Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential

Abstract The present study addresses the effect of intracellular Na + and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT‐expressing cells with gramicidin diminished DA uptake and decreased the potency (increases K i ) of DA in inhibiting the binding of cocaine analog [ 3 H]2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane (CFT). It also compromised the ability of external Na + to reduce DA K i . No substantial effect on DA K i was observed upon gramicidin treatment in Na + ‐free buffer, membrane depolarization with high [K + ] o , or elevation of [Na + ] i with monensin under non‐depolarizing conditions. Elevation of DA K i was greater at more positive potentials when [Na + ] i was raised to a similar level, or at higher [Na + ] i when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA K i was significantly higher but less sensitive to gramicidin than that in wild‐type (WT) cells. In contrast, DA K i in cell‐free membranes was insensitive to Na + , gramicidin, and D313N mutation. The data suggest that (i) intracellular Na + plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell‐free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na + and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA.