Kinetics of the dopamine transporter in Drosophila larva

Dopamine transporters (DAT) regulate neurotransmission and are important in diseases such as addiction and attention deficit hyperactivity disorder. The Drosophila dopamine transporter (dDAT) is analogous to the mammalian DAT, but Michaelis-Menten kinetic parameters have not been characterized in vivo. In this study, dopamine clearance kinetics were measured in a Drosophila larval CNS using an implanted carbon-fiber microelectrode and fast-scan cyclic voltammetry. Dopamine was pressure ejected from a micropipet implanted 15-20 μm from the microelectrode. Clearance of exogenously applied dopamine was significantly reduced in dDAT null (fumin) mutants, and kinetic constants in these mutants were used to determine clearance by other mechanisms including diffusion. After correction for diffusion, the maximal rate of uptake, Vmax, was estimated to be 0.11 ± 0.02 μM/s and Km was 1.3 ± 0.6 μM in wild-type flies. The clearance rate was significantly reduced following treatment with the DAT inhibitor cocaine in wild-type flies, but not in fumin mutants, which indicates that serotonin transporter is not contributing significantly to dopamine clearance in these larvae. Clearance of endogenous dopamine, evoked by optical stimulation in flies expressing Channelrhodopsin2, was similar to clearance of exogenous dopamine, but it was not possible to evoke concentrations that were close to saturation. The ability to quickly assess the role of the dopamine transporter in any Drosophila larva will be useful for future studies of how transporters regulate neurotransmission and to understand the underlying mechanisms of drug addiction.