Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter.

Dopamine transporter (DAT) has been shown to accumulate in filopodia in neurons and non-neuronal cells. To examine the mechanisms of DAT filopodial targeting, we used quantitative live-cell fluorescence microscopy, and compared the effects of the DAT inhibitor cocaine and its fluorescent analog JHC1-64 on the plasma membrane distribution of wild-type DAT and two non-functional DAT mutants, R60A and W63A, that do not accumulate in filopodia. W63A did not bind JHC1-64, whereas R60A did, although less efficiently compared to the wild-type DAT. Molecular dynamics simulations predicted that R60A preferentially assumes an outward-facing (OF) conformation through compensatory intracellular salt bridge formation, which in turn favors binding of cocaine. Imaging analysis showed that JHC1-64-bound R60A mutant predominantly localized in filopodia, whereas free R60A molecules were evenly distributed within the plasma membrane. Cocaine binding significantly increased the density of R60A, but not that of W63A, in filopodia. Further, zinc binding, known to stabilize the OF state, also increased R60A concentration in filopodia. Finally, amphetamine, that is thought to disrupt DAT OF conformation, reduced the concentration of wild-type DAT in filopodia. Altogether, these data indicate that OF conformation is required for the efficient targeting of DAT to, and accumulation in, filopodia.