[3H]GABA Uptake Assays

The [³H]GABA competition uptake assay was performed as previously described [36]. A minor change, however, was introduced for uptake in MDCK II cells, in which 100 nM [³H]GABA was used for a 15 min incubation period at 37 °C instead of 30 nM [³H]GABA and 3 min incubation. Studies related to the inhibition curves with GABA and BPDBA at hBGT1 were obtained both with and without the presence of 10 μM ATP during the [³H]GABA uptake assay.

To examine the influence of Ca²⁺ on the functional activity, tsA201 cells were pre-incubated with 25 μM BAPTA, 25 μM BAPTA/AM, 5 μM U-73122 or 1 μM A804598 in assay buffer for 30 min prior to the [³H]GABA uptake to ensure that the compounds reached their site of action. All compounds, except BAPTA/AM, were also present at the same concentration during the course of the actual [³H]GABA uptake assay.

The [³H]GABA uptake data was normalized to the percentage of total uptake in the individual experiments in the presence of the lowest compound concentration or no compound present. Data presented is the pooled data of at least three independent experiments with three technical replicates. Concentration–response curves (CRCs) were fitted with GraphPad Prism (version 8.2.1, Yosemite, GraphPad Software, San Diego, CA, USA) by non-linear regression to the sigmoidal concentration response model:

where Top is the Top plateau, Bottom is the Bottom plateau, X is the logarithm of the concentration of the compound, LogIC₅₀ is the concentration giving a response halfway between Top and Bottom,and nH is the Hill slope.

And the biphasic model:

where Bottom and Top are the plateaus at the left and right ends of the curve, in the same units as Y. LogIC₅₀_1 and LogIC₅₀_2 are the concentrations that give half-maximal inhibitory effects. nH1 and nH2 are the Hill slopes and Frac is the proportion of maximal response due to the more potent phase.

The best fit for CRCs obtained with the [³H]GABA uptake assay was identified by the extra-sum-of-squares F test (for convenience referred to as F test).