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Crystal structure of a human GABAA receptor

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Abstract

Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.

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Figure 1: Architecture of GABAAR-β3cryst.
Figure 2: Assembly interactions in GABAAR-β3cryst.
Figure 3: Neurotransmitter pocket occupied by the agonist benzamidine.
Figure 4: A conserved glycosylation site interacts with β9–β10 loop residues.
Figure 5: Structural coupling at the ECD–TMD interface.
Figure 6: Structure of the ion channel in a desensitized state.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and the structure factors have been deposited in the Protein Data Bank under the accession code 4COF.

Change history

  • 20 August 2014

    Edits were made to the Extended Data legends, including udpates to reference citations in the Extended Data Fig. 6 legend.

References

  1. Rabow, L. E., Russek, S. J. & Farb, D. H. From ion currents to genomic analysis: recent advances in GABAA receptor research. Synapse 21, 189–274 (1995)

    CAS  PubMed  Article  Google Scholar 

  2. Grenningloh, G. et al. Glycine vs GABA receptors. Nature 330, 25–26 (1987)

    CAS  PubMed  Article  ADS  Google Scholar 

  3. Simon, J., Wakimoto, H., Fujita, N., Lalande, M. & Barnard, E. A. Analysis of the set of GABAA receptor genes in the human genome. J. Biol. Chem. 279, 41422–41435 (2004)

    CAS  PubMed  Article  Google Scholar 

  4. Sigel, E. & Steinmann, M. E. Structure, function, and modulation of GABAA receptors. J. Biol. Chem. 287, 40224–40231 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Yip, G. M. et al. A propofol binding site on mammalian GABAA receptors identified by photolabeling. Nature Chem. Biol. 9, 715–720 (2013)

    CAS  Article  ADS  Google Scholar 

  6. Karlin, A. & Akabas, M. H. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron 15, 1231–1244 (1995)

    CAS  PubMed  Article  Google Scholar 

  7. Miller, P. S. & Smart, T. G. Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol. Sci. 31, 161–174 (2010)

    CAS  PubMed  Article  Google Scholar 

  8. Rudolph, U. & Knoflach, F. Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes. Nature Rev. Drug Discov. 10, 685–697 (2011)

    CAS  Article  Google Scholar 

  9. Li, G. D. et al. Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J. Neurosci. 26, 11599–11605 (2006)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Wallner, M., Hanchar, H. J. & Olsen, R. W. Ethanol enhances α4β3δ and α6β3δ γ-aminobutyric acid type A receptors at low concentrations known to affect humans. Proc. Natl Acad. Sci. USA 100, 15218–15223 (2003)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  11. Belelli, D. & Lambert, J. J. Neurosteroids: endogenous regulators of the GABAA receptor. Nature Rev. Neurosci. 6, 565–575 (2005)

    CAS  Article  Google Scholar 

  12. Brejc, K. et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276 (2001)

    CAS  PubMed  Article  ADS  Google Scholar 

  13. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003)

    CAS  PubMed  Article  ADS  Google Scholar 

  14. Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J. Mol. Biol. 346, 967–989 (2005)

    CAS  PubMed  Article  Google Scholar 

  15. Hilf, R. J. & Dutzler, R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature 452, 375–379 (2008)

    CAS  PubMed  Article  ADS  Google Scholar 

  16. Bocquet, N. et al. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 457, 111–114 (2009)

    CAS  PubMed  Article  ADS  Google Scholar 

  17. Hibbs, R. E. & Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Nury, H. et al. X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. Nature 469, 428–431 (2011)

    CAS  PubMed  Article  ADS  Google Scholar 

  19. Jansen, M., Bali, M. & Akabas, M. H. Modular design of Cys-loop ligand-gated ion channels: functional 5–HT3 and GABA ρ1 receptors lacking the large cytoplasmic M3M4 loop. J. Gen. Physiol. 131, 137–146 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Hansen, S. B., Wang, H. L., Taylor, P. & Sine, S. M. An ion selectivity filter in the extracellular domain of Cys-loop receptors reveals determinants for ion conductance. J. Biol. Chem. 283, 36066–36070 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Sauguet, L. et al. Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels. EMBO J. 32, 728–741 (2013)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Gurba, K. N., Hernandez, C. C., Hu, N. & Macdonald, R. L. GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L γ-aminobutyric acid type A (GABAA) receptor expression and channel gating. J. Biol. Chem. 287, 12083–12097 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Sancar, F. & Czajkowski, C. A GABAA receptor mutation linked to human epilepsy (γ2R43Q) impairs cell surface expression of αβγ receptors. J. Biol. Chem. 279, 47034–47039 (2004)

    CAS  PubMed  Article  Google Scholar 

  24. Epi, K. C. et al. De novo mutations in epileptic encephalopathies. Nature 501, 217–221 (2013)

    Article  ADS  CAS  Google Scholar 

  25. Bergmann, R., Kongsbak, K., Sorensen, P. L., Sander, T. & Balle, T. A Unified model of the GABAA receptor comprising agonist and benzodiazepine binding sites. PLoS ONE 8, e52323 (2013)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  26. Hansen, S. B. et al. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24, 3635–3646 (2005)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Huang, S. et al. Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera. Biochem. J. 454, 303–310 (2013)

    CAS  PubMed  Article  Google Scholar 

  28. Celie, P. H. et al. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41, 907–914 (2004)

    CAS  PubMed  Article  Google Scholar 

  29. Newell, J. G., McDevitt, R. A. & Czajkowski, C. Mutation of glutamate 155 of the GABAA receptor β2 subunit produces a spontaneously open channel: a trigger for channel activation. J. Neurosci. 24, 11226–11235 (2004)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Venkatachalan, S. P. & Czajkowski, C. A conserved salt bridge critical for GABAA receptor function and loop C dynamics. Proc. Natl Acad. Sci. USA 105, 13604–13609 (2008)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  31. Wagner, D. A., Czajkowski, C. & Jones, M. V. An arginine involved in GABA binding and unbinding but not gating of the GABAA receptor. J. Neurosci. 24, 2733–2741 (2004)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Mukhtasimova, N., Free, C. & Sine, S. M. Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor. J. Gen. Physiol. 126, 23–39 (2005)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Rees, D. C., Congreve, M., Murray, C. W. & Carr, R. Fragment-based lead discovery. Nature Rev. Drug Discov. 3, 660–672 (2004)

    CAS  Article  Google Scholar 

  34. Lo, W. Y. et al. Glycosylation of β2 subunits regulates GABAA receptor biogenesis and channel gating. J. Biol. Chem. 285, 31348–31361 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Miller, P. S., Da Silva, H. M. & Smart, T. G. Molecular basis for zinc potentiation at strychnine-sensitive glycine receptors. J. Biol. Chem. 280, 37877–37884 (2005)

    CAS  PubMed  Article  Google Scholar 

  36. Buhr, A. et al. Functional characterization of the new human GABAA receptor mutation β3(R192H). Hum. Genet. 111, 154–160 (2002)

    CAS  PubMed  Article  Google Scholar 

  37. Dellisanti, C. D., Yao, Y., Stroud, J. C., Wang, Z. Z. & Chen, L. Crystal structure of the extracellular domain of nAChR α1 bound to α-bungarotoxin at 1.94 Å resolution. Nature Neurosci. 10, 953–962 (2007)

    CAS  PubMed  Article  Google Scholar 

  38. Lee, W. Y. & Sine, S. M. Principal pathway coupling agonist binding to channel gating in nicotinic receptors. Nature 438, 243–247 (2005)

    CAS  PubMed  Article  ADS  Google Scholar 

  39. Akabas, M. H. Using molecular dynamics to elucidate the structural basis for function in pLGICs. Proc. Natl Acad. Sci. USA 110, 16700–16701 (2013)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  40. Baulac, S. et al. First genetic evidence of GABAA receptor dysfunction in epilepsy: a mutation in the γ2-subunit gene. Nature Genet. 28, 46–48 (2001)

    CAS  PubMed  Google Scholar 

  41. Lape, R., Plested, A. J., Moroni, M., Colquhoun, D. & Sivilotti, L. G. The α1K276E startle disease mutation reveals multiple intermediate states in the gating of glycine receptors. J. Neurosci. 32, 1336–1352 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Xu, M. & Akabas, M. H. Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAA receptor alpha1 subunit. J. Gen. Physiol. 107, 195–205 (1996)

    CAS  PubMed  Article  Google Scholar 

  43. Cymes, G. D., Ni, Y. & Grosman, C. Probing ion-channel pores one proton at a time. Nature 438, 975–980 (2005)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  44. Unwin, N. & Fujiyoshi, Y. Gating movement of acetylcholine receptor caught by plunge-freezing. J. Mol. Biol. 422, 617–634 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Horenstein, J. & Akabas, M. H. Location of a high affinity Zn2+ binding site in the channel of α1β1 γ-aminobutyric acidA receptors. Mol. Pharmacol. 53, 870–877 (1998)

    CAS  PubMed  Google Scholar 

  46. Rajendra, S. et al. Mutation of an arginine residue in the human glycine receptor transforms β-alanine and taurine from agonists into competitive antagonists. Neuron 14, 169–175 (1995)

    CAS  PubMed  Article  Google Scholar 

  47. Young, G. T., Zwart, R., Walker, A. S., Sher, E. & Millar, N. S. Potentiation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc. Natl Acad. Sci. USA 105, 14686–14691 (2008)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  48. daCosta, C. J., Free, C. R., Corradi, J., Bouzat, C. & Sine, S. M. Single-channel and structural foundations of neuronal α7 acetylcholine receptor potentiation. J. Neurosci. 31, 13870–13879 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Shan, Q., Haddrill, J. L. & Lynch, J. W. Ivermectin, an unconventional agonist of the glycine receptor chloride channel. J. Biol. Chem. 276, 12556–12564 (2001)

    CAS  PubMed  Article  Google Scholar 

  50. Jensen, M. L. et al. The β subunit determines the ion selectivity of the GABAA receptor. J. Biol. Chem. 277, 41438–41447 (2002)

    CAS  PubMed  Article  Google Scholar 

  51. Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D 62, 1243–1250 (2006)

    PubMed  Article  CAS  Google Scholar 

  52. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

    CAS  PubMed  Article  ADS  Google Scholar 

  53. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnol. 20, 87–90 (2002)

    CAS  Article  Google Scholar 

  54. Molday, R. S. & MacKenzie, D. Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes. Biochemistry 22, 653–660 (1983)

    CAS  PubMed  Article  Google Scholar 

  55. Oprian, D. D., Molday, R. S., Kaufman, R. J. & Khorana, H. G. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc. Natl Acad. Sci. USA 84, 8874–8878 (1987)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  56. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

    CAS  PubMed  Article  Google Scholar 

  57. Reeves, P. J., Callewaert, N., Contreras, R. & Khorana, H. G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  58. Aricescu, A. R. & Owens, R. J. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Curr. Opin. Struct. Biol. 23, 345–356 (2013)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Tasneem, A., Iyer, L. M., Jakobsson, E. & Aravind, L. Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels. Genome Biol. 6, R4 (2004)

    PubMed  PubMed Central  Article  Google Scholar 

  60. Jansen, M., Bali, M. & Akabas, M. H. Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA ρ1 receptors lacking the large cytoplasmic M3M4 loop. J. Gen. Physiol. 131, 137–146 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  61. Chaudhary, S., Pak, J. E., Gruswitz, F., Sharma, V. & Stroud, R. M. Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells. Nature Protocols 7, 453–466 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Chang, V. T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. Walter, T. S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D 61, 651–657 (2005)

    PubMed  Article  CAS  Google Scholar 

  64. Parker, J. L. & Newstead, S. Current trends in α-helical membrane protein crystallization: an update. Protein Sci. 21, 1358–1365 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Cryst. 43, 186–190 (2009)

    Article  CAS  Google Scholar 

  66. Hibbs, R. E. & Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  67. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. 64, 61–69 (2008)

    CAS  Google Scholar 

  69. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. Bricogne, G. et al. BUSTER version 2.11.2. Cambridge, UK (Global Phasing Ltd., 2011)

  71. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  72. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    CAS  PubMed  Article  Google Scholar 

  73. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    CAS  Article  PubMed  Google Scholar 

  74. Stuart, D. I., Levine, M., Muirhead, H. & Stammers, D. K. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 Å. J. Mol. Biol. 134, 109–142 (1979)

    CAS  PubMed  Article  Google Scholar 

  75. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)

    CAS  PubMed  Article  Google Scholar 

  76. Deprez, C. et al. Solution structure of the E. coli TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain. J. Mol. Biol. 346, 1047–1057 (2005)

    CAS  PubMed  Article  Google Scholar 

  77. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  78. Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLOS Comput. Biol. 8, e1002708 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  79. Hattori, M., Hibbs, R. E. & Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20, 1293–1299 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  80. Fertig, N. et al. Port-a-patch and patchliner: high fidelity electrophysiology for secondary screening and safety pharmacology. Comb. Chem. High Through. Screen. 12, 24–37 (2009)

    Article  Google Scholar 

  81. Saras, A. et al. Histamine action on vertebrate GABAA receptors: direct channel gating and potentiation of GABA responses. J. Biol. Chem. 283, 10470–10475 (2008)

    CAS  PubMed  Article  Google Scholar 

  82. Butts, C. A. et al. Identification of a fluorescent general anesthetic, 1-aminoanthracene. Proc. Natl Acad. Sci. USA 106, 6501–6506 (2009)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

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Acknowledgements

We thank T. Malinauskas, Y. Kong and staff at Diamond Light Source beamlines I03 and I24 for synchrotron assistance; K. Harlos and T. Walter for technical support with crystallization; G. Schertler and J. Standfuss for advice concerning the Rho-1D4 affinity purification method; T. Nakagawa for electron microscopy sample examination; F. Ashcroft, S. Tucker, M. Clausen and P. Proks for access to electrophysiology equipment and assistance with electrophysiological recordings; J. McIlhinney, M. Sansom, L. Carpenter, S. Newstead, I. de Moraes and members of the Aricescu laboratory for discussions; E.Y. Jones, D.I. Stuart and C. Siebold for reading the manuscript. This work was supported by grants from the Wellcome Trust (OXION: Ion channels and Disease Initiative, 084655), the UK Medical Research Council ((MRC) G0700232) and the Royal Society (RG090810). Further support from the Wellcome Trust Core Award Grant Number 090532/Z/09/Z is acknowledged. P.S.M. was a Wellcome Trust OXION Training Fellow. A.R.A. is an MRC Senior Research Fellow.

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Authors and Affiliations

Authors

Contributions

The authors have jointly contributed to the project design, data analysis and manuscript preparation. Experimental work was performed by P.S.M. (protein expression, purification, crystallization, ligand binding assays and electrophysiology) and A.R.A. (crystallography).

Corresponding authors

Correspondence to Paul S. Miller or A. Radu Aricescu.

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Competing interests

A patent application related to the use of benzamidine derivatives as GABAA receptor modulators has been filed by the University of Oxford.

Extended data figures and tables

Extended Data Figure 1 Ligand binding to GABAAR-β3cryst in detergent micelles and in HEK293T cells.

a, Overlays of size-exclusion profiles for equal amounts of purified GABAAR-β3cryst that were pre-heated at different temperatures for 1 h to evaluate protein stability. b, Overlays of size-exclusion profiles of purified GABAAR-β3cryst heated at 66 °C (70% decay temperature) for 1 h in the presence of increasing doses of histamine. Histamine binding protects (stabilizes) the protein in accordance with its affinity, giving a dose–response profile. c, Profiles of GABAAR-β3cryst thermostabilization in detergent micelles by four ligands. The assay was used to evaluate two channel blockers, the insecticide fipronil (26 ± 5 nM) and the convulsant picrotoxin (900 ± 480 nM), and two neurotransmitter agonists, GABA (2.3 ± 0.2 mM) and histamine (400 ± 150 μM); values in brackets represent the EC50 of thermostabilization, n = 4. Note that the GABAAR-β3cryst low sensitivity to GABA is in keeping with that observed for full-length homomeric GABAAR β3 receptors, which are also less sensitive than αβ and αβγ heteromeric GABAARs81. d, e Displacement of bound 1-aminoanthracene (1-AMA) from GABAAR-β3cryst by the anaesthetic etomidate. The fluorescent ligand 1-AMA (5 μM) in the presence of 0.3 μM GABAAR-β3cryst experienced an increase in fluorescence (due to binding in a hydrophobic anaesthetic pocket82) that was displaced by increasing concentrations of etomidate; 50% maximal displacement occurs at 7.1 ± 1.1 μM, n = 5. Peak heights of single traces were measured as the average intensities of peak points between 519 and 533 nm. Equivalent doses of etomidate with the fluorescent ligand 1-AMA (5 μM) in the absence of GABAAR-β3cryst did not displace (reduce) fluorescent signal (not shown). f, g, HEK293T whole-cell patch-clamp recordings of the natural ligand histamine (f), activating an inward current through GABAAR-β3cryst, subsequently blocked by the channel-blocker picrotoxin (fipronil also blocked histamine currents, not shown), and of the anaesthetic propofol, activating an inward current through GABAAR-β3cryst (g). All error bars are ± s.e.m.

Extended Data Figure 2 Structural alignment of pLGIC extracellular domains and AChBP.

The top panel shows structures aligned to the GABAAR-β3cryst ECD, viewed perpendicular to the five-fold pseudo-symmetry axis, from inside the extracellular vestibule. Colour coding: GABAAR-β3cryst in red (PDB 4COF), GluClα in green (PDB 3RIF), ELIC in yellow (PDB 2VL0), GLIC in cyan (PDB 4HFI), AChBP in dark blue (PDB 1UX2), nAChR in violet (PDB 2BG9). Four variable loop regions of functional significance for neurotransmitter binding, signal transduction and receptor assembly have been individually rotated to optimize viewing of the disparities between these structural elements. a, Loop C (β9–β10), capping the neurotransmitter-binding site. b, Loop β6–β7 (Cys-loop in eukaryotic structures), important for ECD–TMD coupling and signal transduction. c, Loop β1–β2, important for ECD–TMD coupling and signal transduction. d, Loop β5–β5′, important for subunit assembly at the ECD level. e, Table showing parameters of structural alignment between the GABAAR-β3cryst ECDs and TMDs, respectively, and equivalent regions in pLGICs as well as the AChBP from Lymnaea stagnalis. Superpositions were performed using the SHP programme (see Methods for details).

Extended Data Figure 3 Sequence alignment of GABAAR-β3cryst with representative human Cys-loop receptor family members and the other pLGICs crystallized to date (ELIC, GLIC and GluClα).

Residue conservation is indicated by black/grey highlights. Residues involved in inter-subunit salt bridges are highlighted in red/blue, sites of N-linked glycosylation are highlighted in orange. Sequence block-highlights indicate the classically defined neurotransmitter-binding loops (A–F, in light cyan), as well as key loops discussed in the manuscript: β1–β2 in purple, β6–β7 (the Cys-loop) in dark green, β8′–β9 loop in light red, M2–M3 loop in yellow, M3–M4 loop in mustard. Dots above the sequence mark residues linked to human diseases (red), binding of anaesthetics (violet), interactions with agonist benzamidine (green) and Tyr 299, whose side-chain conformation appears to contribute to the control of channel desensitization (cyan). Orange hexagons indicate N-linked glycans observed in the GABAAR-β3cryst structure. C-terminal residues on dark blue background represent affinity purification tags. Secondary structure element colouring corresponds to Fig. 1c. The GABAAR-β3cryst residue numbering, shown above the sequence, matches the mature isoform 1 (UniProt entry P28472, Gln 26 becoming Gln 1). Other sequences are from the following Uniprot entries: GBRB2, P47870; GBRB1, P18505; GLRA1, P23415; GLRB, P48167; GBRA1, P14867; GABRG1, Q8N1C3; ACHA1, P02708-2; ACHB1, P11230; ACHD, Q07001; ACHG, P07510; ACHA7, P36544; 5HT3A, P46098; ELIC, P0C7BY7; GLIC, Q7NDN8. GluClα sequence is from PDB 3RIF. To keep the alignment as compact as possible, the following regions of poor conservation were removed: secretion signal sequences; cytoplasmic M3–M4 loops (as annotated in Uniprot), except for the bacterial channels and GluClα; residues 184–192 (GLGPDGQGH) from ACHB1; residues 188–199 (KENRTYPVEWII) from ACHD; residues 187–194 (GQTIEWIF) from ACHG.

Extended Data Figure 4 Solvent-accessible surfaces of GABAAR-β3cryst coloured by electrostatic potential.

a, Outside view of the receptor, perpendicular to the five-fold pseudo-symmetry axis. The exit point of an ECD side tunnel is indicated by a dotted circle (transversal sections in g, h are at this level). b, View from the extracellular side, along the five-fold pseudo-symmetry axis. c, View from the intracellular side, along the five-fold pseudo-symmetry axis. The positively charged region surrounding the central pore originates from the dipoles of the M2 helices. d, Longitudinal cross-section (interior cartoon coloured grey except for pore-lining helices in deep teal), showing electrostatic surface potential inside the pore and in the extracellular vestibule. Arrowheads indicate positions of the transverse cross-sections. A chloride ion bound within the positively charged vestibule belt is shown as a green sphere. The green asterisk marks the exit of an inter-subunit side tunnel. e, f, Transverse section at level of the neurotransmitter-binding site (negatively charged), observed from above and underneath. g, h, Transverse section at level of the ECD tunnels, negatively charged. i, j, Transverse section at level of the anaesthetic (etomidate)-binding site, positively charged.

Extended Data Figure 5 Crystallographic quality control for non-protein elements in the GABAAR-β3cryst structure.

ac, The anion binding site between ECD interfaces (corresponding to Fig. 2c). a, SigmaA-weighted 2Fo – Fc (blue, contoured at 1.5σ) and 2Fo – Fc (green/red contoured at +3σ/−3σ) electron density maps following autoBUSTER refinement in the absence of chloride. b, The same electron density maps and contour levels following refinement in the presence of chloride. c, Final model, showing the chloride coordination sphere. df, Equivalent panels to the ones described above, for the benzamidine ligand bound to the neurotransmitter pocket. gi, Equivalent panels to the ones described above, for the N-linked glycan at site 3 (Asn 149) except that the contour level of the 2Fo – Fc maps is 1σ. Dotted lines in f and i indicate contacts within hydrogen-bonding distance.

Extended Data Figure 6 Binding cavities for intravenous anaesthetics.

a, Side view of a GABAAR-β3cryst surface representation. Dotted lines indicate the planes of the transverse sections shown in b and c. b, Transverse section through the pentamer at the level of 17′ His (His 267), previously found to bind photolabelled propofol5. c, Close-up of tilted transverse section indicated in a, revealing the putative anaesthetic binding pockets in GABAAR-β3cryst in agreement with previous mutagenesis and photolabelling studies with etomidate9 and propofol5 analogues. E, etomidate-binding site; P, propofol-binding site.

Extended Data Figure 7 Assembly interfaces between GABAAR-β3cryst subunits.

a, Top and side (from the vestibule) view of two GABAAR-β3cryst neighbouring subunits, highlighting the nature of inter-subunit contacts between the principal (P) face of one subunit and complementary (C) face of the next (box indicates the region enlarged in e). Residues involved in salt bridges are coloured purple and red, those forming putative hydrogen bonds in cyan and residues forming van der Waals contacts are in orange. b, Analysis of the inter-subunit interfaces between the ECDs and the TMDs. Values shown correspond to the most extensive inter-subunit interface in each PDB entry. The ECD was defined from the N terminus up to one residue C-terminal of the conserved Arg at the end of β10 strands in all pLGICs (Arg 216 in GABAAR-β3cryst). The TMD was defined as all residues beyond this point. c, ‘Open book’ view of the inter-subunit interfaces (subunits were rotated 126° outwards around their long axis, relative to their side orientation in a), with surfaces coloured by the nature of interactions. Dotted lines delineate the trajectory of an inter-subunit side-tunnel. d, Open book view (as above) of the inter-subunit interfaces with surface shaded by degree of conservation among GABAAR and GlyR family members, revealing that key determinants of specificity are located largely in the ECDs where conservation is lower. e, Top-down view at the ECD–TMD interface level, showing key interactions within a single ECD (small oval) and between subunits (large oval). Grey dashed lines indicate putative hydrogen bonds and salt bridges. Boxed residues mark positions of disease mutations discussed in main text.

Extended Data Figure 8 Comparison of the β9–β10 (loop C) conformation, the agonist-binding site and the ligand orientation in GABAAR-β3cryst and equivalent GluClα and AChBP regions.

GABAAR-β3cryst is shown in grey (with blue strands) and its agonist benzamidine in green. Selected N and O atoms are in blue and red, respectively. a, Structural alignment of GABAAR-β3cryst and GluClα (PDB 3RIF, red backbone, with the agonist glutamate in orange). b, Structural alignment of GABAAR-β3cryst and AChBP (PDB 1UV6, red backbone) with agonist carbamylcholine bound (orange backbone). c, Structural alignment of GABAAR-β3cryst and AChBP (PDB 2BYN, red backbone) in apo form. d, Structural alignment of GABAAR-β3cryst and AChBP (PDB 2C9T, red backbone) in an inhibitor toxin-bound form (the toxin was excluded for clarity). Key binding residues from loop B and loop C are presented to highlight interactions with nitrogen atoms. In both a and b, the β9–β10 strand of GABAAR-β3cryst adopts a similar conformation to the closed loop from GluClα-glutamate or AChBP–carbamylcholine. In both c and d, where AChBPs lack agonists, loop C is in an extended, open conformation different from its equivalent in GABAAR-β3cryst. Structural alignments were performed using SHP (see Methods).

Extended Data Figure 9 Analysis of ECD–TMD interfaces and pore-lining M2 helices.

a, Comparative analysis of ECD–TMD interfaces in pLGIC structures reported to date. Values correspond to chain A in each PDB entry. The ECD/TMD boundaries were set between the two residues C-terminal from the Arg that ends the β10 strands in all pLGICs (Arg 216 in GABAAR-β3cryst, see sequence alignment in Extended Data Fig. 3). bf, GABAAR-β3cryst is shown in red (except the M2 helix, in teal). Its ECD was structurally aligned with equivalent regions of GluClα (PDB 3RIF, b), GLIC (PDB 4HFI, c), ELIC (PDB 2VLO, d), nAChR open (PDB 4AQ9, e) and nAChR closed (PDB 4AQ5, f). Alignments reveal relative variations in the ECD–TMD orientation. Structural alignments were performed using SHP (see Methods). g, Comparative analysis of M2 helix curvature, based on individual Cα positions, and pore diameter in GABAAR-β3cryst (desensitized conformation) versus GluCl (PDB 3RIF, open conformation). Residues whose side chains line the pore are highlighted in bold. Pore diameters were calculated at the level of Cα atoms, using Caver (see Methods). *N residues: number of residues involved in ECD–TMD interactions. †Calculated as the difference between total accessible surface areas of isolated and interfacing structures, divided by two, using PISA. ‡Indicates the solvation free energy gain upon formation of the interface. The value is calculated as difference in total solvation energies of isolated and interfacing structures, using PISA. §NH/NSB/NvdW: number of putative hydrogen bonds, salt bridges and additional van der Waals interactions that contribute to the ECD–TMD interface. Rotation of TMD relative to the equivalent region in GABAAR-β3cryst, around the inter-domain ‘effective hinge axis’, following superposition of the A-chain ECDs (calculated using DynDom, http://fizz.cmp.uea.ac.uk/dyndom/). ¶The apparent register shift in the M2 and M3 helices (and connecting M2–M3 loop, which forms a large part of the ECD–TMD interface) in currently available nAChR models may affect the values shown.

Extended Data Table 1 Crystallographic data collection and structure refinement statistics

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Miller, P., Aricescu, A. Crystal structure of a human GABAA receptor. Nature 512, 270–275 (2014). https://doi.org/10.1038/nature13293

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