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.

The D 3 dopamine (DA) 4 receptor, a member of the D2-like family of DA receptors, is expressed in limbic brain regions, both presynaptically on DA neurons as well as postsynaptically. The D3 receptor has gained increasing attention as a target for the treatment of schizophrenia, psycho-stimulant abuse, and DA cell neurodegeneration (1-4). Its restricted central nervous system distribution, relative to D2 receptors, suggests that D3 receptor ligands may have fewer side effects than currently available therapeutic agents.Studies using D3 receptor knock-out mice (5-7) or D3 antisense (8, 9) revealed that D3 receptors regulate extracellular DA in ventral striatum. This effect was attributed to D3 regulation of a long negative feedback loop in which postsynaptic D3 receptors on medium spiny neurons modulate the activity of accumbens output neurons projecting to DA cell bodies in mid-brain (5). However, this hypothesis is incompatible with the effects of DA receptor ligands in tissue preparations in which efferent projections to midbrain DA nuclei are disrupted; modulation of extracellular DA by D3 receptors has been demonstrated in striatal slices (7) and tissue suspensions (10). Pharmacological studies examining the mechanism of such regulation have been precluded by the lack of selective ligands that discriminate between D2 and D3 receptors in vivo (5,(11)(12)(13).DA signaling is terminated by the DA transporter (DAT), an integral membrane protein that re-uptakes DA released into the extracellular space (14). Receptor and second messengerlinked kinase cascades regulate DAT function and cell surface

Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP+ accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP+). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signaling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

J. Neurochem. (2010) 114, 1019–1029. Abstract The serotonin transporter (SERT) mediates clearance of serotonin from the synapse, thereby, regulating extracellular serotonin concentrations. Radioligand uptake techniques are typically used to assess SERT function in tissue and heterologous expression systems. The need for sufficient protein in samples, however, requires use of homogenate preparations, potentially masking effects limited to specific cell populations. 4‐(4‐(dimethylamino)‐styryl)‐N‐methylpyridinium (ASP+) is a fluorescent monoamine transporter substrate that has been used for real‐time monitoring of dopamine and norepinephrine transporter function in single cells. The present live cell imaging studies examine the utility of ASP+ for quantifying human SERT function in HEK293 and neuroblastoma cells. We show rapid membrane binding and intracellular ASP+ accumulation in human SERT‐expressing cells. Accumulation is saturable; dependent on temperature and the presence of sodium and chloride in the media, and attenuated by serotonin. Acute or prolonged exposure of cells to serotonin re‐uptake inhibitors produces a concentration‐dependent decrease in accumulation. Similar effects are produced by protein kinase C activation whereas p38 MAPK activation increases ASP+ accumulation. These data demonstrate the validity of ASP+ as a probe for monitoring SERT function in living cells. Alterations in SERT binding and uptake can be quantified in the same cell and use of a within‐cell design permits analysis of time‐related alterations in SERT function.

The neurotransmitter, dopamine (DA) modulates excitatory and inhibitory neurotransmission in brain regions that control movement, emotion, and reward. Dysregulation of DA transmission is implicated in the etiology of several pathological conditions including Parkinson's disease, drug addiction, and schizophrenia. The DA transporter (DAT) is an integral membrane protein that is a member of the Na + -and Cl ) -dependent cotransporter gene family (Amara and Kuhar 1993). It serves a key role in regulating pre-synaptic DA transmission by clearing DA released into the extracellular space.Systemic administration of the endocannabinoid, anandamide (AEA), increases extracellular DA levels in the nucleus