Date of Award

3-28-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Roxanne A. Vaughan

Abstract

The dopamine transporter (DAT) is an integral membrane protein that clears dopamine (DA) from the synaptic space following neurotransmission. DATs amino acid sequence contains numerous serine, threonine and tyrosine residues, some of which are located in protein kinase consensus sites. While DAT phosphorylation and regulation in response to protein kinase C activation is well documented, evidence suggests that multiple signals and interacting proteins also mediate these processes. Previous research has also demonstrated that DAT regulation is influenced by the presence of the DAT substrates amphetamine (AMPH), methamphetamine (METH) and the uptake blocker cocaine. Using 32PO4 metabolic labeling and [3H]DA uptake assays we analyzed DAT phosphorylation and functional regulation in DAT expressing cells and rat striatal tissue in response to AMPH, METH and cocaine. The application of AMPH or METH increased DAT phosphorylation in both DAT expressing cells and rat striatal tissue and decreased [3H]DA uptake in DAT expressing cells. In contrast, cocaine had no significant effect on DAT phosphorylation or [3H]DA uptake. The finding that the amphetamines can induce DAT phosphorylation demonstrates a previously unknown effect for these drugs which may affect DAT regulation after drug clearance. Recently DAT has been demonstrated to interact with syntaxin 1A. However, the effect of this interaction on the phosphorylation and regulation of DAT has not been reported. To examine the effect of this interaction on DAT we treated rat striatal tissue with botulinum toxin C (BoNT/C), which specifically cleaves syntaxin 1A, and expressed the light chain of BoNT/C (BoNT/C LC) in rDAT PC12 cells expressing syntaxin 1A. DAT phosphorylation in rat striatal tissue was decreased by BoNT/C and [3H]DA uptake in rat striatal tissue and rDAT PC12 cells was increased by applying BoNT/C or expressing BoNT/C LC. In a complementary manner, [3H]DA uptake was decreased by expressing syntaxin 1A in rDAT LLC-PK1 cells which do not endogenously express syntaxin 1A. These results demonstrate a functional interaction between DAT and syntaxin 1A that promotes DATs basal phosphorylation and inhibits DA uptake. Combined with the amphetamine results, these data demonstrate that DAT phosphorylation and regulation can be modulated by protein interactions and endogenously occurring events.

Download

Share

COinS