The Dopamine Transporter (DAT) is a trans-membrane protein that is part of the SLC6 gene family. Protein Kinase C (PKC) has been shown to acutely down regulate DAT via membrane trafficking away from the plasma membrane. The present study was performed to determine the role of PKCβ, a subtype of PKC, in DAT trafficking. Dopamine uptake assays and cell surface protein biotinylations were performed to access the effect of PKCβ’s inhibition. Wild Type DAT function and surface expression were not sensitive to PKCβ inhibition. In contrast, mutating DAT residues 587-589 (DAT 3A) revealed striking PKCβ sensitivity, resulting in significantly increased DAT function and surface expression following 20-minute treatment with the PKCβ inhibitor LY 379196. PC12 cells were subsequently discovered to be PKCβ negative. PC12 cells were then cotransfected with PKCβ and DAT 3A to reproduce the increase in activity seen in SK-NMC cells, but no statistically relevant increase in DA uptake was seen. The fact that the increase in activity was not reproducible in PC12 cells points to an intermediary molecule present in SK-N-MC cells that is needed for the effect. Further experimentation is needed to fully characterize this effect with DAT 3A, including, but not limited to, dopamine internalization and recycling assays.

• This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

Creator

• King, Michael J

Publisher

• Worcester Polytechnic Institute

Identifier

• E-project-042710-175247

Advisor

• Adams, David S.

Year

• 2010

Sponsor

• UMass Medical School

Date created

• 2010-04-27

Resource type

• Major Qualifying Project

Major

• Biochemistry

Rights statement

• In Copyright