Journal: Nature Communications

Article Title: Molecular basis of human GABA transporter 3 inhibition

doi: 10.1038/s41467-025-59066-w

Figure Lengend Snippet: a Sequence alignment between the epitope region of d DAT and h GAT3 WT results in epitope-graft mutant GAT3 EM . Mutations are highlighted in colored boxes. b [ 3 H]-GABA uptake assay to measure inhibition potency of SNAP-5114 comparing GAT3 WT and GAT3 EM . The dot represents a single normalized data point with fit calculated for the average of all data points (three independent measurements ( n = 3), each in triplicates). Source data are provided as a file. c CryoEM map of SNAP-5114-bound GAT3 EM (forest green) with Fab 9D5 (gray) at 3.42 Å. SNAP-5114 in yellow. d CryoEM map of apo GAT3 EM (light blue) with 9D5 (gray) at 3.51 Å.

Article Snippet: Codon-optimized genes encoding full-length wildtype human gat3 ( gat3 WT ) and epitope-grafted gat3 ( gat3 EM ) were synthesized by Twist Biosciences.

Techniques: Sequencing, Mutagenesis, Inhibition

Journal: Nature Communications

Article Title: Molecular basis of human GABA transporter 3 inhibition

doi: 10.1038/s41467-025-59066-w

Figure Lengend Snippet: a Model of GAT3 Apo (light blue). TM helices are labeled with the corresponding number, along with an orthosteric substrate binding pocket (red circle) and extracellular gates (black rectangle). The Cl − ion is depicted as a green sphere. b Structural superposition of GAT3 Apo with inward-open SERT (PDB Id: 7LI8), GlyT1 (PDB Id: 6ZBV), and GAT1 (PDB Id: 7Y7V). The differences in TM1a and ECLs are highlighted with a zoom-in view in the bottom and top insets, respectively. The partial unwinding of the EL4a helical segment in ECL4 leads to a unique conformation of ECL4 in GAT3 compared to other NSS transporters. ECL4 residue E372 interacts with W207 (ECL2) and Y540 (ECL6) and may participate in transport gating. The position of the orthosteric pocket is depicted as a red hexagon. The disulfide bond in GAT3 is depicted as sticks. c The orthosteric pocket of GAT3 Apo . Cyan and green rectangles highlight the ceiling of the orthosteric pocket and Cl − coordination (green sphere). d Zoomed-in view of the extracellular gate (left inset), orthosteric pocket ceiling (right, top inset), and Cl − coordination (right, bottom inset). The color of the inset corresponds to positions in ( a , c ).

Article Snippet: Codon-optimized genes encoding full-length wildtype human gat3 ( gat3 WT ) and epitope-grafted gat3 ( gat3 EM ) were synthesized by Twist Biosciences.

Techniques: Labeling, Binding Assay, Residue

Journal: Nature Communications

Article Title: Molecular basis of human GABA transporter 3 inhibition

doi: 10.1038/s41467-025-59066-w

Figure Lengend Snippet: a Models of GAT3 inhibitor SNAP-5114, and substrate nipecotic acid and GABA. The chemical structures are drawn using ChemDraw (PerkinElmer). b Model of GAT3 (forest green cartoon) in inward-open conformation bound to SNAP-5114 (yellow sticks). Cl − is depicted as a green sphere. The location of TM1a is labeled. c Polar interaction network of SNAP-5114 at the GAT3 orthosteric pocket. d GAT3 residues participating in hydrophobic interactions with SNAP-5114’s piperidine ring (top) and lipophilic domain (bottom) are depicted as sticks.

Article Snippet: Codon-optimized genes encoding full-length wildtype human gat3 ( gat3 WT ) and epitope-grafted gat3 ( gat3 EM ) were synthesized by Twist Biosciences.

Techniques: Labeling

Journal: Nature Communications

Article Title: Molecular basis of human GABA transporter 3 inhibition

doi: 10.1038/s41467-025-59066-w

Figure Lengend Snippet: a Structural superposition of GAT1-tiagabine complex (PDB Id: 7SK2, orange) and SNAP-5114-bound GAT3 (forest green). The differences in TM1a and TM6b are highlighted with arrows, and the corresponding distances are labeled. b Differences in GAT1 and GAT3 orthosteric pocket with bound tiagabine and SNAP-5114, respectively. The non-conserved residues of GAT1 and GAT3 are shown as sticks. c Measurement of SNAP-5114 inhibition potency for wild-type (WT) and mutant GAT3 using [ 3 H]-GABA uptake assay. The experiment was repeated in three biologically independent experiments ( n = 3), each data point was measured in triplicates. The dots represent a single normalized data point with fit calculated for the average of all data points. d The output of [ 3 H]-GABA uptake kinetic assays using GAT3-expressing HEK293F cells. The assay was performed with increasing concentrations of SNAP-5114, preincubated with cells, before the addition of [ 3 H]-GABA. The experiment was repeated three times ( n = 3). The dots represent a single normalized data point with fit calculated for the average of all data points. Source data are provided as a file.

Article Snippet: Codon-optimized genes encoding full-length wildtype human gat3 ( gat3 WT ) and epitope-grafted gat3 ( gat3 EM ) were synthesized by Twist Biosciences.

Techniques: Labeling, Inhibition, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Molecular basis of human GABA transporter 3 inhibition

doi: 10.1038/s41467-025-59066-w

Figure Lengend Snippet: SNAP-5114 (red circle), along with Na + and Cl − ions, bind to GAT3 in outward-open conformation. Upon binding, GAT3 transitions to inward-open via an intermediatory occluded conformation. Na + ions are released into the cytoplasm, whereas the SNAP-5114 and Cl − remain bound. The cryoEM structure of GAT3 in the complex with SNAP-5114 corresponds to this state in which GAT3 obtains inward-open conformation with SNAP-5114 and Cl − bound to the transporter (dashed rectangle).

Article Snippet: Codon-optimized genes encoding full-length wildtype human gat3 ( gat3 WT ) and epitope-grafted gat3 ( gat3 EM ) were synthesized by Twist Biosciences.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural basis for selective inhibition of human GABA transporter GAT3

doi: 10.1101/2025.03.27.645797

Figure Lengend Snippet: a , SR -THAP inhibits [ 3 H]GABA uptake by GAT3 expressed in HEK293T cells with an IC 50 of 25.2 [21.8; 30.4] µM without pre-incubation (blue) and 4.9 [3.2; 7.0] µM with 120 min pre-incubation. b . The enantiomer RS -THAP also inhibits [ 3 H]GABA uptake by GAT3 expressed in HEK293T cells, but with significantly reduced potency, estimated IC 50 values of >600 µM without pre-incubation and >100 µM with pre-incubation. c , [ 3 H]GABA uptake by GAT3 can also be inhibited by non-radioactive GABA, although with minimal observed shift in potency comparing with and without pre-incubation of 120 min, with IC 50 values of 8.5 [6.4; 11] and 5.4 [4.6; 6.4] µM respectively. d , The GlyT1 specific benzoylisoindoline inhibitor Cmpd1 has no effect on GAT3 mediated [ 3 H]GABA transport even at 30 µM. e , Inhibition of [ 3 H]GABA uptake by GAT1 in HEK293T cells by SR -THAP did not reach full inhibition at the highest concentration tested (300 µM); estimated IC 50 of 212 [143; 238] µM. f , Similarly to GAT1, inhibition of [ 3 H]glycine uptake by GlyT1 in HEK293T cells did not reach full inhibition at 300 µM; estimated IC 50 of 123 [104; 147] µM. Data points are shown as individual replicates from n = 3 biological replicates (triplicate measurements) unless othervise stated.

Article Snippet: The human GAT3 complementary DNA sequence was codon optimised and synthetised by GenScript for expression in mammalian cells.

Techniques: Incubation, Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Structural basis for selective inhibition of human GABA transporter GAT3

doi: 10.1101/2025.03.27.645797

Figure Lengend Snippet: Cryo-EM density maps of GAT3 bound to a ) SR -THAP (blue map, contour level = 0.063 in ChimeraX), b ) GABA (green map, contour level = 0.06 in ChimeraX) and c ) in the substrate-free form (light orange map, contour level = 0.03 in ChimeraX). Lipid densities are shown in light yellow. d, e, f , Surface representation of the inhibitor-bound, GABA-bound and substrate-free structures of GAT3 viewed parallel to the membrane. d , Slice view of GAT3 showing the SR -THAP (orange) binding pocket. e , Slice view of GAT3 showing the GABA (green) binding pocket. f , Slice view of GAT3 in the substrate-free state. The extracellular gating residues R75 (TM1 – blue) and D467 (TM10 – not shown), as well as P375 (EL4 – yellow) and F308 (TM6 – green), are shown as sticks in d, e, f .

Article Snippet: The human GAT3 complementary DNA sequence was codon optimised and synthetised by GenScript for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for selective inhibition of human GABA transporter GAT3

doi: 10.1101/2025.03.27.645797

Figure Lengend Snippet: a , b Overall structure of the human GAT3 bound to the selective inhibitor SR -THAP (orange stick) ( a ), and its substrate GABA (green stick) ( b ). Cryo-EM density of SR -THAP (map contour level = 0.072 in ChimeraX) ( a ) and GABA (map contour level = 0.06 in ChimeraX). c, d, e , Close-up view of the binding mode of SR -THAP (orange) to GAT3 ( c ), the binding mode of GABA (blue-grey) to GAT3 ( d ), and the docking pose of RS -THAP (dark grey) to GAT3 ( e ). Protein backbone is shown as ribbons cartoon (azure), chloride ion as green sphere, sodium ion as red sphere and interacting residues within 3 Å are displayed in grey. H-bonds and cation-” interactions are displayed with yellow and green dashed lines, respectively. f , Chemical structure of SR -THAP and of its enantiomer RS -THAP. The three structural elements forming the inhibitor are highlighted in faded orange for the amino acidic moiety R 1 (2-hydroxy-2-pyrrolidin-2-yl)acetic acid); in faded azure for the N-alkyl chain R 2 ; in faded pink for the trimethoxytrityl group R 3 .

Article Snippet: The human GAT3 complementary DNA sequence was codon optimised and synthetised by GenScript for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: Structural basis for selective inhibition of human GABA transporter GAT3

doi: 10.1101/2025.03.27.645797

Figure Lengend Snippet: Occlusion of the extracellular pathway is maintained by a highly conserved interaction network including a salt bridge between R75 and D467 (not shown) and stacking and cation-π interaction of R75 and F308. From the occluded GABA-bound state, the transporter transitions to an inward-open state, by an outward tilt of TM1a as well as partial unwinding of the intracellular part of TM5. The GAT3 selective inhibitor SR -THAP, after diffusion across the plasma membrane, binds GAT3, locking the transporter in an inward-open conformation. The residue E66 in the binding pocket of GAT3, is replaced by a large aromatic tyrosine in GAT1, playing a part in defining the selectivity of SR -THAP for GAT3, against GAT1. Cl − and Na + ions are shown as yellow and purple spheres.

Article Snippet: The human GAT3 complementary DNA sequence was codon optimised and synthetised by GenScript for expression in mammalian cells.

Techniques: Diffusion-based Assay, Clinical Proteomics, Membrane, Residue, Binding Assay