Date of Award

January 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Roxanne A. Vaughan

Abstract

The dopamine transporter (DAT) is an integral membrane protein that reuptakes dopamine (DA) from the extracellular space into the presynaptic neuron. DAT regulates dopaminergic neurotransmission as it maintains homeostatic synaptic DA levels in the brain. Psychostimulants such as cocaine disrupt DA homeostasis as it binds to DAT and prevents reuptake of DA. Excess DA in the synapse leads to prolonged dopaminergic neurotransmission, which is associated with cocaine related euphoria often leading to addiction.

The DAT consists of 12 transmembrane domains (TMs) with N- and C-termini facing the cytoplasm. TMs 1, 3, 6, and 8 make up the core of the protein and the residues from these domains are involved in binding substrates and inhibitors. Although the effect of cocaine binding to DAT is known, the mechanism of DAT-cocaine interaction at the molecular level is still unknown. Therefore, to elucidate how cocaine binds to the DAT and how it is positioned in the binding pocket, we mapped the attachment site of the irreversible binding cocaine analogs, [125I]MFZ 2-24, [125I]RTI 82, and [125I]JHC 2-48. These compounds share a similar cocaine-based core structure, but have analog specific photo activatable side chains that extend from different regions of the cocaine core structure. Upon ultraviolet light activation, the photo activatable phenyl N3 (azido) group forms phenyl nitrene that becomes covalently attached to a residue on the protein, hence irreversible binding cocaine analogs.

Previous studies narrowed the [125I]RTI 82 adduction site to the region surrounding TM6, between Ile291 and Arg344 on human DAT and between Met290 and Lys336 on rat DAT. The [125I]MFZ 2-24 attachment site was localized between residues Ile67 and Leu80 in TM1. To identify the specific amino acid attachment site of these analogs we created several methionine substitution mutants across TMs 1 and 6. This resulted in generation of custom cyanogen bromide (CNBr) cleavage sites. The results from peptide maps of photoaffinity labeled mutants proteolyzed with CNBr narrowed the adduction of [125I]MFZ 2-24 to Asp79 or Leu80 in TM1 and the adduction of [125I]RTI 82 to Phe320 in TM6. Trypsin and CNBr proteolysis of [125I]JHC 2-48 labeled rat DAT indicated a ligand attachment site C-terminal to TM6. Incorporation of three structural analogs to three distinct TM domains demonstrates that the appended azido groups on these analogs identify different faces of the ligand binding pocket. Thus, allowing for triangulation of cocaine orientation in its binding site via computational modeling.

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