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Background:The dopamine transporter establishes synaptic transmitter levels and strength of dopamine neurotransmission. Results: Reciprocal modification of specific DAT phosphorylation and palmitoylation sites dictates steady-state and regulated transport capacity in the absence of trafficking. Conclusion:The balance between phosphorylation and palmitoylation controls DA transport kinetics. Significance: This is a previously unknown mode of transporter regulation that may be a point of dysregulation in dopamine imbalance disorders.

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 co-expressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of twelve 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.

The dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT, respectively) are plasma membrane proteins belonging to the SLC6 family of secondary active transporters and are responsible for clearance of their corresponding substrates dopamine (DA), norepinephrine (NE) and serotonin (5‐HT) from the extraneuronal space during neurotransmission. Each monoamine controls distinct behavioral and physiological functions in the nervous system. DA controls motor functions, mood, reward and cognition. NE regulates arousal, mood, attention, stress‐responsiveness and 5‐HT modulates mood, aggression, motivation, appetite, sleep, cognition and sexual function. Extraneuronal monoamine levels are controlled spatially and temporally by transporter mediated reuptake of released transmitter into presynaptic neurons. Abnormalities in transmitter levels and subsequent neurotransmission are linked to neurological disorders including major depression, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Tourette syndrome, and Parkinson disease, through mechanisms that are incompletely understood. The transporters are sites of action for therapeutic drugs such as methylphenidate, bupropion, selective serotonin and serotonin‐norepinephrine reuptake inhibitors, used to treat these disorders, and are also targets for addictive drugs including cocaine, amphetamine (AMPH), and methylenedioxy methamphetamine (MDMA) that elevate transmitter levels. Several lines of evidence indicate that modulation of transporter phosphorylation, ubiquitylation, and palmitoylation regulate their cell surface expression, activity, trafficking and degradation. We previously reported that rat, mouse and human DATs are modified by S‐palmitoylation, a post‐translational modification in which C16 saturated palmitic acid is added via a thioester linkage to cysteine. These results indicated that DAT palmitoylation has the capacity to impact dopaminergic signaling acutely by regulating DA transport kinetics independent of surface losses and chronically by opposing DAT degradation. We have extended our examination of transporter palmitoylation using acyl‐biotinyl exchange to include SERT and NET finding both transporters are palmitoylated proteins, suggesting potential palmitoylation regulatory mechanisms for NET and SERT analogous to DAT. Furthermore, treatment with the palmitoyl acyltransferase inhibitor 2‐bromopalmitate (2BP), in a heterologous cell system, resulted in decreased overall palmitoylation of these proteins. Inhibition of palmitoylation by 2BP also revealed acute and long‐term differences between SERT and NET stability where loss of palmitoylation is accompanied by a loss of total transporter protein. Collectively, these results indicate that S‐palmitoylation may serve as a potential regulator for NET and SERT properties, sharing some functional consequences as demonstrated with DAT.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A major regulator of synaptic dopaminergic tone is the dopamine transporter (DAT), a presynaptic integral membrane protein whose function is reuptake of dopamine (DA) into the neuron after vesicular neurotransmitter release. DAT is the target of therapeutic and abused psychoactive drugs, and DAT dysfunction is hypothesized to contribute to neurological disorders like attention deficit hyperactive disorder, addiction, and Parkinson disease. DAT function is tightly modulated through binding partners and posttranslational modifications including phosphorylation, glycosylation, and, of importance here, S‐palmitoylation – the labile, enzymatic modification of cysteine sulfhydryl side groups with the fatty acid palmitate, generating a thioester linkage. S‐palmitoylation conveys protein‐specific functions, which can include protein oligomerization membrane microdomain localization, protein maturation, and alleviating transmembrane domain hydrophobic mismatch. DAT palmitoylation acutely increases DA uptake Vmax without altering DAT surface expression and opposes long‐term DAT degradation. As the enzymatic processes of S‐palmitoylation occurs on intracellularly accessible cysteines, we generated cysteine to alanine mutants at each of the intracellular rat DAT cysteine residues (6, 135, 341, 522, and 580). Palmitoylation of each mutant DAT was probed by [3H]palmitic acid metabolic labeling and the acyl‐biotinyl exchange (ABE) method which revealed a 50% loss of labeling only in the C580A mutant. The remaining palmitoylation signal implied the presence of one or more palmitoylation sites. Subsequently, palmitoylation of mutant rDATs possessing a single intracellular Cys residue was assessed. Cys580 and Cys6 displayed similar and significantly increased palmitoylation levels relative to Cys135, Cys341, and Cys522, and mutant rDATs containing both Cys6 and Cys580 had near WT palmitoylation levels indicating Cys6 is a second palmitoylation site. To determine if endogenous palmitoylation is occurring on Cys6, DAT palmitoylation in rat striatal tissue was assessed by ABE after proteolytic or chemical digestion using Asp‐N or CNBr respectively to generate immunospecific N‐terminal DAT fragments. Asp‐N proteolysis releases a peptide containing Cys6 and Cys135 while CNBr digestion releases a peptide that contains Cys6 but not Cys135, and both fragments are detected by an N‐terminal monoclonal DAT antibody. In these experiments, N‐terminal palmitoylated DAT fragments were produced using both digestion methods, strongly suggesting endogenous palmitoylation of rDAT at Cys6 consistent with our mutagenesis results. Additionally, we are assessing transporter surface expression and functional changes incurred by mutation of this novel rDAT palmitoylation site, Cys6. Palmitoylation of this residue may invoke tethering of the cytosolic N‐terminus of DAT to the plasma membrane affecting DAT function and regulation. We are therefore probing for Cys6 palmitoylation mediated tethering of the N‐terminus to the plasma membrane. Interestingly, Cys6 is adjacent to Ser7, a known PKC‐mediated phosphorylation site, and palmitoylation of Cys6 may sterically inhibit phosphorylation of Ser7 and play a significant role in DAT regulation.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research ResourcesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.