Cooperative Ionic Coordination Governs Dopamine Transporter Dynamics and Substrate Translocation: Mechanistic Insights from Molecular Simulations.

Presynaptic reuptake, mediated by the dopamine (DA) transporter (DAT), terminates DAergic neurotransmission and constrains extracellular DA levels. Addictive and therapeutic psychostimulants inhibit DA reuptake and multiple DAT coding variants have been reported in patients with neuropsychiatric disorders. These findings underscore that DAT is critical for DA neurotransmission and homeostasis. DAT surface availability is regulated acutely by endocytic trafficking, and considerable effort has been directed toward understanding mechanisms that govern DAT's plasma membrane expression and postendocytic fate. Multiple studies have demonstrated DAT endocytic recycling and enhanced surface delivery in response to various stimuli. Paradoxically, imaging studies have not detected DAT targeting to classic recycling endosomes, suggesting that internalized DAT targets to either degradation or an undefined recycling compartment. Here, we leveraged PRIME (PRobe Incorporation Mediated by Enzyme) labeling to couple surface DAT directly to fluorophore, and tracked DAT's postendocytic itinerary in immortalized mesencephalic cells. Following internalization, DAT robustly targeted to retromer-positive endosomes, and DAT/retromer colocalization was observed in male mouse dopaminergic somatodendritic and terminal regions. Short hairpin RNA-mediated Vps35 knockdown revealed that DAT endocytic recycling requires intact retromer. DAT also targeted rab7-positive endosomes with slow, linear kinetics that were unaffected by either accelerating DAT internalization or binding a high-affinity cocaine analog. However, cocaine increased DAT exit from retromer-positive endosomes significantly. Finally, we found that the DAT carboxy-terminal PDZ-binding motif was required for DAT recycling and exit from retromer. These results define the DAT recycling mechanism and provide a unifying explanation for previous, seemingly disparate, DAT endocytic trafficking findings.SIGNIFICANCE STATEMENT The neuronal dopamine (DA) transporter (DAT) recaptures released DA and modulates DAergic neurotransmission, and a number of DAT coding variants have been reported in several DA-related disorders, including infantile parkinsonism, attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is also competitively inhibited by psychostimulants with high abuse potential. Therefore, mechanisms that acutely affect DAT availability will likely exert significant impact on both normal and pathological DAergic homeostasis. Here, we explore the cellular mechanisms that acutely control DAT surface expression. Our results reveal the intracellular mechanisms that mediate DAT endocytic recycling following constitutive and regulated internalization. In addition to shedding light on this critical process, these findings resolve conflict among multiple, seemingly disparate, previous reports on DAT's postendocytic fate.

The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [125I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) in TM5 to Met(370/371) in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met(290)-Lys(336) and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.

Binding of Zn(2+) to an endogenous binding site in the dopamine transporter (DAT) leads to inhibition of dopamine (DA) uptake and enhancement of carrier-mediated substrate efflux. To elucidate the molecular mechanism for this dual effect, we expressed the DAT and selected mutants in Xenopus laevis oocytes and applied the two-electrode voltage clamp technique together with substrate flux studies employing radiolabeled tracers. Under voltage clamp conditions we found that Zn(2+) (10 mum) enhanced the current induced by both DA and amphetamine. This was not accompanied by a change in the uptake rate but by a marked increase in the charge/DA flux coupling ratio as assessed from concomitant measurements of [(3)H]DA uptake and currents in voltage-clamped oocytes. These data suggest that Zn(2+) facilitates an uncoupled ion conductance mediated by DAT. Whereas this required substrate in the wild type (WT), we observed that Zn(2+) by itself activated such a conductance in a previously described mutant (Y335A). This signifies that the conductance is not strictly dependent on an active transport process. Ion substitution experiments in Y335A, as well as in WT, indicated that the uncoupled conductance activated by Zn(2+) was mainly carried by Cl(-). Experiments in oocytes under non-voltage-clamped conditions revealed furthermore that Zn(2+) could enhance the depolarizing effect of substrates in oocytes expressing WT. The data suggest that by potentiating an uncoupled Cl(-) conductance, Zn(2+) is capable of modulating the membrane potential of cells expressing DAT and as a result cause simultaneous inhibition of uptake and enhancement of efflux.

The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or "endocytic brake," controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.

A series of 3-(3',4'-dichlorophenyl)-1-indanamine monoamine reuptake blockers have been synthesized in an effort to develop a compound that could be used as a maintenance therapy to treat cocaine abuse. Since the effects of cocaine on dopamine (DA) and serotonin (5HT) transporters are important components of its pharmacological activity, the focus was on nonselective inhibitors of monoamine transport. To reduce or eliminate the abuse potential of a DA reuptake blocker, the compounds were designed to be slow-onset, long-duration prodrugs whose N-demethylated metabolites would have increased activity over the parent compound with the ideal being a parent compound that has little or no activity. To achieve this, pairs of compounds with different groups on the amine nitrogen and with and without an additional N-methyl group were synthesized. All of the synthesized compounds were screened for binding and reuptake at the cloned human DA, 5HT, and norepinephrine (NE) transporters. As previously found, trans isomers are nonselective blockers of DA, 5HT, and NE reuptake, cis isomers with small N-alkyl groups are selective blockers of 5HT reuptake, and tertiary amines of the trans compounds are less potent than the corresponding N-demethylated secondary amines as blockers of DA reuptake. Larger N-alkyl groups in both the trans and cis series were found to reduce activity for the 5HT and NE transporters with less effect at DA transporters. Selected trans compounds were also screened for locomotor activity in mice and generalization to a cocaine-like profile in rats. With intraperitoneal administration, all of the trans isomers showed a slow onset of at least 20 min and an extremely long duration of action in the locomotor assays. Several of the trans compounds also fully generalized to a cocaine-like pharmacological profile. An initial lead compound, the N,N-dimethyl analogue trans-1b, was resolved into chirally pure enantiomers. Surprisingly, both enantiomers were found to have significant affinity for the DA transporter and to cause locomotor activation. This is in contrast to the N-methyl compound in which only the (+)-enantiomer had significant activity. The absolute configuration of the more active enantiomer was determined by X-ray crystallography to be 3R,1S.

Amphetamine is a highly addictive psychostimulant, which is thought to generate its effects by promoting release of dopamine through reverse activation of dopamine transporters. However, some amphetamine-mediated behaviors persist in dopamine transporter knock-out animals, suggesting the existence of alternative amphetamine targets. Here we demonstrate the identification of a novel amphetamine target by showing that in Caenorhabditis elegans, a large fraction of the behavioral effects of amphetamine is mediated through activation of the amine-gated chloride channel, LGC-55. These findings bring to light alternative pathways engaged by amphetamine, and urge rethinking of the molecular mechanisms underlying the effects of this highly-addictive psychostimulant.

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.

genetic variants affecting the reverse transport of DA might influence the risk of common, sporadic forms of autism remains open. It is now widely accepted that rare genetic variants could play an important role in susceptibility to common diseases. In light of Cartier et al.'s results, major resequencing efforts of the SLC6A3 and STX1A genes should be undertaken in large cohorts of individuals with autism, with significant insights into disease biology likely from these results. Another interesting implication of the work by Cartier and colleagues lies in unraveling the therapeutic potential of DA reverse transport in autism. Starting from the observation that rare autism-causing variants are associated with inhibition of reverse transport of DA, it could be hypothesized that psychostimulants may serve as a potential therapeutic aid in autism. Notably, not only have psychostimulants been suggested to be effective for ADHD-like symptoms in autism spectrumdisorderindividuals,buttheprevalenceratesoftheiruseinautism

The reuptake and release of dopamine (DA) can be estimated using in vivo imaging methods by assessing the competition between endogenous DA and an administered exogenous DA transporter (DAT) and D2 receptor (D2 R) radioligand, respectively. The aim of this study was to investigate the comparative roles of DA release vs DA reuptake in the rat striatum with small animal SPECT in relation to l-DOPA-induced behaviors. DAT and D2 R binding, together with behavioral measures, were obtained in 99 rats in response to treatment with either 5 or 10 mg/kg l-DOPA or vehicle. The behavioral parameters included the distance travelled, and durations and frequencies of ambulation, sitting, rearing, head-shoulder motility, and grooming. Data were subjected to a cluster analysis and to a multivariate principal component analysis. The highest DAT binding (i.e., the lowest DA reuptake) was associated with the highest, and the lowest DAT binding (i.e., the highest DA reuptake) was associated with the lowest motor/exploratory activity. The highest and the lowest D2 R binding (i.e., the lowest and the highest DA release, respectively) were merely associated with the second highest and second lowest levels of motor/exploratory activity. These findings indicate that changes in DA reuptake in response to fluctuating DA levels offer a better prediction of motor activity than the release of DA into the synaptic cleft. This dissociation, as reflected by in vivo DAT and D2 R binding data, may be accounted for by the regulatory sensitization meachnisms that occur at D2 R binding sites in response to altered levels of DA. Synapse 70:369-377, 2016. © 2016 Wiley Periodicals, Inc.

Dopamine transporter trafficking is regulated by neutral sphingomyelinase 2/ceramide kinase

Prolonged treatment with pramipexole promotes physical interaction of striatal dopamine D3 autoreceptors with dopamine transporters to reduce dopamine uptake

Efficient clearance of dopamine (DA) from the synapse is key to regulating dopaminergic signaling. This role is fulfilled by DA transporters (DATs). Recent advances in the structural characterization of DAT from Drosophila (dDAT) and in high-resolution imaging of DA neurons and the distribution of DATs in living cells now permit us to gain a mechanistic understanding of DA reuptake events in silico. Using electron microscopy images and immunofluorescence of transgenic knock-in mouse brains that express hemagglutinin-tagged DAT in DA neurons, we reconstructed a realistic environment for MCell simulations of DA reuptake, wherein the identity, population and kinetics of homology-modeled human DAT (hDAT) substates were derived from molecular simulations. The complex morphology of axon terminals near active zones was observed to give rise to large variations in DA reuptake efficiency, and thereby in extracellular DA density. Comparison of the effect of different firing patterns showed that phasic firing would increase the probability of reaching local DA levels sufficiently high to activate low-affinity DA receptors, mainly owing to high DA levels transiently attained during the burst phase. The experimentally observed nonuniform surface distribution of DATs emerged as a major modulator of DA signaling: reuptake was slower, and the peaks/width of transient DA levels were sharper/wider under nonuniform distribution of DATs, compared with uniform. Overall, the study highlights the importance of accurate descriptions of extrasynaptic morphology, DAT distribution, and conformational kinetics for quantitative evaluation of dopaminergic transmission and for providing deeper understanding of the mechanisms that regulate DA transmission.

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced DA efflux is mediated by DAT and results from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT is voltage-dependent, electrogenic, and dependent on intracellular Na(+) concentration in the recording electrode. Intracellular Na(+) fluorescence, as measured by confocal microscopy using a Na(+)-sensitive dye, was enhanced by AMPH application. Furthermore, the ability of AMPH to induce DA efflux was regulated by intracellular Na(+) concentration and correlated with the size of the DAT-mediated, AMPH-induced ion flux across the plasma membrane. In the absence of intracellular Na(+) but the presence of high intracellular Cl(-), AMPH-induced inward currents elicited DA efflux proportionally to their dimension and duration. Thus, we propose that AMPH-induced DA efflux depends on two correlated transporter processes. First, AMPH binds to the DAT and is transported, thereby causing an inward current. Second, because of this AMPH-induced inward current, Na(+) becomes more available intracellularly to the DAT, thereby enhancing DAT-mediated reverse transport of DA.

Using a rapid (0.5 ml/min) flow rate superfusion system, the dopamine (DA) transporter mediated release of DA is further explored, and compared to the depolarization evoked release of DA in rat striatal synaptosomes preloaded with radioactive DA (3H-DA). In this system external DA in the low microM range efficaciously releases the preloaded transmitter, the maximal response being reached at 3 microM DA. The external DA stimulated release is Ca(2+)-independent, Cl(-)-dependent, and blocked by both bupropion and nomifensine. The atypical antidepressant bupropion inhibits 3H-DA accumulation to rat striatal synaptosomes with a calculated IC50 of 1.3 x 10(-6) M. Among DA uptake blockers some are known to act as DA releasing agents. Here we found that the DA uptake blocker nomifensine (30 microM) is unable to modify the baseline release of 3H-DA, whereas bupropion (10 microM) clearly elevates the baseline release of 3H-DA in a Ca(2+)-independent and Cl(-)-dependent manner. The non releasing agent nomifensine blocks the release of 3H-DA induced by bupropion. The Ca(2+)-dependent, high K+ depolarization evoked release of 3H-DA is not modified by nomifensine and does not depend on the external Cl- concentration. When the depolarizing medium contains DA the carrier mediated release of 3H-DA induced by the external DA is additive to the high K+ induced response. A drastic drop in the external Cl- concentration induces 3H-DA release. This release of 3H-DA induced by low external Cl- levels is completely blocked by nomifensine, which only slightly diminished the release of 3H-DA induced by the absence of external Na+. On the basis of these results, it is concluded that: 1) Rapid perfusion flow rates eliminate DA reuptake. 2) DA uptake inhibitors either with or without DA releasing capabilities block the release of DA induced by microM levels of external DA. 3) By preventing translocation of the DA transporter mobile moiety, nomifensine may inhibit the release of DA induced by external DA or bupropion and by drastic drops in the external Cl- concentration. 4) In the absence of nomifensine, the DA transporter works under both resting and depolarized conditions, but in contrast to the GABA transporter (Sitges et al.: Neurochem Res 18:1081-1087, 1993), the DA transporter does not contribute to the amount of the DA released by depolarization. 5) Reversal of the DA uptake carrier is favored by conditions increasing the internal DA levels. 6) Cl- rather than Na+ is a major determinant in 3H-DA movements through the DA transporter.

Cocaine and GBR12909 produce equivalent motoric responses at different occupancy of the dopamine transporter