PHOSPHORYLATION AND REGULATION OF DOPAMINE TRANSPORTER

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The dopamine transporter (DAT) is the primary mechanism which clears extracellular dopamine from the synaptic space. As such, it performs a key role in terminating synaptic transmission and in regulating the concentration of dopamine available for binding to pre- and post-synaptic dopamine receptors. It has recently been discovered that activation of protein kinase C leads to phosphorylation of DATs and to concomitant reductions in dopamine transport, suggesting that DATs undergo functional regulation by phosphorylation. This would provide the neuron with a mechanism for fine temporal and spatial control of extracellular dopamine concentrations, and subsequent downstream dopaminergic neural activity. This property of DAT therefore has the potential to profoundly influence normal dopaminergic neurophysiology, and may also be related to mechanisms of abuse of cocaine or other drugs and dopaminergic neurodegeneration. This study proposes to thoroughly characterize DAT phosphorylation properties and define the relationship between DAT phosphorylation and functional regulation. The specific aims designed to achieve these goals are: 1. Identify sites of PKC-stimulated phosphorylation on DATs. 2. Construct mutants with phosphorylation sites changed to non-phosphate acceptors, and examine the functional consequences of these mutations. 3. Thoroughly characterize phosphorylation and dephosphorylation properties of DAT by identifying the specific kinases and phosphatases which act on the protein. 4. Test for related changes in transport of dopamine, binding of antagonists, and surface expression. 5. Identify the endogenous pathways responsible for in vivo control of DAT phosphorylation.