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Serotonin (5‐HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here, we delineate the molecular mechanisms by which glycogen synthase kinase‐3ß (GSK3ß) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/ß inhibitor CHIR99021, we observed a significant increase in SERT function, Vmax, surface expression with a reduction in 5‐HT Km and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/ß, we used HEK‐293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild‐type hSERT (hSERT‐WT) resulted in a time and dose‐dependent elevation of hSERT function with a concomitant increase in the Vmax and surface transporters because of reduced internalization and enhanced membrane insertion; silencing GSK3α/ß in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/ß phosphorylation site serine at position 48 to alanine in hSERT (hSERT‐S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/ß siRNA. Substantiating these findings, over‐expression of constitutively active GSK3ß with hSERT‐WT, but not with hSERT‐S48A, reduced SERT function, Vmax, surface density, and enhanced transporter phosphorylation. Both hSERT‐WT and hSERT‐S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK, or Src kinase. These findings provide new evidence that GSK3ß supports basal SERT function and trafficking via serine‐48 phosphorylation. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 23-35. d-Amphetamine (AMPH) down-regulates the norepinephrine transporter (NET), although the exact trafficking pathways altered and motifs involved are not known. Therefore, we examined the cellular and molecular mechanisms involved in AMPH-induced NET regulation in human placental trophoblast cells expressing the wild-type (WT)-hNET and the hNET double mutant (DM)-bearing protein kinase C (PKC)-resistant T258A + S259A motif. NET function and surface expression were significantly reduced in cells expressing WT-hNET but not in cells expressing hNET-DM following AMPH treatment. AMPH inhibited plasma membrane recycling of both WT-hNET and hNET-DM. In contrast, AMPH stimulated endocytosis of WT-hNET, and did not affect hNET-DM endocytosis. Although PKC or calcium/calmodulin- dependent kinase-II (CaMKII) inhibition or depletion of calcium failed to block AMPH-mediated down-regulation of WT-hNET, NET-specific blocker desipramine completely prevented AMPH-induced down-regulation. Furthermore, AMPH treatment had no effect on phospho-CaMKII immunoreactivity. The inhibitory potency of AMPH was highest on hNET-DM, intermediary on T258A and S259A single mutants and lowest on WT-hNET. Single mutants exhibited partial resistance to AMPH-mediated down-regulation. AMPH accumulation was similar in cells expressing WT-hNET or hNET-DM. The results demonstrate that reduced plasma membrane insertion and enhanced endocytosis account for AMPH-mediated NET down-regulation, and provide the first evidence that T258/S259 motif is involved only in AMPH-induced NET endocytosis that is desipramine-sensitive, but PKC and CaMKII independent. [ABSTRACT FROM AUTHOR]