Palmitoylation regulates norepinephrine transporter trafficking and expression and is potentially involved in the pathogenesis of postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is an adrenergic signaling disorder characterized by excessive plasma norepinephrine, postural tachycardia, and syncope. The norepinephrine transporter (NET) modulates adrenergic homeostasis via reuptake of extracellular catecholamines and is implicated in the pathogenesis of adrenergic and neurological disorders. Previous research has outlined that NET activity and trafficking is modulated via reversible post-translational modifications like phosphorylation and ubiquitylation. S-palmitoylation, or the addition of a 16-carbon saturated fatty acid, is another post-translational modification responsible for numerous biological mechanisms. In this study, we reveal that NET is dynamically palmitoylated and inhibition of this modification with the palmitoyl acyltransferase (DHHC) inhibitor, 2-bromopalmitate (2BP), results in decreased NET palmitoylation within 90 min of treatment. This result was followed closely with a reduction in transport capacity, cell surface, and total cellular NET expression after 120 min of treatment. Increasing 2BP concentrations and treatment time revealed a nearly complete loss of total NET protein. Co-expression with individual DHHCs revealed a single DHHC enzyme, DHHC1, promoted WT hNET palmitoylation and elevated NET protein levels. The POTS associated NET mutant, A457P, exhibits dramatically decreased transport capacity and cell surface levels which we have confirmed in the current study. In an attempt to recover A457P NET expression we co-expressed the A457P variant with DHHC1 to drive expression as seen with the WT protein but instead saw an increase in NET N-terminal immuno-detectable fragments. Further investigation of A457P NET palmitoylation and surface expression is necessary, but our preliminary novel findings reveal palmitoylation as a mechanism of NET regulation and suggest that dysregulation of this process may contribute to the pathogenesis of POTS.

Post-translational mechanisms in psychostimulant-induced neurotransmitter efflux.

The availability of monoamine neurotransmitters in the brain is under the control of dopamine, norepinephrine, and serotonin transporters expressed on the plasma membrane of monoaminergic neurons. By regulating transmitter levels these proteins mediate crucial functions including cognition, attention, and reward, and dysregulation of their activity is linked to mood and psychiatric disorders of these systems. Amphetamine-based transporter substrates stimulate non-exocytotic transmitter efflux that induces psychomotor stimulation, addiction, altered mood, hallucinations, and psychosis, thus constituting a major component of drug neurochemical and behavioral outcomes. Efflux is under the control of transporter post-translational modifications that synergize with other regulatory events, and this review will summarize our knowledge of these processes and their role in drug mechanisms.

Dopamine transporter membrane mobility is bidirectionally regulated by phosphorylation and palmitoylation.

The primary regulator of dopamine availability in the brain is the dopamine transporter (DAT), a plasma membrane protein that drives reuptake of released dopamine from the extracellular space into the presynaptic neuron. DAT activity is regulated by post-translational modifications that establish clearance capacity through impacts on transport kinetics, and dysregulation of these events may underlie dopaminergic imbalances in mood and psychiatric disorders. Here, using fluorescence recovery after photobleaching, we show that phosphorylation and palmitoylation induce opposing effects on DAT lateral membrane mobility, which may influence functional outcomes by regulating subcellular localization and binding partner interactions. Membrane mobility was also impacted by amphetamine and in polymorphic variant A559V in directions consistent with enhanced phosphorylation. These findings grow the list of DAT properties controlled by these post-translational modifications and highlight their role in establishment of dopaminergic tone in physiological and pathophysiological states.

Palmitoylation Regulates Human Serotonin Transporter Activity, Trafficking, and Expression and Is Modulated by Escitalopram.

In the central nervous system, serotonergic signaling modulates sleep, mood, and cognitive control. During serotonergic transmission, the synaptic concentration of serotonin is tightly controlled in a spatial and temporal manner by the serotonin transporter (SERT). Dysregulation of this process is implicated in the pathogenesis of major-depressive, obsessive-compulsive, and autism-spectrum disorders, which makes SERT a primary target for prescription therapeutics, most notably selective serotonin reuptake inhibitors (SSRIs). S-Palmitoylation, the reversible addition of a 16-carbon fatty acid to proteins, is an increasingly recognized dynamic post-translational modification responsible for modulating protein kinetics, trafficking, and localization patterns in response to physiologic/cellular stimuli. In this study, we reveal that human SERTs are a target for palmitoylation, and using the irreversible palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we have identified several associated functions. Using a lower dose of 2BP in shorter time frames, inhibition of palmitoylation was associated with reductions in SERT Vmax, without changes in Km or surface expression. With higher doses of 2BP for longer time intervals, inhibition of palmitoylation was consistent with the loss of cell surface and total SERT protein, suggesting palmitoylation is an important mechanism in regulating SERT trafficking and maintenance of SERT protein through biogenic or anti-degradative processes. Additionally, we have identified that treatment with the SSRI escitalopram decreases SERT palmitoylation analogous to 2BP, reducing SERT surface expression and transport capacity. Ultimately, these results reveal that palmitoylation is a major regulatory mechanism for SERT kinetics and trafficking and may be the mechanism responsible for escitalopram-induced internalization and ultimately decreased cellular SERT protein levels.

Kappa Opioid Receptor Antagonism Rescues Genetic Perturbation of Dopamine Homeostasis: Molecular, Physiological and Behavioral Consequences.

Aberrant dopamine (DA) signaling is implicated in schizophrenia, bipolar disorder (BPD), autism spectrum disorder (ASD), substance use disorder, and attention-deficit/hyperactivity disorder (ADHD). Treatment of these disorders remains inadequate. We established that the human DA transporter (DAT) coding variant (DAT Val559), identified in individuals with ADHD, ASD, or BPD, exhibits anomalous DA efflux (ADE) that is blocked by therapeutic amphetamines and methylphenidate. As the latter agents have high abuse liability, we exploited DAT Val559 knock-in mice to identify non-addictive agents that can normalize DAT Val559 functional and behavioral effects ex vivo and in vivo. Kappa opioid receptors (KORs) are expressed by DA neurons and modulate DA release and clearance, suggesting that targeting KORs might offset the effects of DAT Val559. We establish that enhanced DAT Thr53 phosphorylation and increased DAT surface trafficking associated with DAT Val559 expression are mimicked by KOR agonism of wildtype preparations and rescued by KOR antagonism of DAT Val559 ex vivo preparations. Importantly, KOR antagonism also corrected in vivo DA release and sex-dependent behavioral abnormalities. Given their low abuse liability, our studies with a construct valid model of human DA associated disorders reinforce considerations of KOR antagonism as a pharmacological strategy to treat DA associated brain disorders.

Palmitoylation regulates human serotonin transporter activity, trafficking, and expression and is modulated by escitalopram.

In the central nervous system, serotonergic signaling modulates sleep, mood, and cognitive control. During neuronal transmission, the synaptic concentration of serotonin is tightly controlled in a spatial and temporal manner by the serotonin transporter (SERT). Dysregulation of serotonergic signaling is implicated in the pathogenesis of major-depressive, obsessive-compulsive, and autism-spectrum disorders, which makes SERT a primary target for prescription therapeutics, most notably selective-serotonin reuptake inhibitors (SSRIs). S-palmitoylation is an increasingly recognized dynamic post-translational modification, regulating protein kinetics, trafficking, and localization patterns upon physiologic/cellular stimuli. In this study, we reveal that human SERTs are a target for palmitoylation, and using the irreversible palmitoyl acyl-transferase inhibitor, 2-bromopalmitate (2BP) we have identified several associated functions. Using a lower dose of 2BP in shorter time frames, inhibition of palmitoylation was associated with reductions in SERT V max , without changes in K m or surface expression. With higher doses of 2BP for longer time intervals, inhibition of palmitoylation was consistent with the loss of cell surface and total SERT protein, suggesting palmitoylation is an important mechanism in regulating SERT trafficking and maintenance of SERT protein through biogenic or anti-degradative processes. Additionally, we have identified that treatment with the SSRI escitalopram decreases SERT palmitoylation analogous to 2BP, reducing SERT surface expression and transport capacity. Ultimately, these results reveal palmitoylation is a major regulatory mechanism for SERT kinetics and trafficking and may be the mechanism responsible for escitalopram-induced internalization and loss of total SERT protein.

The Effect of Short-Term Exposure to Cadmium on the Expression of Vascular Endothelial Barrier Antigen in the Developing Rat Forebrain and Cerebellum: A Computerized Quantitative Immunofluorescent Study.

Clinical and laboratory studies have shown that environmental exposure to cadmium produces damage to several organs, including bones, lungs, and kidneys. The involvement of cadmium in central nervous system (CNS) disorders has also been widely reported, but the precise pathophysiological mechanism is not yet fully understood. Children who were exposed to cadmium during pregnancy are known to suffer from developmental delays, learning difficulties, attention deficit hyperactivity disorder (ADHD), and other cognitive and neurobehavioral deficits. Results from numerous studies suggest that dysfunction of the blood-brain barrier (BBB) structures is an important step in the neurotoxicity of cadmium. A rat-specific BBB marker protein, the endothelial barrier antigen (EBA), has been previously isolated and classified by Sternberger and others. The mouse IgG1 clone, anti-endothelial barrier antigen (anti-EBA), detects a protein triplet (23.5kDa, 25 kDa, and 30kDa) localized to the luminal surface of central and peripheral nervous system (CNS and PNS) vascular endothelial cells with selective permeability barrier functions. This marker has been widely used for characterizing BBB alterations under demyelinating, inflammatory, and other CNS pathologies. Many studies have been published using the rat model system for studying the neurotoxic effect of acute and chronic exposure to cadmium. We applied the indirect immunofluorescent techniques using the anti-EBA antibody in conjunction with the Olympus cellSens computerized image analysis to detect and quantify the surface areas of BBB-competent microvessel profiles in paraformaldehyde-fixed, paraffin-embedded brains of term-delivered young rats after intraperitoneal injection of a single dose of cadmium chloride. We detected a statistically significant reduction in EBA-positive microvessel surface areas in the forebrain (t = 5.86, df = 1789, p-value < 0.001) and cerebellum (t=73.40, df=1337, p < 0.001) of cadmium-treated rats compared to the normal controls. Thus, this study supports the hypothesis that the EBA is a sensitive and measurable indicator for quantitative assessment of the impact of cadmium exposure in the developing rat brain.

Sodium hydrogen exchanger (NHE1) palmitoylation and potential functional regulation.

AIMS: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. MAIN METHODS: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. KEY FINDINGS: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SIGNIFICANCE: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.

BSE can propagate in sheep co-infected or pre-infected with scrapie.

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster.

Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.

Palmitoylation by Multiple DHHC Enzymes Enhances Dopamine Transporter Function and Stability.

The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of dopaminergic neurotransmission. The transporter is subject to fine control that tailors clearance of transmitter to physiological demands, and dysregulation of reuptake induced by psychostimulant drugs, transporter polymorphisms, and signaling defects may impact transmitter tone in disease states. We previously demonstrated that DAT undergoes complex regulation by palmitoylation, with acute inhibition of the modification leading to rapid reduction of transport activity and sustained inhibition of the modification leading to transporter degradation and reduced expression. Here, to examine mechanisms and outcomes related to increased modification, we coexpressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of 12 PATs tested, DAT palmitoylation was stimulated by DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17, with others having no effect. Increased modification was localized to previously identified palmitoylation site Cys580 and resulted in upregulation of transport kinetics and elevated transporter expression mediated by reduced degradation. These findings confirm palmitoylation as a regulator of multiple DAT properties crucial for appropriate DA homeostasis and identify several potential PAT pathways linked to these effects. Defects in palmitoylation processes thus represent possible mechanisms of transport imbalances in DA disorders.

Model systems for analysis of dopamine transporter function and regulation.

The dopamine transporter (DAT) plays a critical role in dopamine (DA) homeostasis by clearing transmitter from the extraneuronal space after vesicular release. DAT serves as a site of action for a variety of addictive and therapeutic reuptake inhibitors, and transport dysfunction is associated with transmitter imbalances in disorders such as schizophrenia, attention deficit hyperactive disorder, bipolar disorder, and Parkinson disease. In this review, we describe some of the model systems that have been used for in vitro analyses of DAT structure, function and regulation, and discuss a potential relationship between transporter kinetic values and membrane cholesterol.

Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity.

Several protein kinases, including protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.

MPP+ decreases store-operated calcium entry and TRPC1 expression in Mesenchymal Stem Cell derived dopaminergic neurons.

Parkinson's disease is a neurodegenerative disorder involving the progressive loss of dopaminergic neurons (DNs), with currently available therapeutics, such as L-Dopa, only able to relieve some symptoms. Stem cell replacement is an attractive therapeutic option for PD patients, and DNs derived by differentiating patient specific stem cells under defined in-vitro conditions may present a viable opportunity to replace dying neurons. We adopted a previously published approach to differentiate Mesenchymal Stem Cells (MSCs) into DN using a 12-day protocol involving FGF-2, bFGF, SHH ligand and BDNF. While MSC-derived DNs have been characterized for neuronal markers and electrophysiological properties, we investigated store-operated calcium entry (SOCE) mechanisms of these DNs under normal conditions, and upon exposure to environmental neurotoxin, 1-methyl, 4-phenyl pyridinium ion (MPP+). Overall, we show that MSC-derived DNs are functional with regard to SOCE mechanisms, and MPP+ exposure dysregulates calcium signaling, making them vulnerable to neurodegeneration. Since in-vitro differentiation of MSCs into DNs is an important vehicle for PD disease modeling and regenerative medicine, the results of this study may help with understanding of the pathological mechanisms underlying PD.

Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding.

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.

Inhibitor mechanisms in the S1 binding site of the dopamine transporter defined by multi-site molecular tethering of photoactive cocaine analogs.

Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2ß position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.

Palmitoylation mechanisms in dopamine transporter regulation.

The neurotransmitter dopamine (DA) plays a key role in several biological processes including reward, mood, motor activity and attention. Synaptic DA homeostasis is controlled by the dopamine transporter (DAT) which transports extracellular DA into the presynaptic neuron after release and regulates its availability to receptors. Many neurological disorders such as schizophrenia, bipolar disorder, Parkinson disease and attention-deficit hyperactivity disorder are associated with imbalances in DA homeostasis that may be related to DAT dysfunction. DAT is also a target of psychostimulant and therapeutic drugs that inhibit DA reuptake and lead to elevated dopaminergic neurotransmission. We have recently demonstrated the acute and chronic modulation of DA reuptake activity and DAT stability through S-palmitoylation, the linkage of a 16-carbon palmitate group to cysteine via a thioester bond. This review summarizes the properties and regulation of DAT palmitoylation and describes how it serves to affect various transporter functions. Better understanding of the role of palmitoylation in regulation of DAT function may lead to identification of therapeutic targets for modulation of DA homeostasis in the treatment of dopaminergic disorders.

Phosphorylation mechanisms in dopamine transporter regulation.

The dopamine transporter (DAT) is a plasma membrane phosphoprotein that actively translocates extracellular dopamine (DA) into presynaptic neurons. The transporter is the primary mechanism for control of DA levels and subsequent neurotransmission, and is the target for abused and therapeutic drugs that exert their effects by suppressing reuptake. The transport capacity of DAT is acutely regulated by signaling systems and drug exposure, providing neurons the ability to fine-tune DA clearance in response to specific conditions. Kinase pathways play major roles in these mechanisms, and this review summarizes the current status of DAT phosphorylation characteristics and the evidence linking transporter phosphorylation to control of reuptake and other functions. Greater understanding of these processes may aid in elucidation of their possible contributions to DA disease states and suggest specific phosphorylation sites as targets for therapeutic manipulation of reuptake.

Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics.

The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders.

Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3ß-(p-chlorophenyl)tropane-2ß-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.