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

Neurosci Lett. 2001 Aug 31;309(3):173-6. doi: 10.1016/s0304-3940(01)02058-4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Methyl-4-phenylpyridinium / metabolism
  • Animals
  • Carrier Proteins / biosynthesis*
  • Cell Line / metabolism*
  • Diffusion
  • Dopamine / metabolism
  • Dopamine Plasma Membrane Transport Proteins
  • GABA Plasma Membrane Transport Proteins
  • Humans
  • Kidney
  • Membrane Glycoproteins / biosynthesis*
  • Membrane Proteins / biosynthesis*
  • Membrane Transport Proteins / biosynthesis*
  • Nerve Tissue Proteins*
  • Neurotransmitter Uptake Inhibitors / metabolism*
  • Organic Anion Transporters*
  • Rats
  • Serotonin / metabolism
  • Serotonin Plasma Membrane Transport Proteins
  • gamma-Aminobutyric Acid / metabolism

Substances

  • Carrier Proteins
  • Dopamine Plasma Membrane Transport Proteins
  • GABA Plasma Membrane Transport Proteins
  • Membrane Glycoproteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Neurotransmitter Uptake Inhibitors
  • Organic Anion Transporters
  • SLC6A1 protein, human
  • SLC6A4 protein, human
  • Serotonin Plasma Membrane Transport Proteins
  • Slc6a1 protein, rat
  • Slc6a4 protein, rat
  • Serotonin
  • gamma-Aminobutyric Acid
  • 1-Methyl-4-phenylpyridinium
  • Dopamine