Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, following their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We find that the protein Flotillin-1/Reggie-2 (Flot1) is required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we have identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 is also required to localize DAT within plasma membrane microdomains in stable cell lines, and is essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.