γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.
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We thank K. Villers and C. Hanson for technical support for recombinant protein expression in mammalian and Sf9 cells; C. Cato and R. Oania for general laboratory support; S. Khan for helpful discussions; T. Osinski, J. Chu and B. D. Kim at the USC Center for Advanced Research Computing (CARC) for support with computing resources; and D. J. Slotboom (University of Groningen) and K. Pande (LBNL) for comments and suggestions on the manuscript. This research was supported by a US National Institutes of Health grant R35 GM127086 (to V.C.). We acknowledge the Center of Excellence for Nano Imaging (CNI) at the University of Southern California for microscope time.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Protein purification of GAT1 for cryo-EM analysis and [3H]-GABA uptake assay results for quantification of relative uptake rates.
a, Size-exclusion chromatography on a Superose 6 Increase (10/300) column shows a monodisperse peak at a size of approximately 100 kDa, accommodating the protein, as well as the LMNG/CHS detergent micelle. b, SDS-PAGE prior to cryo-EM sample preparation, confirms the high purity of the final sample (> 95%). For gel source data, see Supplementary Fig. 4. c, Thermal stability assay as described in the methods section. Changing the detergent from DDM to LMNG resulted in an increase of Tm by 12 °C. AU, arbitrary units. d, Scintillation proximity assay of GAT1 purified from Sf9 cells, showing specific competition of [3H]-GABA with tiagabine, presented as all data points, overlaid with a nonlinear regression fit, resulting in an IC50 of 290 ± 60 nM. The experiment was performed three times independently with similar results. e, Absolute counts of a representative [3H]-GABA uptake experiment from constructs used for probing tiagabine binding pocket. (Bottom) Comparison of expression levels, based on relative fluorescence of whole cells compared to wildtype GAT1, and maximum uptake values, relative to wildtype GAT1 in above experiment
Extended Data Fig. 2 Cryo-EM reconstruction of full-length, wild-type human GAT1 in complex with tiagabine.
a, Workflow of cryo-EM processing of GAT1 in complex with tiagabine in the inward-open conformation with representative micrograph, picking templates and 2D classes. Data processing was entirely performed in cryoSPARC57. After motion correction, CTF estimation and particle picking, the dataset was sorted using 2D classification, followed by 3D ab initio reconstruction and heterogenous refinement for further clean-up of the particle stack. Finally, 3D reconstructions were performed using non-uniform refinement58 and local refinement, which yielded a final map of 3.8 Å resolution. b, Data quality of the final reconstruction, illustrated as a local resolution map ranging from 3 - 4.8 Å, gold-standard fourier shell correlation plot (masked and unmasked) and angular sampling of the final reconstruction
Extended Data Fig. 3 Atomic model and cryo-EM Coulomb potential map of the human GAT1 complex with tiagabine.
The overall structure of the GAT1 (cyan) complex with bound tiagabine (salmon) (right) and magnified views of individual transmembrane helices (left) are shown in the cryo-EM map (grey mesh) at 5 sigma.
a, Sequence alignment of GABA transporters: human GAT1, GAT2, GAT3 (Uniprot: P30531, Q9NSD5, P48066). Green boxes highlight conserved residues observed in the substrate binding pocket and red boxes highlight non-conserved residues. b, AlphaFold55 prediction of GAT1 and GAT2 in the outward-open conformation with tiagabine superimposed with substrate from LeuT (PDB ID: 4HOD33), highlighting non-conserved residues between the two subtypes in the substrate-binding pocket. c, AlphaFold55 prediction of GAT1 outward-open conformation, superimposed with experimentally determined inward-open cryo-EM structure, shows the small relative transition that tiagabine bound to GAT1 would have to undergo for the proposed induced-fit mechanism.
a, Superposition of outward-open dDAT26 (PDB ID: 4XP1, yellow, residues F53, Y142, S320 in stick representation) with outward-open SERT30 (PDB ID: 6DZY, red, residues F105, Y121, S336 in stick representation). b, Superposition of outward-open dDAT26 (PDB ID: 4XP1, yellow, residues F53, Y142, S320 in stick representation) with inward-open GAT1 (this study, residues F70, Y86, S295 in stick representation). c, Superposition of inward-open GAT1 structure (this study, residues F70, Y86, S295 in stick representation) with inward-open GlyT131 (PDB ID: 6ZBV, blue, residues F126, Y142, S371 in stick representation). a–c are shown to confirm the conformational state (inward-open) of GAT1, together with the conformation of the Na+ and Cl− coordinating residues suggesting likely empty ion binding pockets. d–f, Superimposed structures of inward-open conformation SERT, GlyT1 and GAT1, highlighting differences in the relative orientation of TM1a. g,h, Superposition to GlyT1 and SERT, showing differences in extracellular gate. GlyT1 shows a very similar network of interactions to GAT1, while the inward-open SERT structure shows markedly larger distances between the corresponding residues. Numbers correspond to the shortest atomic distances, between the respective residues, in Å.
Extended Data Fig. 6 Structural comparison of GAT1 (green) with GlyT1 (blue) and SERT (pink) inward-open structures.
a, Superposition of overview models between three structures. b, Focus on the intracellular gate, showing differences in TM6b, TM7, TM1a and TM5. c–e, Minor differences between GAT1, GlyT1 and SERT in TM12, TM9, TM10 and TM8 with respective residues highlighted in stick representation: G457 highlights the unique residue in GAT1, potentially leading to additional flexibility of TM10. The interacting residues between ibogaine and SERT TM8 A441-G442-L443 show a subtle helix break, which is unique among the compared sequences and structures.
a, Time series showing the shortest observed distance among heavy atoms of the respective GAT1 residues to the 3-methyl-2-thienyl moiety of tiagabine. Numbers in parentheses are the mean and standard deviation of the respective distances in MD simulations. See Supplementary Figs. 1 and 2 for time series of individual trajectories and Supplementary Fig. 3 for histograms of these distances. b, Snapshots of tiagabine binding site for one of the trajectories, taken every 200 ns and spanning 1000 ns. Tiagabine from the cryo-EM structure is colored dark green, MD simulations light green, GAT1 cryo-EM structure in dark pink, GAT1 MD simulations in light pink. c, Root mean square fluctuations (r.m.s.f.) per residue in GAT1. Solid line shows the average of 10 independent trajectories at each residue position; shading refers to 95% confidence interval (n = 10). d, Root mean square deviation (r.m.s.d.) of GAT1 Cα in the 1 µs simulations. The r.m.s.d. are calculated with the protein in trajectories superimposed on the protein in the first frame. e, Root mean square deviation (r.m.s.d.) of tiagabine heavy atoms in the 1 µs simulations. The r.m.s.d. are calculated with the protein in trajectories superimposed on the protein in the first frame.
Blue circle highlights nipecotic acid moiety, sand colored circle highlights bis(3-methyl-2-thienyl) tail.
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Motiwala, Z., Aduri, N.G., Shaye, H. et al. Structural basis of GABA reuptake inhibition. Nature 606, 820–826 (2022). https://doi.org/10.1038/s41586-022-04814-x