The serotonin transporter (SERT) is regulated by various signaling mechanisms that may operate to maintain appropriate levels of synaptic serotonin (5-HT). We demonstrate that one of the mitogen-activated protein kinases (MAPKs), p38 MAPK, regulates SERT.

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. -(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASPϩ accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D 2 R stimulation. These experiments demonstrate that D 2S R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D 2 R. Together, these results suggest a novel mechanism by which D 2S R autoreceptors may regulate DAT in the central nervous system. Dopamine (DA) is the predominant catecholamine neurotransmitter in the central nervous system. Dysregulation of DA neurons has been implicated in the pathogenesis of Parkinson's disease, schizophrenia, and drug addiction (Sotnikova et al., 2006). Extracellular DA levels are primarily regulated by the DA transporter (DAT), an integral membrane protein that is a member of the Na ϩ /Cl Ϫ -dependent J.J., A.Z., and T.S.S. contributed equally to this work.

Here we report a significant reduction in native NET function and surface expression in these cells following phorbol ester (␤-PMA) treatment. The ␤-PMA-mediated down-regulation of NET occurs by a rapid sequestration of NETs from the plasma membrane and is calciumindependent. Reversible biotinylation experiments revealed a significant enhancement of NET endocytosis following ␤-PMA treatment. Chemical treatments and expression of dominant negative mutants of dynamin 1 and 2 failed to prevent the ␤-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 ␤-PMA-mediated NET sequestration. Discontinuous sucrose density gradient centrifugation revealed NET in the lipid raft fractions. Following ␤-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. NET,1 which belongs to GAT1/NET family of Na ϩ /Cl Ϫ -dependent transporters, is selectively expressed on noradrenergic nerve terminals and exerts spatial and temporal control over the actions of NE (1, 2). NET is also an important target for various antidepressants and psychostimulants (3). Functional silencing of NET through genetic manipulations results in altered behavior when animals are stressed or challenged pharmacologically (hypersensitivity to psychostimulants and opiates) (4, 5). NE uptake sites and NET activity are also compromised in cardiovascular diseases and brain disorders (6 -8). A point mutation in NET (A457P in exon 9 of the NET gene) has been shown to be associated with orthostatic intolerance (9), whereas another study shows the absence of this specific mutation in all the orthostatic intolerance patients examined (10). The A457P mutation disrupts surface expression of mutant and wild type NETs when coexpressed in Chinese hamster ovary cells (11).Diverse stimuli, including neuronal activity, peptide hormones, and second messengers elevated after receptor activation, regulate NET. For example, activation of PKC downregulates the activity of NET and results in redistribution of NET from the plasma membrane (12, 13). Activation of PKC leads to a reduction in amine transport capacity, revealed by a change in maximal transport with little or no change in substrate affinity (12)(13)(14). Similarly, inhibition of protein phosphatase 1/2A (PP1/PP2A) reduces amine transport activity, and stimulates transporter phosphorylation (15). PP2Ac exists in physical...

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 approximately 3.6 x 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-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD) 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 uptake 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.