Abstract
γ-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, is recycled through specific GABA transporters (GATs). GAT1, which is mainly expressed in the presynaptic terminals of axons, is a potential drug target of neurological disorders due to its essential role in GABA transport. Here we report four cryogenic electron microscopy structures of human GAT1, at resolutions of 2.2–3.2 Å. GAT1 in substrate-free form or in complex with the antiepileptic drug tiagabine exhibits an inward-open conformation. In the presence of GABA or nipecotic acid, inward-occluded structures are captured. The GABA-bound structure reveals an interaction network bridged by hydrogen bonds and ion coordination for GABA recognition. The substrate-free structure unwinds the last helical turn of transmembrane helix TM1a to release sodium ions and substrate. Complemented by structure-guided biochemical analyses, our studies reveal detailed mechanism of GABA recognition and transport, and elucidate mode of action of the inhibitors, nipecotic acid and tiagabine.
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Data availability
The UniProt accession codes for the sequences of human GAT1, GAT2, GAT3 and BGT1 are P30531, Q9NSD5, P48066 and P48065, respectively. Atomic coordinates of GAT1 in the substrate-free state, or the presence of GABA, or inhibitors in various conditions (GAT1A/G/N/T) have been deposited in the PDB (http://www.rcsb.org) under the accession codes 7Y7V (https://doi.org/10.2210/pdb7Y7V/pdb), 7Y7W (https://doi.org/10.2210/pdb7Y7W/pdb), 7Y7Y (https://doi.org/10.2210/pdb7Y7Y/pdb) and 7Y7Z (https://doi.org/10.2210/pdb7Y7Z/pdb), respectively. The corresponding EM maps have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/), under the accession codes EMD-33671, EMD-33672, EMD-33674 and EMD-33675, respectively. Previously resolved structures used in this study are under the accession codes 4US3 (inward-occluded Trp-bound MhsT structure), 6XWM (inward-occluded Phe-bound LeuT structure), 7LIA (outward-open serotonin-bound SERT structure), 4XP1 (outward-open dopamine-bound dDAT structure), 7LI8 (inward-open apo SERT structure), 3TT3 (inward-open apo LeuT structure), 6ZPL (inward-open cmpd1-bound GlyT1 structure) and 7SK2 (inward-open tiagabine-bound GAT1 structure) in the PDB. Source data are provided with this paper.
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Acknowledgements
We thank F. Yang and X. Li for technical support during EM data collection. We thank the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for providing the cryo-EM facility support and the computational facility support. This work was funded by the National Key R&D Program of China (2020YFA0509301, C.Y.), Beijing Nova Program (Z201100006820039, C.Y.), Beijing Frontier Research Center for Biological Structure, Beijing Advanced Innovation Center for Structural Biology, State Key Laboratory of Membrane Biology, Tsinghua University Initiative Scientific Research Program, and Start-up funds from Tsinghua-Peking Center for Life Sciences and Tsinghua University.
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C.Y. and Y.Y. conceived the project. Y.Y., C.Y. and A.Z. designed all experiments. A.Z., J.H., Y.Y. and J.T. performed the experiments. A.Z., F.K., C.Y. and J.L. contributed to the structure determination. All authors analyzed the data and contributed to manuscript preparation. C.Y., A.Z. and Y.Y. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Biochemical characterization of recombinantly expressed human GAT1.
a, SEC purification of the human GAT1 in the presence of 0.02% (w/v) DDM from one independent experiment. b, Peak fractions of SEC purification were further examined by Coomassie blue staining of SDS–PAGE. GAT1 appeared in two smeared bands due to different extent of glycosylation. The experiments were repeated independently more than 3 times with consistent results. c, The time course analysis of the transport activity of GAT1. Data are presented as mean with SD of three independent experiments. d, Determination of the kinetic parameters of GAT1 for the transport of GABA. Data are presented as mean with SD of three independent experiments and were fitted using the Michaelis–Menten non-linear fitting method, yielding Km at 27.51 ± 3.86 μM and Vmax at 248.6 ± 9.33 nmol/mg/min. Source data are provided.
Extended Data Fig. 2 Data processing of different GAT1 datasets.
a, The flowchart for GAT1T data processing. Details can be found in Methods. b, The flowchart for GAT1A/G/N data processing. Details can be found in Methods.
Extended Data Fig. 3 Cryo-EM analysis of GAT1 in different states.
a, Gold-standard Fourier shell correlation (FSC) curve for the 3D refinement of the overall structure of GAT1A/G/N/T, respectively. b, Angular distribution of the particles used for the final reconstructions. c, FSC curves of the refined models versus the sharpen maps (black); of the models refined against the first half maps versus the same maps (purple); and of the models refined against the first half maps versus the second half maps (green). The small difference between the purple and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting. d, Local resolution of the sharpen maps shows in the front view and longitudinal section view. Red dashed lines label the location of GABA, nipecotic acid or tiagabine in the map of GAT1G/N/T, respectively.
Extended Data Fig. 4 Representative EM densities of GAT1.
a, Representative EM maps of GAT1A transmembrane helices and EM densities are contoured at 5 σ. b, EM maps of TM1a in GAT1A, GAT1G and GAT1T, respectively. EM densities are contoured at 4 σ. c, EM map of the disulfide bond between C164 and C173 in GAT1A and EM densities are contoured at 5 σ.
Extended Data Fig. 5 Sequence alignment of human GAT1-3 and BGT1.
Secondary structural elements of hGAT1 are indicated above the sequence alignment. Invariant and highly conserved amino acids are shaded yellow and grey, respectively. The residues that are hydrogen-bonded with GABA, or chelating Na+ at the Na1 and Na2 sites and Cl– in hGAT1 are indicated by orange star, magenta, red and green circles, respectively. The residues which form disulfide bond are indicated by brown triangles. The glycosylation site residues are indicated by cyan squares. The UniProt (https://www.uniprot.org) IDs for the aligned proteins are: hGAT1 (P30531), hGAT2 (Q9NSD5), hGAT3 (P48066) and hBGT1 (P48065). Sequences are aligned with ClustalW.
Extended Data Fig. 6 Water molecules determined in the structures of GAT1.
a, Water molecules in the structure of GAT1G in an inward-occluded state. Waters are shown as marine spheres. b, Waters in the structure of GAT1A in an inward-open state. c, The structural waters that mediated hydrogen bonds with GABA or GAT1 residues. d, Extracellular gating residues in GAT1G. The hydrogen bonds formed by Arg69, Gln291, Asp451 and Ser456 are indicated by gold dashed lines. Cation-π interaction is indicated by black dashed line. The hydrogen bonds formed by the water cluster are indicated by cyan dashed lines.
Extended Data Fig. 7 Structural comparison of GAT1G to substrate-bound prokaryotic NSS members.
a, Overall structural comparison between the cryo-EM structures of GABA-bound GAT1G, inward-occluded Trp-bound MhsT (PDB code: 4US3), inward-occluded Phe-bound LeuT (PDB code: 6XWM). The intracellular permeation pathway is occluded by TM1a colored marine among the three structures. TM5 colored maroon shows different geometry among the structures. b-d, Comparison of Na1 and Na2 site in GABA-bound GAT1G (b), Trp-bound MhsT (c) and Phe-bound LeuT (d). The sodium ions are colored magenta, and the ion coordination is colored maroon. e-g, Comparison of substrates binding mode in GABA-bound GAT1G (e), Trp-bound MhsT (PDB code: 4US3) (f) and Phe-bound LeuT (PDB code: 6XWM) (g). The yellow dashed lines indicate hydrogen bonds and ion coordination of the substrate.
Extended Data Fig. 8 Structural comparison of GAT1G to eukaryotic NSS members.
a-d, Comparison of Cl– binding site in GABA-bound GAT1G (a), serotonin-bound SERT (PDB code: 7LIA) (b), dopamine-bound dDAT (PDB code: 4XP1) (c) and Cmpd1-bound GlyT1 (PDB code: 6ZPL) (d). The chloride ions are colored green, and the ion coordination is colored dark green. The tetra-coordination of Cl– is strictly conserved among the four structures. e-g, Comparison of substrates binding mode in GABA-bound GAT1G (e, colored silver in f and g), serotonin-bound SERT (PDB code: 7LIA) (f) and dopamine-bound dDAT (PDB code: 4XP1) (g). Monoamine substrates like serotonin and dopamine are located in the cleft formed by TM3 and TM8 compared to GABA.
Extended Data Fig. 9 Structural comparison of inward-open NSS members.
a, Shown here are superimposed structures of inward-open substrate-free GAT1A (colored purple), inward-open tiagabine-bound GAT1T (colored yellow), inward-open apo SERT (PDB code: 7LI8) (colored salmon), inward-open apo LeuT (PDB code: 3TT3) (colored pink) and inward-open Cmpd1-bound GlyT1 (PDB code: 6ZPL) (colored marine). TM1a adopts a different orientation in different structures. b, Shown here are superimposed structures of inward-open substrate-free GAT1A (colored purple), inward-open tiagabine-bound GAT1T (colored yellow) and inward-open tiagabine-bound GAT1 reported by Gati’s group (PDB code: 7SK2) (colored cyan). The compound tiagabine is located in the same central pocket in both structures. Coordination of the nipecotic acid moiety is similar, while the two 3-methyl-2-thienyl rings cannot be accurately compared due to their relative flexibility (right panel).
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Source data
Source Data Figs. 3c and 5a,b and Extended Data Fig.1a,c–d
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Source Data Extended Fig. 1b
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Zhu, A., Huang, J., Kong, F. et al. Molecular basis for substrate recognition and transport of human GABA transporter GAT1. Nat Struct Mol Biol 30, 1012–1022 (2023). https://doi.org/10.1038/s41594-023-00983-z
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DOI: https://doi.org/10.1038/s41594-023-00983-z
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