Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and selfadministration without impacting cocaine-induced behaviours. To study VMAT’s role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H þ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

Our data lead to a model for how pharmacologically relevant concentrations of amphetamines increase extracellular dopamine: (1) DAT imports and concentrates amphetamines in the cytoplasm. (2) Cytoplasmic amphetamines (and endogenous dopamine) are accumulated into vesicles by VMAT in a H þ – antiport process that diminishes vesicular H þ concentration. (3) Diminished vesicular DpH promotes dopamine redistribution from vesicles into the cytoplasm through a mechanism that is still unclear. Vesicle deacidification alters the protonation state of luminal dopamine, which might be sufficient to increase its diffusion across the membrane. However, this mechanism does not readily explain protonophore-induced efflux of MPP þ from vesicles64, since this compound is a quaternary ammonium and thus pH cannot alter MPP þ ’s protonation state. Whether VMAT itself might be a route of dopamine release from vesicles11,65 requires further study. (4) The redistributed cytoplasmic dopamine subsequently effluxes out of the cell through DAT via amphetamine-stimulated reverse transport. Our previous work demonstrated that phosphorylation of DAT is essential for dopamine efflux66 and for locomotor behaviour induced by amphetamine but not for the actions of methylphenidate, a competitive non-substrate inhibitor of DAT20,21. These results are consistent with the inference from our rodent behavioural data that competitive inhibition at DAT by amphetamines is not sufficient to produce behavioural effects at the concentrations tested. By the same logic, stimulation of dopamine synthesis by TH and/or inhibition of dopamine catabolism by monoamine oxidases, both of which can elevate cytoplasmic dopamine concentrations6, are insufficient in themselves to produce amphetamines’ acute behavioural effects. These data highlight the functional coupling of DAT and VMAT as critical to amphetamines’ actions in vivo. Because amphetamines are also substrates of norepinephrine transporter and serotonin transporter, and VMAT is present in adrenergic and serotonergic neurons6, the tandem actions of plasma membrane transporters and VMAT are likely important for amphetamine-induced release of other monoamines as well. Furthermore, our results demonstrate the first application of a novel experimental system that can be used to develop important new insights into the physiology of intact monoaminergic vesicles.

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Nat Commun. 2016 Feb 16;7:10652. doi: 10.1038/ncomms10652.