Dual mode of stimulation by the β‐carboline ZK 91085 of recombinant GABAA receptor currents: molecular determinants affecting its action

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Abstract
In electrophysiological measurements the β‐carboline ethyl 6‐benzyloxy‐β‐carboline‐3‐carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant α1β2γ2 GABAA receptors and binds with high affinity (IC50=1.5 nM) to the [3H]‐flunitrazepam site. Flumazenil was able to partially counteract the current modulation. These observations indicate an action of ZK 91085 at the benzodiazepine binding site. At the dual subunit combination α1β2, which lacks the γ subunit required for benzodiazepine modulation, we still observed a potentiation of GABA currents. Thus ZK 91085 acts via an additional site on the channel. At the subunit combination α1β1, ZK 91085 potentiation is strongly reduced as compared to α1β2. In binding studies, ZK 91085 was able to decrease [35S]‐TBPS binding in α1β2γ2 and α1β2 but not in α1β1. This selectivity of ZK 91085 for receptors containing the β2 isoform over those containing the β1 isoform is reminiscent of the action of loreclezole. To identify amino acid residues important for the second type of modulation, we functionally compared wild type α1β2 and mutant receptors for stimulation by ZK 91085. The mutation β2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation β2Y62L increased stimulation by ZK 91085 3–4 fold, locating an influencing entity of the second type of action of ZK 91085 at an α/β subunit interface. Structural intermediates of ZK 91085 and the β‐carboline abecarnil, the latter of which only slightly potentiated GABA currents in α1β2, were analysed to determine structural requirements for modulation. ZK 91085 thus allosterically stimulates the GABAA receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical β‐carbolines, that confer negative allosteric modulation through the benzodiazepine site. British Journal of Pharmacology (1999) 127, 1231–1239; doi:10.1038/sj.bjp.0702639