terminal regions with the αl counterparts

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Abstract
1. The benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype of gamma-aminobutyric acidA (GABAA) receptors is distinguishable from that of the alpha 1 beta 2 gamma 2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15-1788, which behaves as an antagonist in the alpha 1 beta 2 gamma 2 subtype. 2. The point mutation of Arg 100 of the alpha 6 subunit to histidine (the corresponding residue in alpha 1) has been shown to enable the alpha 6 beta 2 gamma 2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15-1788 to enhance GABA-induced Cl- currents. 3. Here we identified the segment of P161 to L187 of alpha 6 to contain the functional region responsible for the agonistic action of Ro 15-1788. Its replacement with the corresponding alpha 1 sequence abolished the ability of Ro 15-1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U-92330 and 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4. In addition we have learned that several residues in the N-terminal region of alpha 6, such as R100, V142 and N143, have the ability to influence GABA-dependent Cl- current induction probably by allosterically modulating low affinity GABA sites.