The overall goal of this post-doctoral fellowship is to determine the effects of protein kinase C (PKC) on dopamine transporter (DAT) activity and trafficking. The proposed experiments will test the hypotheses that (1) PKC activation initiates internalization and/or inactivation of DAT at the membrane surface and (2) modulation of DAT function by PKC occurs via a network of proteins. PKC activation attenuates the function of the human DAT (hDAT) expressed in Xenopus oocytes. First, we will further characterize the effects of PKC on hDAT function ([3H]-DA uptake and DAT- associated currents) and then examine PKC's effects on hDAT subcellular localization (biotinylation/immunoblotting). Next, we will study PKC-induced modulation of hDAT activity/trafficking in oocytes co-expressing hDAT and candidate proteins. We will then selectively inactivate these proteins to determine if they are necessary for PKC-mediated regulation. Finally, we will attempt to identify essential structural motifs of hDAT involved in PKC- mediated regulation. A leucine heptad repeat exists in hDAT that may be involved in protein-protein interactions. Thus, we will study PKC regulation in a mutated hDAT in which the leucine heptad repeat has been removed. These experiments will enhance our understanding of mechanisms by which hDAT, a critical component in dopaminergic neurotransmission, can be regulated.
|Doolen, S; Zahniser, N R (2001) Protein tyrosine kinase inhibitors alter human dopamine transporter activity in Xenopus oocytes. J Pharmacol Exp Ther 296:931-8|
|Zahniser, N R; Doolen, S (2001) Chronic and acute regulation of Na+/Cl- -dependent neurotransmitter transporters: drugs, substrates, presynaptic receptors, and signaling systems. Pharmacol Ther 92:21-55|