Abstract
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.
| Original language | English |
|---|---|
| Journal | Neurochemistry International |
| Volume | 73 |
| Pages (from-to) | 107-12 |
| Number of pages | 6 |
| ISSN | 0197-0186 |
| DOIs | |
| Publication status | Published - Jul 2014 |
Keywords
- Biotinylation
- Down-Regulation
- HEK293 Cells
- Humans
- Kinetics
- Membrane Proteins
- Neurons
- Primary Cell Culture
- Raphe Nuclei
- Serotonin
- Serotonin Plasma Membrane Transport Proteins
- Transfection