GABA A receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA A receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA A receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

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Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABA A receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an un biased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepinebinding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design. GABA A receptors (GABA A Rs) are the major inhibitory transmitter receptors in the brain and the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anesthetics and convulsants 1 . These receptors are chloride ion channels composed of five subunits that can belong to different subunit classes. A total of 19 GABA A receptor subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-© 2012 Nature America, Inc. All rights reserved. * margot.ernst@meduniwien.ac.at. author contributions L.R., W.S., G.F.E., I.J.P.d.E., C.d.G. and M.E. contributed to the design and evaluation of experiments. Docking and virtual screening experiments were performed by L.R. (supervised by G.F.E.) and C.d.G. GABA A R models were generated and evaluated by M.E. and M.M. Radioligand and electrophysiological experiments were performed by Z.V. The manuscript was written by W.S., L.R., G.F.E., I.J.P.d.E., C.d.G. and M.E., and all authors commented on and helped to revise the text. Competing financial interestsThe authors declare no competing financial interests. Additional informationSupplementary information and chemical compound information are available online at http://www.nature.com/ naturechemicalbiology/. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Correspondence and requests for materials should be addressed to M.E. Accession codes. PDB: the previously determined crystal structures for A. californica AChBP, L. stagnalis AChBP, mouse nAChR, a nematode glutamategated chloride channel, an nAChR structure and four AChBP structures are deposited under accession codes 2BYQ, 1UW6, 2QC1, 3RIF, 2BG9, 1I9B, 2BYN, 2BYR and 2BYS, respectively. Europe PMC Funders GroupAuthor Manuscript Nat Chem Biol. Author manuscript; available in PMC 2012 June 06. Europe PMC Funders Author ManuscriptsEurope PMC Fun...

GABA A receptorsmediatetheactionofmanyclinicallyimportantdrugsinteractingwithdifferentbindingsites.Forsomepotentialbindingsites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular ␣1ϩ␤3Ϫ interface, which is homologous to the benzodiazepine binding site at the ␣1ϩ␥2Ϫ interface. On screening of Ͼ100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3Ϫc]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at ␣1␤3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at ␣1␤3␥2 and ␣1␤3 receptors. By mutating pocket-forming amino acid residues at the ␣1ϩ and the ␤3Ϫ side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethiosulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect of CGS 9895. The inhibition was not caused by a general inactivation of GABA A receptors, because the GABA-enhancing effect of ROD 188 or the steroid ␣-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the ␣ and ␤ subunit types forming the interface. CGS 9895thusrepresentsthefirstprototypeofdrugsmediatingbenzodiazepine-likemodulatoryeffectsviathe␣ϩ␤ϪinterfaceofGABA A receptors. Sincesuchbindingsitesarepresentat␣␤,␣␤␥,and␣␤␦receptors,suchdrugswillhaveamuchbroaderactionthanbenzodiazepinesandmight become clinical important for the treatment of epilepsy.

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