Substantial loss of substrate by diffusion during uptake in HEK-293 cells expressing neurotransmitter transporters

Human embryonic kidney 293 (HEK-293) cells stably transfected with the huma n serotonin (5-HT) or dopamine transporter (hSERT, hDAT), or the rat GABA t ransporter GAT-1 were incubated with saturating concentrations of transport er substrates (hSERT: [H-3]5-HT, [H-3]N-methyl-phenyl-pyridinium (MPP+); hD AT: [H-3]dopamine, [H-3]MPP+; rGAT: [H-3]GABA). Uptake velocities decreased significantly over time for [H-3]5-HT and [H-3]dopamine (already visible a t 1 min), but not for [H-3]MPP+ or [H-3]GABA. In efflux experiments cells w ere preloaded and substrate diffusion into the medium was studied following the addition of appropriate uptake inhibitors. Fractional effluxes were (% min(-1)) 1.27, 0.72, 0.27 and 0.08 for [H-3]5-HT, [H-3]dopamine, [H-3]MPP and [H-3]GABA, respectively. The results suggest that in uptake experiment s the more lipophilic substrates [H-3]5-HT and [H-3]dopamine leave the cell s by diffusion already after a short time (1 min) of accumulation. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.