Defense Date

12-9-2005

Graduation Date

Fall 2005

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Pharmacology-Toxicology

School

School of Pharmacy

Committee Chair

Christopher K. Surratt

Committee Member

Paula A. Witt-Enderby

Committee Member

David A. Johnson

Committee Member

Wilson S. Meng

Committee Member

John A. Pollock

Keywords

dopamine transporter, structure function studies

Abstract

A long-standing postulate holds that cocaine inhibits DAT-mediated dopamine transport via competition with dopamine for formation of an ionic bond with the DAT transmembrane 1 aspartic acid residue 79. A recent study from our laboratory indicated that mutation of this aspartate to glutamate (D79E) had little or no effect on dopamine affinity or dopamine uptake inhibition potencies for WIN35,428 and cocaine, and decreased WIN35,428 affinity by only 3 fold (Wang et al., 2003). The study cast doubt on the requirement of a dopamine-D79 ion pair, but did not address whether the residue plays a role in recognizing the cocaine pharmacophore. In the present study, DAT inhibitors containing variations of three primary components of this pharmacophore- the positively charged tropane nitrogen atom, the seven-carbon tropane ring itself, and the aromatic substituent at the tropane C-3 position- were assessed for binding affinity and dopamine uptake inhibition at the same D79 DAT mutants. Only inhibitors with modifications of the phenyl ring substituent of cocaine, i.e. benztropine and its analogs, displayed considerably altered dopamine uptake inhibition potency as a function of the D79E mutation. These observations may suggest that the side chain of the D79 residue is important for the recognition of the aromatic components of DAT ligands.

Furthermore, we investigated the influence of cell passage number, the density of the cell monolayer and the effect of varying DAT expression levels by manipulation of transfection conditions on DAT function. It was observed that the DUIPs of cocaine, mazindol, methylphenidate, and benztropine fluctuated as a function of DAT-CHO cell passage number. The binding affinities of these DAT inhibitors, however, remained static. Also, the DUIP of cocaine fluctuated as a result of variations in DAT cell surface expression. It is therefore conceivable that an unidentified cellular mediator modulates DAT inhibitor DUIP but not binding affinity at the DAT protein.

Format

PDF

Language

English

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