[3H]GABA Uptake Assay

The [³H]GABA competition uptake assay was performed as previously described (Kvist et al., 2009; Al-Khawaja et al., 2014). Briefly, cells were seeded in white 96-well plates (White Opaque Tissue Culture (TC) plated 96-well Microplate) pre-coated with PDL. Medium was removed on the following day and cells washed with 100 µL/well assay buffer (HBSS supplemented with 20 mM HEPES, 1 mM CaCl₂ and 1 mM MgCl₂, pH 7.4). Then, 75 µL/well assay buffer containing 30 nM [³H]GABA and various concentrations of test compounds were added to the cells and incubated for 3 min at 37°C. The cells were washed 3 times with 100 µL/well ice-cold assay buffer, 150 µL/well MicroScint™20 was subsequently added before the plate was shaken for at least 1 h. The plate was counted in a Packard TopCounter microplate scintillation counter (PerkinElmer) for 3 min per well. Typical counts per minute for the different GATs were in the range of: wt hBGT1 (100–4,000), BGT1 Q299 (100–1,000), hBGT E52Y (150–500), BGT1 E52A (150–8,000), BGT1 E52A + Q299L (150–500), wt GAT3 (200–8,000), GAT3 L314Q (100–1,500), wt GAT1 (100–10,000) and wt GAT2 (150–7,500). The [³H]GABA uptake data for concentration-response curves was normalized to total uptake in the presence of the lowest concentration of test compound, while data for single test concentrations was normalized to total uptake. Data presented is the pooled data of at least three independent experiments with three technical replicates if not stated otherwise in figure legends. Concentration–response curves were fitted by non-linear regression to the sigmoidal concentration-response model with GraphPad Prism (version 9.0.0, GraphPad Software, San Diego, CA, United States) as described (Lie et al., 2020).