Abstract
The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. In addition to DA release and availability of synaptic DA receptors, timing and magnitude of DA neurotransmission depend on extracellular DA-level regulation by the dopamine transporter (DAT), the membrane expression and trafficking of which are highly dynamic. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane. Broadly, these findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, thus having an impact on the capacity for DAT to regulate extracellular DA levels.
Highlights
The dopaminergic system is essential for cognitive processes, including reward, attention and motor control
To determine whether AMPHinduced dopamine transporter (DAT) internalization was the result of a psychostimulantspecific action or may be, in part, due to activation of voltagesensitive mechanisms, we performed live cell total internal reflection fluorescence microscopy (TIRFM) of yellow fluorescent protein-tagged DAT (YPF-DAT) in Human Embryonic Kidney (HEK) cells when perfused with only extracellular solution, 10 mM AMPH or 100 mM KCl (Fig. 1), which depolarized cells by 13.5 and 35.7 mV, respectively (Fig. 1e)
The presence of DAT at the cell membrane is crucial in the regulation of DAergic signalling, timing and magnitude throughout the brain, and any alteration in the functional capacity of the transporter may significantly have an impact on neurological functions in which DA is involved
Summary
The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane These findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, having an impact on the capacity for DAT to regulate extracellular DA levels. Previous studies using striatal synaptosomes have revealed a reduction in DA uptake in depolarized (elevated KCl) conditions[30,31], while in vitro preparations have suggested elevated DAT function at hyperpolarized states[13] It is not known whether these changes in functional capacity arise from changes in ionic driving forces, essential for DA transport, changes in DAT protein presence at the membrane or both. We use confocal and total internal reflection fluorescence microscopy (TIRFM), biochemistry, electrophysiology and optogenetics to demonstrate the degree to which surfacemembrane DAT levels are shaped by and sensitive to MP changes
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