The projects described in this dissertation revolve around the functional consequences experienced by the transport protein for the neurotransmitter dopamine resulting from alterations to its palmitoylation condition or exposure to Parkinson disease-inducing transport substrates. This membrane-resident transport protein, pragmatically termed the dopamine transporter, relocates dopamine from extracellular areas of receptor sites of action to intracellular sequestration; the dopamine transporter is a powerful mediator of dopamine signaling. As such, genetic, toxicant, or chronic breakdown of dopamine transporter function is associated with multiple psychological abnormalities. Biomedical research has produced several pharmacotherapies for maladies like depression, attention deficit/hyperactivity disorder, and addiction, some of which produce their therapeutic profiles by modulating dopamine transporter function. The potential for pharmacological manipulation of the dopamine transporter is, however, not without its dark side. Dopamine’s critical role in the neurotransmission of reward and pleasure render the dopamine transporter a favorite target of illicit, addictive drugs of abuse like cocaine and several flavors of amphetamine. All of these factors make intimate understanding of the many mechanisms involved in the dopamine transporter’s function relevant not only to mental, but also societal health. This dissertation explores aspects of dopamine transporter mechanistic regulation which, once more thoroughly understood, may be modified to allow finer control over transporter operation, generating novel approaches to mental health treatment.
The first study investigates site identification and functional characterization of post-translational modification of the dopamine transporter by a lipid moiety, palmitic acid. Palmitic acid, hexadecanoic acid in IUPAC nomenclature, is a saturated 16 carbon fatty acid whose attachment to proteins is termed S-palmitoylation. This lipidation process is executed by an array of enzymes belonging to the acyl transferase class of the gene name zDHHC. Importantly, S-palmitoylation is reversible: a protein’s palmitoylation status can change in response to cell stimuli or the palmitoylated protein’s activation. As palmitate is of an aliphatic nature, its attachment creates a hydrophobic protein microenvironment around the site of its augmentation which propitiates its insertion into likewise hydrophobic loci – usually membranes – which induces a protein-specific functional outcome. A combination of dopamine transporter proteolysis, site-directed mutagenesis, acyl-biotinyl exchange, surface biotinylation, and forward and reverse dopamine transport assays implicate two N-terminal cysteine residues as sites of palmitate incorporation, in addition to the previously analyzed C-terminal site, and reveal a role for this lipid modification in dopamine transporter-mediated dopamine efflux.
The second project seeks to further understand the dopamine transporter’s contribution to Parkinson disease. The hallmark of this disease is a loss of motor coordination precipitated by selective death of nigro-striatal dopamine neurons and concomitant depletion of dopamine neurotransmission in the movement planning and execution region of the brain – the striatum. The selective loss of these neurons directly correlates with dopamine transporter expression; indeed, even amongst dopamine neuronal pathways, the nigro-striatal fiber, which is lost to the greatest extent of these, has the highest transporter expression. It is for this reason the dopamine transporter has been a focus of Parkinson disease research. This study utilizes the dopamine transporter substrates 6-hydroxydopamine and 1-methyl-4-phenylpyridinium, which induce cell death through a panoply of biochemical mechanisms and are used to generate Parkinsonian symptoms in animal models, to probe for aberrant dopamine transporter function and post-translational modification. This inquiry revealed that, though these compounds induce cell death through similar mechanisms, their dopamine transporter-specific effects are quite different. Interestingly, 1-methyl-4-phenylpyridinim is a strong inducer of dopamine uptake downregulation and dopamine efflux, a phenomenon now implicated in Parkinson disease onset, while 6-hydroxydopamine mitigates this efflux event as well as attenuates transporter phosphorylation.
Overall, this dissertation argues for the existence of N-terminal palmitoylation of the dopamine transporter, that palmitoylation is an additional contributor to the dopamine efflux paradigm, that transporter-mediated efflux may contribute to Parkinson disease onset, and that some of the transporter-specific effects of 6-hydroxydopamine may be exploited to alleviate neuropsychiatric maladies associated with aberrant dopamine efflux
