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 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: [(3)H]5-HT, [(3)H]N-methyl-phenyl-pyridinium (MPP+); hDAT: [(3)H]dopamine, [(3)H]MPP(+); rGAT: [(3)H]GABA). Uptake velocities decreased significantly over time for [(3)H]5-HT and [(3)H]dopamine (already visible at 1 min), but not for [(3)H]MPP(+) or [(3)H]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 [(3)H]5-HT, [(3)H]dopamine, [(3)H]MPP(+) and [(3)H]GABA, respectively. The results suggest that in uptake experiments the more lipophilic substrates [(3)H]5-HT and [(3)H]dopamine leave the cells by diffusion already after a short time (1 min) of accumulation.
Scholze
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P
Pharmacological Institute, University of Vienna, Waehringer Strasse 13a, 1090 Vienna, Austria. petra.scholze@univie.ac.at
Sitte
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HH
Singer
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EA
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis*; D002460:Cell Line / Q000378:metabolism*; D004058:Diffusion; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D007668:Kidney; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000096:biosynthesis*; D026901:Membrane Transport Proteins / Q000096:biosynthesis*; D009419:Nerve Tissue Proteins; D014179:Neurotransmitter Uptake Inhibitors / Q000378:metabolism*; D027361:Organic Anion Transporters; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014179:Neurotransmitter Uptake Inhibitors; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493415:Slc6a1 protein, rat; C493232:Slc6a4 protein, rat; D012701:Serotonin; D005680:gamma-Aminobutyric Acid; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine