Physical and functional interactions between the serotonin transporter and the neutral amino acid transporter ASCT2

The activity of serotonergic systems depends on the re-uptake of extracellular serotonin via its plasma membrane serotonin transporter (SERT), a member of the Na+/Cl--dependent solute carrier 6 family. SERT is finely regulated by multiple molecular mechanisms including its physical interaction with intracellular proteins. The majority of previously identified SERT partners that control its functional activity are soluble proteins, which bind to its intracellular domains. SERT also interacts with trans-membrane proteins, but its association with other plasma membrane transporters remains to be established. Using a proteomics strategy, we show that SERT associates with ASCT2, a member of the solute carrier 1 family co-expressed with SERT in serotonergic neurons and involved in the transport of small neutral amino acids across the plasma membrane. Co-expression of ASCT2 with SERT in HEK-293 cells affects glycosylation and cell surface localization of SERT with a concomitant reduction in its 5-HT uptake activity. Conversely, depletion of cellular ASCT2 by RNA interference enhances 5-HT uptake in both HEK-293 cells and primary cultured mesencephalon neurons. Mimicking the effect of ASCT2 down-regulation, treatment of HEK-293 cells and neurons with the ASCT2 inhibitor D-threonine also increases 5-HT uptake. Moreover, D-threonine does not further enhance the maximal velocity of 5-HT uptake in cells depleted of ASCT2. Collectively, these findings provide evidence for a complex assembly involving SERT and a member of another solute carrier family, which strongly influences the subcellular distribution of SERT and the re-uptake of 5-HT.