Journal
NEUROCHEMISTRY INTERNATIONAL
Volume 73, Issue -, Pages 107-112Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.neuint.2014.01.005
Keywords
Serotonin transporter; Monoamine transporters; Neurotransmitter:sodium symporters; Serotonin; Trafficking; Internalization; ELISA; Raphe neurons
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Funding
- National Institute of Health [P01 DA 12408]
- The Danish Medical Research Council
- University of Copenhagen BioScaRT Program of Excellence
- Lundbeck Foundation Center for Biomembranes in Nanomedicine
- Novo Nordisk Foundation
- Fabrikant Vilhelm Pedersen og Hustrus Mindelegat
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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 K-m value observed for 5-HT uptake. The 5-HT-induced reduction in surface signal reached maximum within 40-60 min 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 V-max 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. (C) 2014 Elsevier Ltd. All rights reserved.
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