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

P Scholze, HH Sitte, EA Singer - Neuroscience letters, 2001 - Elsevier
P Scholze, HH Sitte, EA Singer
Neuroscience letters, 2001Elsevier
Human embryonic kidney 293 (HEK-293) cells stably transfected with the human serotonin
(5-HT) or dopamine transporter (hSERT, hDAT), or the rat GABA transporter GAT-1 were
incubated with saturating concentrations of transporter substrates (hSERT:[3H] 5-HT,[3H] N-
methyl-phenyl-pyridinium (MPP+); hDAT:[3H] dopamine,[3H] MPP+; rGAT:[3H] GABA).
Uptake velocities decreased significantly over time for [3H] 5-HT and [3H] dopamine
(already visible at 1 min), but not for [3H] MPP+ or [3H] GABA. In efflux experiments cells …
Human embryonic kidney 293 (HEK-293) cells stably transfected with the human serotonin (5-HT) or dopamine transporter (hSERT, hDAT), or the rat GABA transporter GAT-1 were incubated with saturating concentrations of transporter substrates (hSERT: [3H]5-HT, [3H]N-methyl-phenyl-pyridinium (MPP+); hDAT: [3H]dopamine, [3H]MPP+; rGAT: [3H]GABA). Uptake velocities decreased significantly over time for [3H]5-HT and [3H]dopamine (already visible at 1 min), but not for [3H]MPP+ or [3H]GABA. In efflux experiments cells were 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 [3H]5-HT, [3H]dopamine, [3H]MPP+ and [3H]GABA, respectively. The results suggest that in uptake experiments the more lipophilic substrates [3H]5-HT and [3H]dopamine leave the cells by diffusion already after a short time (1 min) of accumulation.
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