Regulate Dopamine Transporter Function Research Articles

Inhibition of LRRK2 kinase activity rescues deficits in striatal dopamine physiology in VPS35 p.D620N knock-in mice

Dysregulation of dopamine neurotransmission profoundly affects motor, motivation and learning behaviors, and can be observed during the prodromal phase of Parkinson’s disease (PD). However, the mechanism underlying these pathophysiological changes remains to be elucidated. Mutations in vacuolar protein sorting 35 (VPS35) and leucine-rich repeat kinase 2 (LRRK2) both lead to autosomal dominant PD, and VPS35 and LRRK2 may physically interact to govern the trafficking of synaptic cargos within the endo-lysosomal network in a kinase-dependent manner. To better understand the functional role of VPS35 and LRRK2 on dopamine physiology, we examined Vps35 haploinsufficient (Haplo) and Vps35 p.D620N knock-in (VKI) mice and how their behavior, dopamine kinetics and biochemistry are influenced by LRRK2 kinase inhibitors. We found Vps35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12 and Rab29. In contrast, Vps35 haploinsufficiency reduces phosphorylation of Rab12. While striatal dopamine transporter (DAT) expression and function is similarly impaired in both VKI and Haplo mice, that physiology is normalized in VKI by treatment with the LRRK2 kinase inhibitor, MLi-2. As a corollary, VKI animals show a significant increase in amphetamine induced hyperlocomotion, compared to Haplo mice, that is also abolished by MLi-2. Taken together, these data show Vps35 p.D620N confers a gain-of-function with respect to LRRK2 kinase activity, and that VPS35 and LRRK2 functionally interact to regulate DAT function and striatal dopamine transmission.

Bile Acids Gate Dopamine Transporter Mediated Currents.

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764–25773; Cheng and Bahar, Structure, 2015, 23, 2171–2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

Gβγ subunit activation promotes dopamine efflux through the dopamine transporter.

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.

Discovering Palmitoylation Sites on the Dopamine Transporter

The dopamine transporter (DAT) controls synaptic dopamine (DA) levels by reuptake after presynaptic neuronal vesicular release. Parkinson disease, addiction, and ADHD are neurological disorders associated with abnormal DA homeostasis causing DAT dysregulation to be hypothesized as a contributing factor in these disorders. Several binding partner interactions and posttranslational modifications combine to regulate DAT function. One such modification is S‐palmitoylation, the reversible, catalytic addition of palmitate to cysteine via a thioester linkage. S‐palmitoylation can regulate protein‐protein interactions, protein trafficking, transmembrane domain tilting, and membrane stability, and DAT palmitoylation increases DA transport Vmax without altering DAT surface expression and opposes DAT degradation. Because S‐palmitoylation is thought to be an intracellular process, we generated five cytosolic accessible cysteine to alanine mutants at residues Cys6, Cys135, Cys341, Cys522, and Cys580 and analyzed them for palmitoylation by [3H]palmitic acid metabolic labeling. A significant decrease in labeling was seen in only the C580A mutant versus WT control; however, approximately fifty percent of the signal remained, implying the presence of additional S‐palmitoylation site(s). To this end the more sensitive acyl‐biotinyl exchange (ABE) method was used to reanalyze these same mutants, and similar results were obtained confirming Cys580 as a major palmitoylation site with similar palmitoylation reduction in the C580A DAT and approximately 50% remaining residual palmitoylation. To further assess palmitoylation at individual cysteines, all but one of the intracellular cysteines were mutated to alanine, leaving a single cysteine available for palmitoylation. ABE analysis of these mutants showed significantly elevated palmitoylation of DATs containing Cys6 or Cys580 relative to those containing Cys135, Cys341, or Cys522 and mutants containing both Cys6 and Cys580 displayed near WT levels of palmitoylation. To assess if endogenous N‐terminal palmitoylation is present, we performed peptide mapping of rat striatal DAT using endoproteinase Asp‐N digestion prior to ABE and epitope specific immunoblotting. Asp‐N DAT cleavage occurs at Asp174, producing a characteristic 19 kDa peptide fragment containing Cys6 and Cys135 when blotted with a DAT N‐terminal antibody. This analysis revealed palmitoylation of this fragment, indicating one or both of these residues is palmitoylated suggesting further analysis is necessary to distinguish between these sites. Together these results provide evidence supporting DAT palmitoylation at Cys6, implying the potential for tethering of the distal N‐terminus to the plasma membrane. Additionally, Cys6 is adjacent to a known PKC‐mediated phosphorylation site, Ser7, suggesting the potential for interplay between phosphorylation and palmitoylation at these sites and a role for these modifications in DAT regulation.Support or Funding InformationSupported by National Institute on Drug Abuse grants R15 DA031991 (J.D.F.) and R01 DA13147 (R.A.V.) and NIH grants P20GM103442/P20RR016471 (to UND).

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing

The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na(+) or Cl(-) ion. Although isosmotic substitution of extracellular Na(+) ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl(-) ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons.

HIV-1 Tat Protein Decreases Dopamine Transporter Cell Surface Expression and Vesicular Monoamine Transporter-2 Function in Rat Striatal Synaptosomes

The dopamine (DA) transporter (DAT) and vesicular monoamine transporter (VMAT2) proteins interact as a biochemical complex to regulate dopaminergic neurotransmission. We have reported that HIV-1Tat(1-86) decreases the specific [(3)H]DA uptake and [(3)H]WIN 35,428 binding sites without a change in total DAT immunoreactivity in rat striatum (Zhu et al., 2009b). The present study determined the effects of Tat on DAT phosphorylation and trafficking, and vesicular [(3)H]DA uptake. Pre-incubation of rat striatal synaptosomes with the protein kinase C (PKC) inhibitor bisindolylmaleimide I (1μM) completely blocked Tat(1-86)-induced reduction of [(3)H]DA uptake, indicating that Tat regulates DAT function through a PKC-dependent mechanism. After exposure of synaptosomes to Tat(1-86) (1μM), DAT immunoreactivity was decreased in plasma membrane enriched fractions (P3) and increased in vesicle-enriched fractions (P4) relative to controls without change in total synaptosomal fractions (P2), suggesting that Tat-induced inhibition of DA uptake is attributable to DAT internalization. Although both DAT and VMAT2 proteins are essential for the regulation of DA disposition in synapse and cytosol, Tat inhibited the specific [(3)H]DA uptake into vesicles (P4) and synaptosomes (P2) by 35% and 26%, respectively, inferring that the inhibitory effect of Tat was more profound in VMAT2 protein than in DAT protein. Taken together, the current study reveals that Tat inhibits DAT function through a PKC and trafficking-dependent mechanism and that Tat impacts the dopaminergic tone by regulating both DAT and VMAT2 proteins. These findings provide new insight into understanding the pharmacological mechanisms of HIV-1 viral protein-induced dysfunction of DA neurotransmission in HIV-infected patients.

Akt-Dependent and Isoform-Specific Regulation of Dopamine Transporter Cell Surface Expression

Dopamine (DA) is a neurotransmitter implicated in multiple functions, including movement, cognition, motivation, and reward. The DA transporter (DAT) is responsible for clearing extracellular DA, thereby terminating DA neurotransmission. Previously, it has been shown that insulin signaling through protein kinase B/Akt regulates DAT function by fine-tuning DAT cell surface expression. Importantly, specific Akt isoforms (e.g., Akt1, Akt2) serve distinct physiological functions. Here, we demonstrate using isoform-specific Akt inhibitors that basal activity of Akt2, rather than Akt1, regulates DAT cell surface expression. Since Akt2 activation is mediated by insulin, these data further implicate insulin signaling as an important modulator of DAT function and dopaminergic tone.

Dysregulation of Dopamine Transporters via Dopamine D2Autoreceptors 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 D(2) receptor (D(2)R)-mediated inhibition of DA synthesis and release, and DA transporter (DAT)-mediated DA clearance. D(2)Rs 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 D(2)R provides support for hDAT A559V-mediated ADE. We determine in hDAT A559V a pertussis toxin-sensitive, CaMKII-dependent phosphorylation mechanism that supports D(2)R-driven DA efflux. These studies identify a signaling network downstream of D(2)R activation, normally constraining DA action at synapses, that may be altered by DAT mutation to impact risk for DA-related disorders.

Ethanol Alters Endosomal Recycling of Human Dopamine Transporters

Dynamic membrane trafficking of the monoamine dopamine transporter (DAT) regulates dopaminergic signaling. Various intrinsic and pharmacological modulators can alter this trafficking. Previously we have shown ethanol potentiates in vitro DAT function and increases surface expression. However, the mechanism underlying these changes is unclear. In the present study, we found ethanol directly regulates DAT function by altering endosomal recycling of the transporter. We defined ethanol action on transporter regulation by [(3)H]DA uptake functional analysis combined with biochemical and immunological assays in stably expressing DAT HEK-293 cells. Short-term ethanol exposure potentiated DAT function in a concentration-, but not time-dependent manner. This potentiation was accompanied by a parallel increase in DAT surface expression. Ethanol had no effect on function or surface localization of the ethanol-insensitive mutant (G130T DAT), suggesting a trafficking-dependent mechanism in mediating the ethanol sensitivity of the transporter. The ethanol-induced increase in DAT surface expression occurred without altering the overall size of DAT endosomal recycling pools. We found ethanol increased the DAT membrane insertion rate while having no effect on internalization of the transporter. Ethanol had no effect on the surface expression or trafficking of the endogenously expressing transferrin receptor, suggesting ethanol does not have a nonspecific effect on endosomal recycling. These results define a novel trafficking mechanism by which ethanol regulates DAT function.

Regulation of dopamine transporter activity by carboxypeptidase E

BackgroundThe dopamine transporter (DAT) plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT) can directly interact with other cellular proteins and regulate DAT function and trafficking.ResultsHere, we have identified that carboxypeptidase E (CPE), a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization.ConclusionTaken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity.

Activity‐dependent regulation of the dopamine transporter is mediated by Ca2+/calmodulin‐dependent protein kinase signaling

The mechanisms regulating expression of the dopamine transporter are poorly understood. We tested the hypothesis that neuronal activity is one of the non-genetic determinants of dopamine transporter abundance. Sustained changes in neuronal activity caused by tetrodotoxin and 4-aminopyridine altered the dopamine uptake and abundance of dopamine transporter and its mRNA in rat mesencephalic cultures. The altered neuronal activity caused by these two drugs is accompanied by changes in intracellular calcium concentrations and Ca(2+)/calmodulin-dependent protein (CaM) kinase II activity in dopamine neurons. Chronic treatment with an L-type calcium channel blocker (nifedipine) or CaM kinase inhibitor (KN93) decreased dopamine transporter-mediated uptake and occluded the effects of tetrodotoxin and 4-aminopyridine. These data suggest that neuronal activity can regulate dopamine transporter function and abundance via calcium/CaM kinase II signaling.

Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells

The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.

The dopamine transporter proteome

Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT-mediated re-uptake system limits not only the intensity but also the duration of DA actions at presynaptic and postsynaptic receptors. This protein is the primary target for cocaine and amphetamine, both highly addictive and major substances of abuse worldwide. DAT is also the molecular target for therapeutic agents used in the treatment of mental disorders, such as attention deficit hyperactivity disorder and depression. Given the role played by the DAT in regulation of DA neurotransmission and its contribution to the abuse potential of psychostimulants, it becomes not only important but also necessary to understand the functional regulation of this protein. To investigate the cellular and molecular mechanisms associated with DAT function and regulation, our laboratory and others have embarked on a systematic search for DAT protein-protein interactions. Recently, a growing number of proteins have been shown to interact with DAT. These novel interactions might be important in the assembly, targeting, trafficking and/or regulation of transporter function. In this review, I summarize the main findings obtained from the characterization of DAT-interacting proteins and discuss the functional implications of these novel interactions. Based on these new data, I propose to use the term DAT proteome to explain how interacting proteins regulate DAT function. These novel interactions might help define new mechanisms associated with the function of the transporter.

Rapid regulation of dopamine transporter function by substrates, blockers and presynaptic receptor ligands

The extracellular actions of dopamine are terminated primarily through its binding to dopamine transporters and translocation back into dopamine neurons. The transporter thereby serves as an optimal target to regulate dopamine neurotransmission. Although acute pharmacological blockade of dopamine transporters is known to reversibly inhibit transporter function by preventing the binding of its endogenous substrate dopamine, it recently has become clear that dopamine transporter substrates, such as amphetamines, and blockers, such as cocaine, also have the ability to rapidly and persistently regulate transporter function after their direct pharmacological effect has subsided. Presynaptic receptor ligands can also regulate dopamine transporter function. This has been investigated most extensively for dopamine D2 receptors, but there is also evidence for regulation by γ-aminobutyric acid (GABA) GABA B receptors, metabotropic glutamate, nicotinic acetylcholine, serotonin, σ 2- and κ-opioid receptors. The focus of this review is the rapid, typically reversible, regulation of dopamine transporter velocity by substrates, blockers and presynaptic receptor ligands. The research discussed here suggests that a common mechanism through which these different classes of compounds regulate transporter activity is by altering the cell surface expression of dopamine transporters.