Dopamine efflux via wild-type and mutant dopamine transporters: Alanine substitution for proline-572 enhances efflux and reduces dependence on extracellular dopamine, sodium and chloride concentrations

The dopamine transporter (DAT) can mediate not only inward uptake of dopamine, but also its outward efflux by mechanisms that have been only partially elucidated. DAT-dependent dopamine efflux can be studied kinetically and apparent substrate affinity and V_max values determined. We now report that wild-type DAT displays apparent affinities for efflux more than 300-fold lower than those for uptake. Efflux rates are enhanced by increased extracellular concentrations of dopamine or amphetamine and by lowered extracellular concentrations of Na⁺ or Cl^-. Alanine substitutions for six proline residues located in or near DAT transmembrane domains increase apparent affinity and decrease V_max values for dopamine efflux mediated by these mutant transporters. Mutant 12P572A displays increased DAT efflux with reduced dependence on ion or dopamine concentrations. These data add to evidence for the specificity of transporter-mediated efflux processes and begin to elucidate DAT candidate domains that may be preferentially involved with efflux activities.