Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, β-, γ-, δ-, ε-, ρ-, θ- and π-subunits
Cryo-electron microscopy structures are used to identify mechanisms underlying distinct features of extrasynaptic type A γ-aminobutyric acid receptors.
Type A GABA (GABAA) receptors belong to the pentameric ligand-gated ion channel (pLGIC) superfamily, which includes mammalian nicotinic acetylcholine receptors (nAChRs), serotonin type 3A receptors and glycine receptors, as well as other non-mammalian homologues
We reconstituted purified α1β3 receptor pentamers into lipid nanodiscs
α-Cobratoxin (α-CBTx) blocks muscle nAChRs to paralyse prey, but has also been shown to act with reduced potency as an inhibitor of GABAA receptors in recombinant expression systems
α-CBTx does not overlap with and directly antagonize GABA binding (Fig.
The outward motion of loop C, which is usually associated with antagonist binding to pLGICs, is larger than the one caused by the competitive antagonist bicuculline
The divalent transition metal cation Zn2+ is a non-competitive inhibitor of αβ and αβγ GABAA receptors
The Zn2+ site comprises a triad of His267 side chains from the pore-lining M2 helices of the three β3 subunits (Fig.
Previous GABAA receptor structures have shown how blockade is achieved at the intracellular end of the pore, for example, by picrotoxinin or cations
Comparison of the α-CBTx–Zn2+, GABA–Zn2+ and GABA-bound structures reveals the activation pathway. GABA binding at the two β–α sites induces realignment of the corresponding β-subunit ECDs (chains B and E) and clockwise translation of the β1–β2 and β6–β7 base loops above the transmembrane domain (TMD) (Extended Data Fig.
At the level of the TMD, without Zn2+ bound, the M2–M3 loops (which link the top of channel-lining α-helix 2 to helix 3) of the two GABA-bound β-subunits switch from ‘inward’ to ‘outward’ (Fig.
With all the M2–M3 loops in the outward position, each of the five channel-lining M2 helices, one contributed by each subunit around the pore, tilt and laterally translate outwards (Extended Data Fig.
Given that extrasynaptic αβ and αβδ receptors have low gating efficacy
Further evidence for reduced channel opening was obtained by measuring the probability of activation Top-down views of ECDs (top row) and cross sections of TMDs (helices shown as black circles) at the level of the 9′ Leu gate (bottom row) for αβ and αβγ receptors. Pore leucines are represented by black ‘fronds’ projecting from the innermost α-helix, M2. In response to GABA, the two binding β-subunits are the principal responders, with their ECDs twisting similarly anticlockwise (black arrows) for both αβ receptors and αβγ receptors. The downstream reaction of the TMD is limited in the αβ receptor and the 9′ gate remains mostly closed (red and orange circles), whereas for αβγ receptors, the TMD response is greater and the 9′ gate opens (green).
The structures we present here explain key aspects of the molecular pharmacology of α1β3 GABA receptors, including the mode of α-CBTx antagonism and the signature property for αβ receptors of high-sensitivity Zn2+ channel blockade. Despite the ECDs and M2–M3 loops responding to GABA, a more upright β-subunit M2 helix occupying the γ-subunit position results in the 9′ Leu pore gate remaining mostly closed. This provides a molecular explanation for the comparatively low
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
The protein sequences used were: human GABAAR α1 (mature polypeptide numbering 1–416, QPSL…TPHQ; Uniprot P14867) and human GABAAR β3 (mature polypeptide numbering 1–447, QSVN…YYVN; Uniprot P28472). The α1 intracellular M3–M4 loop amino acids 313–391 (RGYA…NSVS) were substituted by the SQPARAA sequence
Four-hundred millilitres of HEK 293S-GnTI- cells (which yield proteins with truncated N-linked glycans, Man5GlcNAc2
The receptor was double purified against first the SBP tag and then the 1D4-tag to only purify receptors containing one of each of the alternatively SBP or 1D4 tagged α1 subunits. The β3 subunit was transfected in excess relative to the α1 subunit, at 2:1, to ensure that the double-purified material consisted of only receptors comprising two α1 subunits and three β3 subunits, as previously proposed
On-bead nanodisc reconstitution was performed
Nb25 was produced exactly as described
All cryo-EM data presented here were collected in the Department of Biochemistry, University of Cambridge and all data collection parameters are given in Extended Data Table
Model building was carried out in Coot
Whole-cell responses were recorded in patch clamp experiments from HEK 293 cells transiently transfected with human GABAA α1β3 (WT), α1GLIVIβ3BRIL (α1β3cryo-EM; see ‘Constructs’) or α1β3γ2 (WT). HEK 293 cells were grown in DMEM supplemented with 10% v/v fetal bovine serum, 100 U ml−1 penicillin-G and 100 µg ml−1 streptomycin (37 °C; 95% air/5% CO2), and transfected using a calcium-phosphate precipitation method with α1:β3:GFP or α1:β3:γ2:GFP cDNAs in a ratio of 1:1:1 or 1:1:3:1, respectively, 12–24 h before experimentation. Recordings were performed with cells continuously perfused with Krebs solution composed of (mM): 140 NaCl, 4.7 KCl, 1.2 MgCl2, 2.52 CaCl2, 11 Glucose and 5 HEPES (pH 7.4; ~300 mOsm). Patch pipettes (TW150F-4; WPI; 3–4 MΩ) were filled with an internal solution containing (mM): 140 KCl, 1 MgCl2, 11 EGTA, 10 HEPES,1 CaCl2, 2 K-ATP (pH 7.2; ~305 mOsm). Drugs were applied to cells using fast Y-tube application, where Zn2+ and histamine were pre-applied before co-application with GABA. Cells were voltage-clamped at −40 mV with an Axopatch 200B amplifier (Molecular Devices), currents were digitized at 50 kHz via a Digidata 1322A (Molecular Devices), filtered at 5 kHz (−36 dB), and acquired using Clampex 10.2 (Molecular Devices). Series resistance was compensated at 60–70% (lag time 10 μs).
For free Zn2+ concentration experiments, the Zn2+ chelator tricine was used to precisely control Zn2+ concentration and eliminate background Zn2+ contamination. Krebs solution was supplemented with 10 mM tricine and pH corrected to 7.4. The free Zn2+ concentrations were calculated according to the equation: [Zn]free = (
Peak current responses and desensitization rates were obtained using Clampfit 10.2 (Molecular Devices). The EC50 and IC50 values were obtained by curve fitting concentration response data from individual experiments to the Hill equation (
Single GABA-activated channel currents were recorded in outside-out patches from transfected HEK 293 cells at –70 mV holding potential. Channel currents were recorded using an Axopatch 200B and filtered at 5 kHz (4-pole Bessel filter) before digitizing at 20 kHz with a Digidata 1322A. The fixed time resolution of the system was set at 80 μs. WinEDR was used for analysing single channel data. The single-channel current was determined from compiling channel current amplitude histograms and fitting Gaussian components to define the mean current, s.d. and the total area of the component. The single-channel conductance was calculated from the mean unitary current and the difference between the patch potential and GABA current reversal potential. Individual open and closed dwell times were measured using a 50% threshold cursor applied to the main single channel current amplitude in each patch. The subsequent detection of open and closed events formed the basis of an idealized single channel record used for compiling the dwell time distributions. Frequency distributions were constructed from the measured individual open and closed times and analysed by fitting a mixture of exponentials, defined by:
HEK 293T cells used for electrophysiology and HEK 293S GnTI− cells used for protein production for cryo EM were obtained from ATCC. Further authentication of cell lines was not performed for this study. Mycoplasma testing was not performed for this study.
Further information on research design is available in the
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We thank L. Cooper for training in cryo-EM grid preparation and for performing grid clipping; J. Stayaert and R. Aricescu for advice on, access to and use of Mb25; R. Aricescu and S. Masiulis for valuable discussions and training in sample preparation; and E. Seiradake for provision of the pHLsec-SBP plasmid. This work was supported by a Department of Pharmacology new lab start-up fund, the University of Cambridge Isaac Newton and Wellcome Trust Institutional Strategic Support Fund, and Academy of Medical Sciences Springboard Award (SBF004\1074). Electrophysiology work in the T.G.S. laboratory was funded by an MRC programme grant (MR/T002581/1) and Wellcome Trust Collaborative Award (217199/Z/19/Z). The cryo-EM facility receives funding from the Wellcome Trust (206171/Z/17/Z; 202905/Z/16/Z) and University of Cambridge.
structural interpretation. A.A.W. performed protein purification. M.M. and T.G.S. designed and analysed the electrophysiological experiments, which were performed by M.M. and V.D. S.W.H. and D.Y.C. performed cryo-EM data acquisition and processing. P.S.M. performed construct design, protein purification, cryo-EM sample preparation, atomic model building and structural interpretation. P.S.M. wrote the manuscript with input from all other authors.
Atomic model coordinates for α-CBTx–Zn2+, GABA–Zn2+ and GABA-bound structures have been deposited in the Protein Data Bank with accession codes
The authors declare no competing interests.
For the three structures, α-CBTx/Zn2+, GABA/Zn2+, and GABA-bound, a map on the left is coloured by local resolution (see
Cryo-EM data collection, refinement and validation statistics for GABA, GABA/Zn2+, α-CBTx/Zn2+ Cryo-EM data collection, refinement and validation statistics for GABA, GABA/Zn2+, α-CBTx/Zn2+
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