Understanding Addiction, Depression, and Autism Spectrum Disorder through Structure-Function Analyses of the Dopamine and Serotonin Transporters

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 CaMKIIalpha- 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).