Bi-directional transport of GABA in human embryonic kidney (HEK-293) cells stably expressing the rat GABA transporter GAT-1.
1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.
Sitte
Harald H
HH
Institute of Pharmacology, University of Vienna, 1090 Vienna, Austria.
Singer
Ernst A
EA
Scholze
Petra
P
D000613:Aminobutyrates / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D018756:GABA Antagonists / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D014179:Neurotransmitter Uptake Inhibitors / Q000494:pharmacology; D009557:Nipecotic Acids / Q000494:pharmacology; D027361:Organic Anion Transporters; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D018079:Receptors, GABA / Q000378:metabolism; D000078308:Tiagabine; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000493:pharmacokinetics
D000613:Aminobutyrates; D002352:Carrier Proteins; D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014179:Neurotransmitter Uptake Inhibitors; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; D018079:Receptors, GABA; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; D014316:Tritium; C030278:nipecotic acid; C005959:2,4-diaminobutyric acid; D005680:gamma-Aminobutyric Acid; D010042:Ouabain; D000078308:Tiagabine