Date of Award

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

The dopamine transporter (DAT) is a neuronal presynaptic transmembrane protein that clears released dopamine (DA) from the synaptic space, regulating the neurotransmitter concentration and availability, DAT and the related serotonin (SERT) and norepinephrine (NET) transporters belong to the SLC6 family of Na+ and CI- dependent symporters, and are major targets for the action of several drugs, including the psychostimulant cocaine. DAT is predicted to possess 12 transmembrane spanning domains (TMs), with both N- and C-termini located intracellularly. Extensive research efforts to gain insight on the molecular aspects of DAT have been attempted, but the three-dimensional arrangement of the protein as well as the molecular mechanisms involved in substrate translocation remain undiscovered.

Cocaine and other structurally diverse compounds bind to DAT with high affinity and inhibit its transport activity, but their binding site within the protein and the mechanism by which they block DA transport remain unclear. Two distinct ligand interaction sites at transmembrane domains (TMs) 1-2 and 4-6 have been identified using irreversible uptake-blockers. The current studies explore the incorporation site of the novel cocaine analogue [125I]MFZ-2-24, which is structurally similar to the previously characterized [125 I]RTI 82, but the reactive azido (N3) group is differentially positioned within the cocaine pharmacophore.

Trypsin and cyanogen bromide (CNBr) proteolytic maps of [125I]MFZ-2-24 labeled rat and human DATs, followed by epitope-specific immunoprecipitation were used to localize the incorporation site of the ligand to a 13 amino acid stretch in TM1, between residues I67 and L80. This highly conserved region harbors the functionally essential D79 and residues involved in substrate and inhibitor binding. In marked contrast, incorporation of [125I]RTI 82 occurs in TM6, demonstrating that differential positioning of the N 3 group on the cocaine pharmacophore leads to distinct incorporation patterns. This further indicates that TMs 1 and 6 are in dose proximity three dimensionally and participate in the reversible binding of cocaine to DAT.

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