Madhur Shetty

Date of Award

January 2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

First Advisor

James D. Foster

Abstract

The dopaminergic system is a regulatory system of the brain controlling various motor, cognitive and behavioral activities. Neurotransmitter dopamine (DA) plays an important role in modulating brain circuits controlling functions of this system with dopamine transporter (DAT) maintaining its homeostasis. Abnormalities in this homeostasis and/or nucleotide polymorphisms in the DAT structure leads to its association with a wide range of neurological and neuropsychiatric disorders, with attention-deficit hyperactivity disorder (ADHD) being one of them.

Disrupting the normal function of DAT, many psychostimulants such as amphetamine (AMPH) upon being transported via DAT into the pre-synaptic neuron alters DAT properties triggering N-terminal DAT phosphorylation associated DA efflux.

Recently identified, ADHD associated human DAT (hDAT) single nucleotide polymorphism (SNP), A559V, displayed anomalous DA efflux (ADE). Our results showed A559V hDAT and its rat homologue, A558V rat DAT (rDAT), displaying AMPH independent increased phosphorylation, including T53 site (human equivalent being S53), a proline directed phosphorylation site specific for rDAT, further unaffected by AMPH, which may support ADE observed for this polymorphism. These SNPs also showed reciprocal decreased palmitoylation status. With these modifications impacting DAT properties, we found phosphorylation driven increased membrane raft localization for A559V hDAT and other palmitoylation deficient mutants. These membrane rafts serve as a site for localization of phosphorylated DAT and a platform for DA efflux. These mutants also showed increased lateral membrane mobility, which was reciprocally decreased for phosphorylation deficient mutants (T53A rDAT, S7A hDAT and S53A hDAT) which have increased palmitoylation.

Further, studies confirmed C581 to be a palmitoylation site in humans, with C581A hDAT being a palmitoylation deficient mutant having elevated phosphorylation with membrane microdomain and mobility properties similar to A559V hDAT.

For A559V hDAT, the close proximity of Val substitution to the palmitoylation site could cause a structural alteration in DAT transmembrane spanning domain (TMD) 12 helical structure. The bulkier substitution may mechanistically hinder C581 palmitoylation causing its movement away from DAT core region, lose of flexibility, altered DAT membrane localization, mobility and function. We believe other phosphorylation or palmitoylation deficient mutants might show similar unknown mechanisms, reciprocally regulating post-translational modifications.

With palmitoylation helping in membrane raft partitioning, stabilization of membrane anchoring and integral membrane protein interaction, we demonstrate palmitoylation to be a factor for DAT membrane mobility. We believe the palmitate group affects DAT’s interaction with binding partners and cholesterol and that its deficiency leads to increased lateral membrane mobility. This palmitoylation status could be the driving force for phosphorylation driven increased localization of phosphorylated DAT in membrane raft microdomains, leading to increased interaction with binding partners, serving as a platform for phosphorylation-dependent DA efflux either by AMPH-stimulation or by polymorphism.

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