Defense Date

7-20-2006

Graduation Date

Summer 2006

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Pharmacology-Toxicology

School

School of Pharmacy

Committee Chair

Christopher K. Surratt

Committee Member

David A. Johnson

Committee Member

Wilson S. Meng

Keywords

abuse, addiction, affinity, dopamine, dopamine transporter, potency

Abstract

Cocaine and other dopamine transporter (DAT) inhibitors block synaptic dopamine uptake by binding to the DAT. Cocaine, a powerful psychostimulant, potentiates the dopamine mediated signal transduction in the reward areas of the brain resulting in physical dependence on the drug. Amphetamines are psychotomimetics that act as substrates at the DAT and potentiate dopaminergic neurotransmission leading to addiction. It was previously reported that a conservative glutamate-for-aspartate substitution in transmembrane 1 domain of the rat DAT protein (D79E) significantly decreased the binding affinities of several classical DAT inhibitors, but had little or no effect on the dopamine uptake inhibition potencies (DUIPs) of these blockers. This finding implied that different DAT sites/conformations/populations may be responsible for dopamine uptake and for high affinity binding of inhibitors. In the present study, the DUIPs of cocaine and other DAT ligands at wildtype DAT CHO cells fluctuated as a function of cell age, while their binding affinities remained static. Curiously, neither the DUIP nor the binding affinity of amphetamine fluctuated with cell age. The DUIP fluctuations of cocaine also extended to the N2A neuronal cells, demonstrating its physiological relevance. The loss of correlation between DUIP and binding affinity at DAT might be a unique property of DAT blockers that is not exhibited by DAT substrates. A plausible explanation for the DUIP fluctuations with cell age is the existence of two different DAT populations on the cell surface, the relative distribution of which changes with cell age under the influence of intracellular events such as DAT-protein interactions or alterations in the phosphorylation state. Several proteins interact with the N- and C-terminal regions of the DAT. Studies employing GFP fusion polypeptides of the DAT N- or C-terminal fragments, which would act as decoys by intercepting DAT modulators, showed that the DAT N- or C-terminal interactions with intracellular proteins do not appear to be responsible for the DUIP shift. The role of phosphorylation state changes of DAT in DUIP fluctuations was also investigated. Studies employing a mutant DAT lacking the first 20 N-terminal amino acids demonstrated that N-terminal phosphorylation of DAT, specifically involving the first five serines is not associated with the DUIP shift.

Format

PDF

Language

English

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