![]() ![]() Although amphetamines are less lipophilic (for example, Log P of amphetamine=1.41) 15, they have been hypothesized both to enter vesicles by diffusion as well as by VMAT-mediated transport 2. This is the mechanism of action for weak bases such as chloroquine, which is lipophilic enough to diffuse across lipid bilayers (Log P=4.72) 11, 12, 13, 14. Amphetamines are weak bases (pK a 8.8–9.9) and have been proposed to decrease the vesicular ΔpH by buffering luminal free protons, thereby depleting vesicles of stored dopamine and indirectly blocking substrate import. This ΔpH, generated by the vacuolar H +-ATPase (V-ATPase), not only drives loading of monoamines into the vesicle lumen by VMAT, but also promotes intravesicular retention of monoamines through their protonation 11. A third proposed mechanism is that amphetamines deplete synaptic vesicle dopamine stores through mechanisms akin to lipophilic weak bases and protonophores 2, 10, which degrade the vesicular pH gradient (ΔpH). Others have inferred that amphetamines are substrates of VMAT that drive carrier-mediated exchange of vesicular monoamines into the cytoplasm 8, 9. ![]() Whether amphetamines also act directly on VMAT to redistribute dopamine from vesicles into the cytoplasm has been debated, and numerous mechanisms have been proposed 6.Īmphetamines interact with VMAT in vitro, leading some investigators to conclude that they act as non-substrate inhibitors that elevate cytoplasmic dopamine by simply blocking its accumulation into vesicles and thus making more available for efflux 7. Dopamine is synthesized in the cytosol and concentrated into synaptic vesicles ∼10 5-fold ( ∼0.1 M intraluminal dopamine) relative to cytoplasmic dopamine ( ∼1 μM) by the vesicular monoamine transporter (VMAT) 3, 4, 5. How amphetamines mobilize dopamine from vesicles to the cytoplasm for subsequent efflux is less clear. Amphetamines’ psychostimulant effects are generally thought to result from increased extracellular dopamine mediated by efflux of cytoplasmic dopamine through the dopamine transporter (DAT) 2. Prescribed and illicit amphetamines, including amphetamine and methamphetamine derivatives, are some of the most widely used and abused drugs: Total prescriptions number over 15 million yearly in the US, with ∼56 million users globally 1. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. This amphetamine-induced deacidification requires VMAT function and results from net H + antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. 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. To study VMAT’s role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. ![]()
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