Serotonin-induced down-regulation of cell surface serotonin transporter.
The serotonin transporter (SERT) terminates serotonergic signaling and enables refilling of synaptic vesicles by mediating reuptake of serotonin (5-HT) released into the synaptic cleft. The molecular and cellular mechanisms controlling SERT activity and surface expression are not fully understood. Here we demonstrate that the substrate 5-HT itself causes acute down-regulation of SERT cell surface expression. To assess surface SERT expression by ELISA, we used a SERT variant (TacSERT) where the N-terminus of SERT was fused to the intracellular tail of the extracellularly FLAG-tagged single-membrane spanning protein Tac. In stably transfected HEK293 cells, 5-HT caused a dose-dependent reduction in TacSERT surface signal with an EC50 value equivalent to the Km value observed for 5-HT uptake. The 5-HT-induced reduction in surface signal reached maximum within 40-60min and was blocked by the selective SERT inhibitor S-citalopram. 5-HT-induced reduction in SERT expression was further supported by surface biotinylation experiments showing 5-HT-induced reduction in wild type SERT plasma membrane levels. Moreover, preincubation with 5-HT lowered the Vmax for 5-HT uptake in cultured raphe serotonergic neurons, indicting that endogenous cell-surface resident SERT likewise is down-regulated in the presence of substrate.
Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, The Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Trine Nygaard
TN
Jørgensen
Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, The Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Peter Møller
PM
Christensen
Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, The Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address: gether@sund.ku.dk.
Ulrik
U
Gether
D019921:Biotinylation; D015536:Down-Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D061251:Primary Cell Culture; D011903:Raphe Nuclei / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D014162:Transfection
D008565:Membrane Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin
ELISA; Internalization; Monoamine transporters; Neurotransmitter:sodium symporters; Raphe neurons; Serotonin; Serotonin transporter; Trafficking