Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, following their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We find that the protein Flotillin-1/Reggie-2 (Flot1) is required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we have identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 is also required to localize DAT within plasma membrane microdomains in stable cell lines, and is essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells.By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At Ϫ60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 M) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at Ϫ60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

The dopamine (DA) transporter (DAT) mediates the removal of released DA. DAT is the major molecular target responsible for the rewarding properties and abuse potential of the psychostimulant amphetamine (AMPH). AMPH has been shown to reduce the number of DATs at the cell surface, and this AMPHinduced cell surface DAT redistribution may result in longlasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wildtype human DAT (WT-hDAT). However, AMPH did not induce cytosolic redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn 2ϩ ) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn 2ϩ and AMPH consistently reduced WThDAT trafficking but stimulated cytosolic redistribution of Y335A-hDAT. Furthermore, direct intracellular application of AMPH, via a whole-cell patch pipette, stimulated the trafficking of Y335A-hDAT. Taken together, these data suggest that the DAT transport cycle is not required for AMPH-induced downregulation and that an increase of intracellular AMPH is an essential component of DAT redistribution. Dopamine (DA) is a monoaminergic neurotransmitter involved in the control of locomotion, cognition, reward, and emotion (Iversen, 1971;Giros and Caron, 1993). The DA transporter (DAT) coordinates the spatial and temporal regulation of dopaminergic neurotransmission by mediating the reuptake of DA into presynaptic neurons. Although diffusion and enzymatic degradation contribute to reducing the synaptic concentration of DA, knockout studies have established DAT as the primary mechanism controlling extracellular DA levels (Giros et al., 1996;Jones et al., 1998). The regulatory mechanisms governing DAT are critical to DA signaling/ homeostasis. Altered dopaminergic signaling has been implicated in multiple brain disorders, including Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder, and addiction (Jucaite, 2002).DAT belongs to the Na ϩ /Cl Ϫ -dependent transporter gene family, which also consists of plasmalemmal carriers for the other monoamines, norepinephrine, epinephrine, and serotonin. Modulation of transporter surface expression may constitute a dynamic component of DA clearance (Melikian and Buckley, 1999;Loder and Melikian, 2003;Sorkina et al., 2005). Indeed, the clearance efficiency of DAT is determined not only by the transport rate of an individual transporter but also by the number of functional transporters on the cell surface. DAT trafficking has been shown to be regulated by receptor signaling, as well as by direct activation of protein

The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.