Dopamine transporter affects cellular localization of α‐synuclein

Abstract

The dysregulation of dopamine transporter (DAT) and over‐expression of α‐synuclein have been implicated in Parkinson's disease. We and others have shown a direct interaction between DAT and α‐synuclein. The objective of this study is to elucidate the localization of this interaction and to examine whether α‐synuclein over‐expression may affect the viability of dopaminergic neurons. We employed complementary approaches to examine the location of this interaction and how it may alter the activity of either protein in two model systems: heterologous expression system over‐expressing fluorescently labeled DAT and α‐synuclein as well as primary culture of mice DA neurons. α‐synuclein over‐expression in DAT cells decreases DAT‐mediated substrate uptake. Dialysis of α‐synuclein into DA neurons produced a DAT‐mediated, Cl‐dependent depolarizing current. To examine the locale of DAT/α‐synuclein interaction we used biochemistry and microscopy approaches including fluorescence recovery after photo‐bleaching (FRAP) and Forster resonance energy transfer (FRET). Biotinylation and FRET results suggest a direct DAT/α‐synuclein interaction at the plasma membrane. FRAP results suggest a transient interaction between the two proteins as indicated by a decrease in only α‐synuclein mobility. Ongoing experiments examine whether excess α‐synuclein influences the biology of dopaminergic neurons.