Date of Award

January 2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

L. Keith Henry

Abstract

The dopamine (DAT) and serotonin (SERT) transporters are monoamine neurotransmitter transporters (MATs) responsible for the reuptake of dopamine (DA) or serotonin (5-HT) from the synapse following vesicular release, effectively regulating synaptic neurotransmission. Blockade of these transporters by antagonists such as psychostimulant drugs or transporter mutations that affect function can compromise DA or 5-HT homeostasis and impact fundamental brain processes including movement, emotion, behavior, motivation, and memory. To address these issues, my dissertation research focused on: (1) identifying and characterizing the cocaine binding site in DAT, (2) determining the influence of membrane depolarization on DAT trafficking, (3) investigating antidepressant metabolite action in an antidepressant-insensitive mouse model, and (4) understanding the link between the structural and functional changes induced by mutations in SERT associated with autism spectrum disorder.

Cocaine binding site in DAT – Through the development and utilization of several high affinity, photoactivatable cocaine ligands we identified a cocaine binding site in the core of DAT, a site that overlaps with the putative DA binding site, supporting a competitive mechanism for cocaine inhibition of DA uptake. (Chapters II-IV)

Influence of membrane depolarization on DAT trafficking – Trafficking of mature DAT to and from the cell membrane is a highly regulated process. Within this process, we demonstrated that membrane depolarization alone could induce a CaMKIIα- and dynamin-dependent rapid reversible reduction in membrane DAT. (Chapter V)

Characterization of antidepressant metabolites in the blockade of 5-HT reuptake – As studies of the antidepressant selective serotonin reuptake inhibitors (SSRIs) have revealed discrepancies between acute and chronic dosing treatments, we evaluated the sensitivity of SSRI metabolites in a mouse model of depression and identified a role for metabolites in antidepressant administration that may confound study conclusions. (Chapter VI)

Autism-associated SERT mutations – Previously, several rare SERT coding variants were identified in humans with autism spectrum disorder that augment 5-HT transport function. We studied the structure of these variants and discovered alterations in SERT tertiary structure, which likely impact the catalytic activity or surface expression of SERT. (Chapter VII)

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