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The serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary targets for antidepressants for therapeutic intervention of mood disorders. Our previous studies have identified the involvement of several signalling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether Akt/PKB (protein kinase) regulates SERT function is not known. In the present study, we made the novel observation that inhibition of Akt resulted in the down-regulation of SERT function through the regulation of SERT trafficking and phosphorylation. Akt inhibitor Akt X {10-(4′-[N-diethylamino)butyl]-2-chlorophenoxazine} reduced the endogenously phosphorylated Akt and significantly decreased 5-HT uptake and 5-HT-uptake capacity. Furthermore, SERT activity is also reduced by siRNA down-regulation of total and phospho-Akt levels. The reduction in SERT activity is paralleled by lower levels of cell-surface SERT protein, reduced SERT exocytosis with no effect on SERT endocytosis and accumulation of SERT in intracellular endocytic compartments with the most prominent localization to late endosomes and lysosomes. Akt2 inhibitor was more effective than Akt1 inhibitor in inhibiting SERT activity. Inhibition of downstream Akt kinase GSK3α/β (glycogen synthase kinase α/β) stimulates SERT function. Akt inhibition leads to a decrease in SERT basal phosphorylation. Our results provide evidence that Akt regulates SERT function and cell-surface expression by regulating the intracellular SERT distribution and plasma membrane availability, which perhaps may be linked to SERT phosphorylation state. Thus any changes in the activation of Akt and/or GSK3α/β could alter SERT-mediated 5-HT clearance and subsequently serotonergic neurotransmission. [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]

The effects of norepinephrine in the brain and periphery are terminated primarily by active reuptake of the catecholamine via cocaine- and amphetamine- sensitive norepinephrine transporters (NETs). Activation of protein kinase C (PKC) down-regulates NET by sequestering it from the plasma membrane, although the underlying mechanism is not yet known. Previously, we showed robust expression of endogenous NETs in rat placental trophoblasts (Jayanthi, L. D., Vargas, G., and DeFelice, L. J. (2002) Br. J. Pharmacol. 135, 1927–1934). Here we report a significant reduction in native NET function and surface expression in these cells following phorbol ester (β-PMA) treatment. The β-PMA-mediated down-regulation of NET occurs by a rapid sequestration of NETs from the plasma membrane and is calcium independent. Reversible biotinylation experiments revealed a significant enhancement of NET endocytosis following β-PMA treatment. Chemical treatments and expression of dominant negative mutants of dynamin 1 and 2 failed to prevent the β-PMA effect, suggesting a clathrin-independent pathway. In contrast, treatment with the cholesterol-disrupting agent filipin, which blocks caveolae/lipid raft-mediated internalization, completely blocked the β-PMA-mediated NET sequestration. Discontinuous sucrose density gradient centrifugation revealed NET in the lipid raft fractions. Following β-PMA treatment, there was reduced NET levels in the lipid raft fractions suggesting that cholesterol-rich lipid rafts mediate PKC-triggered NET internalization. Metabolic labeling and immunoprecipitation studies revealed that NET phosphorylation is stimulated severalfold by PKC activation and protein phosphatase 1/2A inhibition. Together, these findings demonstrate for the first time that in trophoblasts (i) PKC activation regulates NET function and surface expression by an enhanced internalization process that is lipid raft-mediated and (ii) PKC and protein phosphatase(s) modulation regulates NET phosphorylation. [ABSTRACT FROM AUTHOR]

D3 dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this modulation remain ill defined. The dopamine transporter, which terminates dopamine transmission via reuptake of released neurotransmitter, is regulated by receptor- and second messenger-linked signaling pathways. Whether D3 receptors regulate dopamine transporter function is unknown. We addressed this issue using a fluorescent imaging technique that permits real time quantification of dopamine transporter function in living single cells. Accumulation of the fluorescent dopamine transporter substrate trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (ASP+) in human embryonic kidney cells expressing human dopamine transporter was saturable and temperature-dependent. In cells co-expressing dopamine transporter and D3 receptors, the D2/D3 agonist quinpirole produced a rapid, concentration-dependent, and pertussis toxin-sensitive increase of ASP+ uptake. Similar agonist effects were observed in Neuro2A cells and replicated in human embryonic kidney cells using a radioligand uptake assay in which binding to and activation of D3 receptors by [³H]dopamine was prevented. D3 receptor stimulation activated phosphoinositide 3-kinase and MAPK. Inhibition of either kinase prevented the quinpirole-induced increase in uptake. D3 receptor activation differentially affected dopamine transporter function and subcellular distribution depending on the duration of agonist exposure. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression and increased dopamine transporter exocytosis. In contrast, prolonged agonist exposure reduced uptake and transporter cell surface expression. These results demonstrate that D3 receptors regulate dopamine transporter function and identify a novel mechanism by which D3 receptors regulate extracellular dopamine concentrations. [ABSTRACT FROM AUTHOR]

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of ∼3.6 × 10-9 cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-β2AR (β32 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-β-cyclodextrin (mβCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine u ptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity. [ABSTRACT FROM AUTHOR]