title: Dual mode of stimulation by the beta-carboline ZK 91085 of recombinant GABA(A) receptor currents: molecular determinants affecting its action.
abstract: In electrophysiological measurements the beta-carboline ethyl 6-benzyloxy-beta-carboline-3-carboxylate (ZK 91085) acts as a positive allosteric modulator on rat recombinant alpha1beta2gamma2 GABA(A) 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 alpha1beta2, which lacks the gamma 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 alpha1beta1, ZK 91085 potentiation is strongly reduced as compared to alpha1beta2. In binding studies, ZK 91085 was able to decrease [35S]-TBPS binding in alpha1beta2gamma2 and alpha1beta2 but not in alpha1beta1. This selectivity of ZK 91085 for receptors containing the beta2 isoform over those containing the beta1 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 alpha1beta2 and mutant receptors for stimulation by ZK 91085. The mutation beta2N265S, that abolishes loreclezole effects, also abolishes ZK 91085 stimulation. The mutation beta2Y62L increased stimulation by ZK 91085 3-4 fold, locating an influencing entity of the second type of action of ZK 91085 at an alpha/beta subunit interface. Structural intermediates of ZK 91085 and the beta-carboline abecarnil, the latter of which only slightly potentiated GABA currents in alpha1/beta2, were analysed to determine structural requirements for modulation. ZK 91085 thus allosterically stimulates the GABA(A) receptor through two sites of action: the benzodiazepine site and the loreclezole site in contrast to classical beta-carbolines, that confer negative allosteric modulation through the benzodiazepine site.
authors.affiliation: Department of Pharmacology, University of Bern, Switzerland.
authors.forename: U
authors.initials: U
authors.lastname: Thomet
authors.forename: R
authors.initials: R
authors.lastname: Baur
authors.forename: P
authors.initials: P
authors.lastname: Scholze
authors.forename: W
authors.initials: W
authors.lastname: Sieghart
authors.forename: E
authors.initials: E
authors.lastname: Sigel
mesh_terms: D000818:Animals; D002243:Carbolines / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005445:Flunitrazepam / Q000378:metabolism; D018757:GABA Modulators / Q000494:pharmacology*; D017354:Point Mutation; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects*; D011994:Recombinant Proteins / Q000187:drug effects; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D002243:Carbolines; D018757:GABA Modulators; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C121467:ethyl 6-benzyloxy-beta-carboline-3-carboyxlate; D005445:Flunitrazepam