Date of Award

6-12-2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Roxanne A. Vaughn

Abstract

The dopamine transporter (DAT) is a transmembrane spanning protein responsible for the clearance of dopamine from the dopaminergic synapse. Uptake blockers such as cocaine inhibit transport resulting in elevated levels of dopamine in the synapse. DAT is predicted to contain twelve transmembrane spanning domains (TMs) connected by alternating intra- and extracellular loops, however, the spatial arrangement of these regions is unknown. Extracellular loop 2 (EL2) is composed of approximately 80 amino acids and connects TM3 and TM4, two regions associated with uptake blocker binding. Using limited proteolysis we analyzed the aqueous accessibility of EL2 in the presence or absence of DAT uptake blockers. Epitope-specific immunoblot analysis and deglycosylation experiments indicate that trypsin and endoproteinase Asp-N cleaved rat DAT (rDAT) at Arg218 and Asp174 in EL2. Binding of uptake blockers, but not substrates reduced protease sensitivity 100-1000 fold. Uptake blocker-induced protease resistance is stereospecific, Na+ dependent, and occurs at nanomolar ligand concentrations indicating that this change in protease sensitivity is the result of uptake blocker binding. To examine the functional role of EL2, proteolyzed rDAT was analyzed for binding using the cocaine analog, [3H]2β-carbomethoxy-3β-(4-fluorophenyl) tropane ([3H]CFT), and [3H]dopamine transport activity. Both binding and transport activity decreased relative to the extent of digestion, indicating that the integrity of EL2 is required for protein function. To refine our examination we used the substituted cysteine accessibility method (SCAM) to assess the aqueous accessibility and function of EL2. Using a human DAT (hDAT) mutant that is resistant to the thiol-reactive compound, 2-(Trimethyl-ammonium) ethyl methanethio-sulfonate (MTSET), the residues flanking R219 (Y216-L229) were replaced in DAT EL2 one at a time with cysteines. Reaction of MTSET with residues E218C-H223C decreased DAT binding and transport activity 15-30% confirming their aqueous accessibility and importance for DAT function. Coincubation of the R219C mutant with cocaine and MTSET blocked the MTSET-induced reduction in binding, supporting our hypothesis that uptake blockers affect the aqueous accessibility of this region. Together with the protease studies, these data are consistent with uptake blockers inducing conformational changes that significantly alter the hydrophilic accessibility of EL2.

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