Fig. 4 |. Proposed two-step mode of tiagabine inhibition.

a, Results of [³H]-GABA saturation uptake assays using wild-type GAT1–eGFP-expressing HEK293S cells, in the absence (0 μM in blue) and the presence of tiagabine (0.125 μM in red, 0.25 μM in green and 0.5 μM in purple) without pre-incubation before uptake. Curves were calculated from n = 3 biologically independent experiments, each performed in triplicate measurements. Data points are averages of each independent triplicate experiment, with non-linear regression fits showing characteristic results for competitive inhibition. b, Results of [³H]-GABA saturation uptake assays using wild-type GAT1–eGFP-expressing HEK293S cells, in the absence (0 μM in blue) and the presence of tiagabine (0.125 μM in red, 0.25 μM in green and 0.5 μM in purple) with pre-incubation before uptake. Curves were calculated from n = 3 biologically independent experiments, each performed in triplicate measurements. Data points are averages of each independent triplicate experiment, with non-linear regression fits showing characteristic results for non-competitive (mixed-type) inhibition. c, Proposed mode of inhibition of GAT1 by tiagabine. The initial conformation is based on the AlphaFold⁵⁵ prediction of the GAT1 outward-open conformation, with superimposed substrates Na⁺ (purple), Cl⁻ (green) and tiagabine (salmon) based on the outward-open LeuT structure (PDB ID: 4HOD³³). Initially, tiagabine and ions are bound to the substrate-binding site in the outward-open conformation, resulting in the conformational rearrangement of GAT1 to the inward-open conformation, accompanied by the release of ions, as tiagabine ultimately stalls the transporter in the inward-open conformation.