Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential.
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]2beta-carbomethoxy-3beta-(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.
Chen
Nianhang
N
Department of Psychiatry, New York University School of Medicine, New York, New York, USA. nianhang@med.nyu.edu
Reith
Maarten E A
ME
D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology; D008536:Meglumine / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology
D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D006096:Gramicidin; D008536:Meglumine; D008094:Lithium; D012964:Sodium; D011188:Potassium; D004298:Dopamine