Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABAA receptor). Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABAA receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1β2γ2 GABAA receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.
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We thank D. Cawley for antibody production, X. Bai for electron microscopy discussion, P. Emsley for guidance on glycosylation tools in Coot, W. Costello for initial construct screening and all members of the Hibbs laboratory for discussion. Single-particle cryo-EM data were collected at the University of Texas Southwestern Medical Center Cryo-Electron Microscopy Facility, which is supported by the CPRIT Core Facility Support Award RP170644. We thank D. Nicastro and D. Stoddard for support in facility access and data acquisition. R.W. acknowledges support from the Sara and Frank McKnight Fund for Biochemical Research and the NIH (T32GM008203). R.E.H. is supported by a McKnight Scholar Award, The Welch Foundation (I-1812) and the NIH (DA037492, DA042072, and NS095899).
Nature thanks G. Akk, P. Corringer, A. Evers and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Cryo-EM constructs (γ2 affinity tag not shown) are numbered starting with the first residue of the mature protein. Sequences aligned (UniProt or PDB accession codes): Homo sapiens α1 GABAA (HS, P14867), H. sapiens β2 GABAA (P47870), H. sapiens γ2 GABAA (P18507), H. sapiens GABAA β3 (4COF), H. sapiens glycine α3 (5CFB), Danio rerio glycine α1 (DR, 3JAE), Caenorhabditis elegans α (CE, 3RHW), H. sapiens α4 nAChR (5KXI), H. sapiens β2 nAChR (5KXI) and Mus musculus 5-HT3 receptor (MM, 4PIR). α-helices (cylinders), β-strands (arrows), and inserted linker (cyan) are indicated.
a, FSEC of GABAA receptor with and without Fab bound, and SDS–PAGE analysis of a representative purification (from n > 10 purifications). b, Saturation binding assay with [3H]-flumazenil. Single-site binding fits for receptor alone and receptor plus Fab both exhibited a Hill slope of ~1 (0.97 and 0.89, respectively). Plotted results are from a representative experiment performed in triplicate. n = 3 independent experiments. Data are shown as mean ± s.d. for a representative triplicate measurement. c, Competition of 10 nM [3H]-flumazenil with diazepam. Calculated Ki for diazepam assumes a Kd for [3H]-flumazenil of 7.7 nM. n = 2 independent experiments in triplicate. Data are shown as mean ± s.d. for a representative triplicate measurement. d, Dose–response experiments in the presence or absence of Fab. HEK cells were transfected with cryo-EM constructs and patch-clamped with or without pretreatment with 1 µM Fab for 1 min. Hill slopes are 1.7 and 1.4 with and without Fab, respectively. Published EC50 values for GABA range from 6.6 µM–107 µM71,72,73,74. n = 3 experiments from different cells. Data are plotted as mean ± s.d. e, Whole-cell patch-clamp recording of long application of cryo-EM ligands at concentrations used in cryo-EM samples to assess conformational state at equilibrium. The two traces shown are from one continuous recording; in between the two responses, Fab was added to 1 µM for one minute to saturate all receptor sites before second application of GABA and flumazenil (including Fab). n = 3 independent experiments. f–g, Docking of diazepam at the benzodiazepine-binding site based on superposition of benzodiazepine rings. The phenyl ring of diazepam would orient towards the membrane, possibly forming π–π-stacking interactions with Y58 on the complementary subunit. Similar to flumazenil, the halogen of diazepam could interact with H102, suggesting that this contact is conserved broadly among benzodiazepines and flumazenil. This orientation is largely consistent with predictions from a modelling and docking study75, and distinct from that suggested by affinity labelling76. In this latter prediction, the diazepam phenyl group orients away from the membrane and would require local reorganization of side chains to avoid atomic clashes. h–j, Structural details of Fab–α1 interaction. Labelled residues are on the α-subunit. i, Top view. j, Side view.
a, Representative cryo-electron micrograph of the GABAA receptor–Fab complex. n = 5,594 images. b, Images of selected 2D classes from reference-free 2D classification by Relion. c, Overview of the image processing procedure (see Methods).
a, Angular distribution histogram of GABAA receptor conformation A particle images. b, Fourier shell correlation (FSC) of conformation A maps before (black) and after (blue) masking. c, Local resolution of the GABAA receptor estimated by ResMap. d–f, as in a–c but for GABAA receptor conformation B.
a, Data collection and refinement statistics. b, c, FSC curves for cross-validation between the maps and models of both conformation A (b) and conformation B (c). FSC curves for final model versus summed map (whole) in black, for model versus half map in green (work), and for model versus half map not used for refinement in blue (free).
a–e, Cryo-EM density map of the GABAA receptor conformation A for a representative of each subunit. f–h, Cryo-EM density segments of loop C in α1, β2 and γ2-subunits. i–k, Cryo-EM density segments of M2 helix in α1, β2 and γ2-subunits. l–n, Cryo-EM density maps of ligand binding sites: flumazenil (l), two GABA binding sites (m, n).
a–e, Cryo-EM density map of the GABAA receptor conformation B for a representative of each subunit; chain IDs are in parentheses. f–h, Cryo-EM density segments of loop C in α1, β2 and γ2-subunits. i–k, Cryo-EM density segments of M2 helix in α1, β2 and γ2-subunits. l–n, Cryo-EM density maps of ligand-binding sites: flumazenil (l), two GABA binding sites (m, n).
a–e, Subunits of conformation A are compared to the corresponding subunit from conformation B. f–j, Superposition of subunits within conformation A. k–o, Superposition of subunits within conformation B. Chimera MatchMaker was used to generate alignments; r.m.s.d. values in Å are for Cα atoms over entire subunit. Chain IDs are in parentheses.
a, Cartoon of permeation pathway for conformation A. A single β2-subunit is removed for clarity. Purple spheres indicate pore diameters >5.6 Å; yellow is >2.8 Å and <5.6 Å; red is <2.8 Å. b, Same as a but for conformation B. c, Pore diameters for conformation A (red) and conformation B (black). The zero value along the y axis of the plot is aligned with the α-carbon of the −2′ position of conformation B. d–m, Side view of two adjacent subunits in conformations A (d–h) and B (i–m). The view is from the periphery of the receptor towards the pore axis. Cholesterol at the interface is shown in yellow in d, i and k. Cartoon pentagons (bottom) are coloured to illustrate all subunits composing the displayed interface; subunits not participating in the displayed interface are grey. Principal (+) and complementary (−) sides of the displayed interface are labelled on each pentagon. n, Analysis of the subunit interfaces of both conformations using PDBePISA server69.
a, b, Top and side view of the TMD of conformation A with density for the γ2-subunit shown. c, d, As in a, but for conformation B. e, Transmembrane domain superposition of conformation A (subunits in colour) over conformation B (grey). α-Helices are represented as cylinders. f–j, Superposition of single subunit TMD in conformation A (coloured) with its corresponding subunit in conformation B (grey). k–r, Superpositions of the four non-γ-subunits. Top and bottom rows contain the same superpositions in different representations. Conformation B is shown in all panels with α-subunits in green and β-subunits in blue. Reference structures include the glycine receptor with ivermectin bound (3JAF)77, glutamate-gated chloride channel with ivermectin bound (3RHW)78 and the GABAA β3 homopentamer (4COF)16.
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