Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Roxanne Vaughan

Abstract

The dopamine transporter (DAT) is an integral membrane protein that mediates the reuptake of synaptic dopamine and thus regulates the spatio-temporal dynamics of dopaminergic neurotransmission. DAT is also the target of powerful addictive psychostimulants, including cocaine and methamphetamine that resulted in dysregulation of dopaminergic signaling. Complex control of DAT is exerted by various regulatory processes, including posttranslational modifications. We previously found that DAT is palmitoylated via the addition of a 16-carbon palmitic acid moiety to Cys 580. Pharmacological and mutagenic studies determined that depalmitoylation conditions led to reductions in Vmax and transporter levels, suggesting palmitoylation controls one or more functions of DAT. Palmitoylation is a dynamic and reversible process catalyzed by palmitoyl acyltransferases (PATs) and depalmitoylation is catalyzed by palmitoyl-protein thioesterases (PPTs). PAT enzymes have been further classified as DHHC enzymes based on a conserved cysteine rich domain and the active site sequence Asp-His-His-Cys. Currently, 23 PAT enzymes have been identified in the human genome with some associated with dopaminergic diseases such as schizophrenia; however, the enzymes that act on DAT are unknown. Therefore, we hypothesized that specific PAT enzymes will drive DAT palmitoylation, controlling expression, DA transport, and membrane lateral mobility of the transporter. To address this question, we co-expressed a specific subset of neuronally-expressed PATs individually with DAT and assessed DAT palmitoylation, total and surface expression, transport activity, and lateral membrane mobility. Additional studies were also conducted to investigate psychostimulant regulation of DAT palmitoylation.

Palmitoylation assessed by acyl-biotin exchange revealed that the neuronal PATs, DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17 increased DAT palmitoylation at Cys580, while several others had no effect, indicating a level of substrate specificity for actions against DAT. We observed a correlation between increased DAT palmitoylation and increased total DAT expression, consistent with a role for palmitoylation opposing DAT degradation. DAT palmitoylation also led to enhanced DA uptake with no effect on surface levels, suggesting that palmitoylation also increases transport capacity via an alteration of DAT transport kinetics. Our results also indicated that DAT palmitoylation increased the time for recovery after photobleaching, suggesting that increased palmitoylation decreases the membrane lateral mobility of the transporter. Lastly, we observed treatment with methamphetamine but not cocaine resulted in decreased DAT palmitoylation in both heterologous cells and in animal models, suggesting that DAT palmitoylation is regulated by psychostimulants. Palmitoylation thus plays an important role in both short- and long-term regulation of DAT by controlling multiple functions. Therefore, this modification could potentially affect neurotransmitter clearance in dopaminergic disorders where palmitoylation may be impacted.

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