Roxanne A. Vaughan, Ph.D.
Project: Dopamine Transporter Regulation.
Mentor: Roxanne A. Vaughan, Ph.D.
Professor, Department of Biomedical Sciences
Research Project: The dopamine transporter, DAT, is a synaptic protein that drives reuptake of dopamine from the synapse into the presynaptic neuron, and is the major mechanism for regulation of DA neurotransmission. DAT activity is regulated by phosphorylation of multiple serine and threonine residues, driven by multiple protein kinases, but many major questions remain as to the mechanisms underlying the responses. Regulatory mechanisms linked to phosphorylation are induced not only by exogenous activators of Protein Kinase C (PKC), but also by amphetamine and methamphetamine, indicating the involvement of these abused drugs in endogenous regulatory mechanisms. More recently we have extended our studies to regulatory processes of the synthetic cathinones methcathinone (MCAT) and methylenedioxypyrovalerone (MDPV). These psychoactive and addictive compounds are of major concern in the drug abuse community, but their mechanisms of action are currently poorly understood.
Preliminary work in our lab has shown that pretreatment of DAT-expressing cells with MCAT and MDPV induce DAT regulatory responses and affect phosphorylation of DAT residue Threonine (T)53. We have generated a phospho-specific antibody against T53 for analysis of this site, and our proposed project for the SURF student will be to characterize this phosphorylation process for dose and time courses of responses to MCAT and MDPV, using previously characterized responses to amphetamine or PKC as controls. As time permits, the student will determine levels DAT transport activity in parallel conditions and assess DAT regulatory responses in a phosphorylation-null T53A mutant. The results will indicate if MCAT and MDPV exert physiological regulatory dysfunction in DAT as part of their overall actions.
This project is suitable for execution by an undergraduate student in that the constructs and assay conditions have been worked out, and the questions to be answered are scientifically important. The study will introduce a student to many pharmacological principles related to DAT and drug abuse, as well as to basic scientific principles in experimental methodology.
Techniques: The SURF student will learn DAT SDS-PAGE and pan/phosphospecific immunoblotting, DAT uptake assays and pharmacological principles, cell culture and transfection, statistical analysis, and hypothesis testing. The student will also be given exposure to DAT literature, and the chance to write up research findings for posters or papers.
Student Independence: The SURF student will have independence to execute daily experiments, with assistance as needed, and to analyze results for discussion with Dr. Vaughan. The student will be able to suggest experimental directions as findings dictate and formulate plans for execution of ideas.
