Development of a screening assay for the SLC6A15/B0AT2 transporter

SLC6A15 belongs to the family of neurotransmitter transporters which also includes the antidepressant-binding transporters for serotonin and norepinephrine. The protein encoded by SLC6A15, B0AT2, has been shown to transport neutral amino acids. Recently, a study by Kohli et al. showed a genetic association between hSLC6A15 SNPs and increased stress susceptibility in depressive patients. These findings were further supported by animal experiments. In order to better understand the function of SLC6A15/B0AT2 we aim to find specific ligands to pharmacologically modulate its function. Towards this goal, HEK293 cells stably overexpressing hSLC6A15 were generated and the transporter activity was measured in a 3H-proline uptake assay. Saturation analysis showed a KM = 1.6 ± 0.2 mM and a V_max = 4679 ± 164 pmol/min/106 cells. The 3H-uptake activity increased linearly for the first 20minutes. It was shown to be strictly sodium dependent, unaffected by DMSO concentrations up to 0.3% and to be optimal at pH 7.5. The optimized conditions were adapted to a 384-well plate format to allow a throughput sufficient for screening. Inhibition experiments with proline and leucine showed an IC50 = 257±53µM and 210 ± 30 µM, respectively. The functionality of the transporter was further validated by patch clamp experiments. Treatment with 10 mM proline reversibly induced inward currents at −50 mV in HEK293 cells overexpressing SLC6A15 but not in untransfected controls.