Namita Sen

N-Terminal Phosphorylation of the Dopamine Transporter Is Required for Amphetamine-Induced Efflux

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.

Calmodulin Kinase II Interacts with the Dopamine Transporter C Terminus to Regulate Amphetamine-Induced Reverse Transport

Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

D2 Receptors Regulate Dopamine Transporter Function via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent and Phosphoinositide 3 Kinase-Independent Mechanism

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D2 dopamine receptors (D2R) has been reported. The high affinity of DA and other DAT substrates for the D2R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP+) with live cell imaging techniques to identify the role of two D2R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D2R regulation of DAT. Addition of the D2/D3 receptor agonist quinpirole (0.1–10 μM) to human embryonic kidney cells coexpressing human DAT and D2 receptor (short splice variant, D2SR) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP+ accumulation. The D2/D3 agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP+ accumulation. D2SR activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP+ 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 D2R stimulation. These experiments demonstrate that D2SR 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 D2R. Together, these results suggest a novel mechanism by which D2SRautoreceptors may regulate DAT in the central nervous system.

Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after 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 found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was 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.

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca2+]i from internal stores compared with AMPH. [Ca2+]i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca2+]i and for the enhanced effects of METH on [Ca2+]i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca2+ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.

Syntaxin 1A Interaction with the Dopamine Transporter Promotes Amphetamine-Induced Dopamine Efflux

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 -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 μM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/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.

Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3

The herbicide paraquat (PQ) has increasingly been reported in epidemiological studies to enhance the risk of developing Parkinsons disease (PD). Furthermore, case-control studies report that individuals with genetic variants in the dopamine transporter (DAT, SLC6A) have a higher PD risk when exposed to PQ. However, it remains a topic of debate whether PQ can enter dopamine (DA) neurons through DAT. We report here a mechanism by which PQ is transported by DAT: In its native divalent cation state, PQ2+ is not a substrate for DAT; however, when converted to the monovalent cation PQ+ by either a reducing agent or NADPH oxidase on microglia, it becomes a substrate for DAT and is accumulated in DA neurons, where it induces oxidative stress and cytotoxicity. Impaired DAT function in cultured cells and mutant mice significantly attenuated neurotoxicity induced by PQ+. In addition to DAT, PQ+ is also a substrate for the organic cation transporter 3 (Oct3, Slc22a3), which is abundantly expressed in non-DA cells in the nigrostriatal regions. In mice with Oct3 deficiency, enhanced striatal damage was detected after PQ treatment. This increased sensitivity likely results from reduced buffering capacity by non-DA cells, leading to more PQ+ being available for uptake by DA neurons. This study provides a mechanism by which DAT and Oct3 modulate nigrostriatal damage induced by PQ2+/PQ+ redox cycling.

Dysregulation of Dopamine Transporters via Dopamine D2 Autoreceptors Triggers Anomalous Dopamine Efflux Associated with Attention-Deficit Hyperactivity Disorder

The neurotransmitter dopamine (DA) modulates brain circuits involved in attention, reward, and motor activity. Synaptic DA homeostasis is primarily controlled via two presynaptic regulatory mechanisms, DA D2 receptor (D2R)-mediated inhibition of DA synthesis and release, and DA transporter (DAT)-mediated DA clearance. D2Rs can physically associate with DAT and regulate DAT function, linking DA release and reuptake to a common mechanism. We have established that the attention-deficit hyperactivity disorder-associated human DAT coding variant Ala559Val (hDAT A559V) results in anomalous DA efflux (ADE) similar to that caused by amphetamine-like psychostimulants. Here, we show that tonic activation of D2R provides support for hDAT A559V-mediated ADE. We determine in hDAT A559V a pertussis toxin-sensitive, CaMKII-dependent phosphorylation mechanism that supports D2R-driven DA efflux. These studies identify a signaling network downstream of D2R activation, normally constraining DA action at synapses, that may be altered by DAT mutation to impact risk for DA-related disorders.

A pincer-like configuration of TM2 in the human dopamine transporter is responsible for indirect effects on cocaine binding

The second transmembrane segment (TM2) of DAT and other neurotransmitter transporters has been proposed to play a role in oligomerization as well as in cocaine binding. In an attempt to determine whether TM2 contributes to the binding site and/or transport pathway of DAT, we mutated to cysteine, one at a time, 25 residues in TM2 - from Phe98 to Gln122 - in an appropriate DAT background construct. Four of the mutants, F98C, G110C, P112C, and E117C, did not express at the cell surface, and G121C was inactive, despite its presence on the cell surface. Of the 21 mutants that expressed, none of the substituted cysteines reacted with MTSEA biotin-CAP, and none of the 20 functional mutants was sensitive to MTSEA or MTSET. Thus, TM2 does not appear to be water-accessible, based both on the lack of functional effects of charged MTS derivatives, and on the biochemical determination of lack of reaction with a biotinylated MTS derivative. This leads to the conclusion that TM2 does not contribute directly to the substrate-binding site or the transport pathway, and suggests that the observed effect of mutations in this region on cocaine binding is indirect. Three mutants, M106C, V107C and I108C, were crosslinked by treatment with HgCl(2). This crosslinking was inhibited by the presence of the cocaine analogue MFZ 2-12, likely due to a conformational rearrangement in TM2 upon inhibitor binding. However, the lack of crosslinking of cysteines substituted for Leu99, Leu113 and Leu120 - three of the residues that along with Met106 form a leucine heptad repeat in TM2 - makes it unlikely that this leucine repeat plays a role in symmetrical TM2 dimerization. Importantly, a high-resolution structure of LeuT, a sodium-dependent leucine transporter that is sufficiently homologous to DAT to suggest a high degree of structural similarity, became available while this manuscript was under review. We have taken advantage of this structure to explore further and interpret our experimental results in a rigorous structural context.