Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

Radovan Spurnya, Joachim Ramerstorferb, Kerry Pricec, Marijke Bramsa, Margot Ernstb, Hugues Nuryd, Mark Verheije, Pierre Legrandf, Daniel Bertrandg, Sonia Bertrandg, Dennis A. Doughertyh, Iwan J. P. de Esche, Pierre-Jean Corringerd, Werner Sieghartb, Sarah C. R. Lummisc, and Chris Ulensa,1 aDepartment of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, Catholic University of Leuven, 3000 Leuven, Belgium; bDepartment of Biochemistry and Molecular Biology of the Nervous System, Medical University of Vienna, A-1090 Vienna, Austria; cDepartment of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom; dPasteur Institute, G5 Group of Channel-Receptor, Centre National de la Recherche Scientifique, 75724 Paris, France; eDepartment of Medicinal Chemistry, VU University Amsterdam, 1081 HV, Amsterdam, The Netherlands; fSOLEIL Synchrotron, 91192 Gif sur Yvette, France; gHiQScreen, CH 1211 Geneva, Switzerland; and hCalifornia Institute of Technology, Pasadena, CA 91125 AUTHOR SUMMARY

Ligand-gated ion channels play X-ray crystal structure of ELIC an important role in fast elec- in complex with GABA, which trochemical signaling in the reveals important features of brain. Pentameric ligand-gated agonist recognition. For exam- ion channels are activated by ple, the ligand-binding site is specific neurotransmitters formed by highly conserved (ligands), including acetylcho- aromatic residues, which form line (ACh), serotonin (5-HT), an electron-rich environment γ glycine (Gly), and -amino- Fig. P1. (Left) Cross-section through a surface representation of the that stabilizes the cationic amino butyric acid (GABAA/C). Differ- ELIC crystal structure obtained in the presence of excess GABA and moiety of GABA. To probe the ent receptors contain an a low concentration of flurazepam. One GABA molecule is shown as importance of such cation–π extracellular domain that spe- green spheres at the interface between two neighboring subunits. interactions, we introduced non- cifically recognizes one of these One subunit forms the principal face (shown in yellow), and the other naturally occurring amino acid neurotransmitters and opens subunit forms the complementary face (shown in blue). Flurazepam is residues into ELIC and de- a transmembrane channel upon shown as red spheres and is localized at an intrasubunit benzo- termined the affinity of GABA diazepine site that faces the channel vestibule in ELIC (marked by ligand binding. The GABAA/C a gray square). (Right) The ELIC crystal structure obtained in the for each derivative. A serial and Gly receptors selectively presence of equal amounts of GABA and bromo-flurazepam. Under decrease in agonist potency with transport chloride ions, which these conditions, bromo-flurazepam displaces GABA and occupies the increasing fluorination is hyperpolarize the cell membrane intersubunit benzodiazepine site (marked by a gray square). indicative of a cation–π inter- and reduce electrical excitability. action. We observed a clear Here, we showed that a bacterial correlation between agonist receptor, ELIC, is similarly activated by GABA and is modulated potency and the cation-π–binding ability of phenylalanine by therapeutically important psychoactive drugs, benzodiaze- derivatives incorporated into two positions of the binding site, pines. These findings reveal aspects of the mechanism of action namely, loop B and C aromatics. This result is consistent with the of mammalian neurotransmitter signaling. GABA-binding conformation in the crystal structure, which GABAA receptors serve as a target for a therapeutically im- places the amino moiety almost equidistant between the loop B portant class of psychoactive drugs, namely, benzodiazepines, and C aromatic residues in the binding pocket. which are widely prescribed because of their anxiolytic, anti- Next, we determined crystal structures of ELIC in complex convulsant, muscle relaxant, and sedative hypnotic effects (1). with different benzodiazepine-site ligands, namely, flurazepam, Benzodiazepines, such as diazepam (Valium), bind to the bromo-flurazepam, and zopiclone; zopiclone is a more recently GABAA receptor at a site that is separate (allosteric) from the developed nonbenzodiazepine hypnotic. We observe that, site (orthosteric) for the agonist GABA. Benzodiazepines derive depending on their concentrations, benzodiazepines occupy two their effects from a fundamental molecular property, which is to possible sites on ELIC (Fig. P1). We discovered an intrasubunit modulate channel opening when GABA is bound to the re- site adjacent to the GABA-recognition site, but it faces the ceptor. This action of benzodiazepines, positive allosteric mod- channel vestibule. Using cysteine-scanning mutagenesis, we ulation, causes a reduction of electrical excitability in the central demonstrated that this site is involved in the potentiating effects nervous system, ultimately inducing sedation, anxiolysis, and sleep. Author contributions: W.S., S.C.R.L., and C.U. designed research; R.S., J.R., K.P., M.B., M.V., The molecular interactions required for recognition of GABA P.L., D.B., S.B., S.C.R.L., and C.U. performed research; M.V., S.B., D.A.D., P.-J.C., S.C.R.L., and and benzodiazepines at their respective receptor-binding sites C.U. contributed new reagents/analytic tools; R.S., J.R., K.P., M.B., M.E., H.N., M.V., P.L., have been studied extensively using various approaches, in- D.B., S.B., D.A.D., I.J.P.d.E., W.S., S.C.R.L., and C.U. analyzed data; and R.S., J.R., K.P., M.E., cluding photo-affinity labeling and mutagenesis experiments. M.V., D.B., I.J.P.d.E., P.-J.C., W.S., S.C.R.L., and C.U. wrote the paper. However, mechanistic details of their molecular recognition are The authors declare no conflict of interest. still unclear. One major reason is that structural data for this This Direct Submission article had a prearranged editor. therapeutically important class of receptors is lacking. Freely available online through the PNAS open access option. Here, we report that a recently identified bacterial ligand- Data deposition: The structures of ELIC in complex with GABA and flurazepam, zopiclone, gated ion channel, ELIC, is activated by GABA and modulated and Br-flurazepam have been deposited in the Protein Data Bank, www.pdb.org (PDB ID by certain benzodiazepines with effects that are similar to human codes 4A96, 4A97, and 4A98, respectively). 1To whom correspondence should be addressed. E-mail: [email protected]. GABAA receptors. Similar to recent results published by Zim- mermann and Dutzler (2), ELIC was found to be activated by See full research article on page E3028 of www.pnas.org. relatively high concentrations of GABA. We determined the Cite this Author Summary as: PNAS 10.1073/pnas.1208208109.

17752–17753 | PNAS | October 30, 2012 | vol. 109 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1208208109 Downloaded by guest on October 1, 2021 of flurazepam on ELIC. A second intersubunit site partially investigate a possible functional role of the intrasubunit site in PNAS PLUS overlaps the GABA site and likely corresponds to a low-affinity human GABAA receptors. benzodiazepine-binding site in GABAA receptors that mediate Our present study offers a structural view of how GABA and the inhibitory effects of the benzodiazepine flurazepam. The benzodiazepines are recognized at multiple allosteric binding existence of multiple binding sites for GABA and benzodiaze- sites; this structure helps explain the multisite modulation in the pines further substantiates a multisite model of allosteric class of pentameric ligand-gated ion channels. modulation in this family of channels. The discovery of an intrasubunit benzodiazepine site facing the vestibule could help 1. Sieghart W (1995) Structure and pharmacology of gamma-aminobutyric acidA receptor subtypes. Pharmacol Rev 47(2):181–234. in understanding unexplained actions of benzodiazepines on 2. Zimmermann I, Dutzler R (2011) Ligand activation of the prokaryotic pentameric GABAA receptors. However, further studies are warranted to ligand-gated ion channel ELIC. PLoS Biol 9(6):e1001101. PHARMACOLOGY

Spurny et al. PNAS | October 30, 2012 | vol. 109 | no. 44 | 17753 Downloaded by guest on October 1, 2021