title: Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations.
abstract: Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction.
authors.affiliation: Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, Illinois 61656, USA. nhc@uic.edu
authors.forename: Nianhang
authors.initials: N
authors.lastname: Chen
authors.forename: Judy
authors.initials: J
authors.lastname: Rickey
authors.forename: Maarten E A
authors.initials: ME
authors.lastname: Reith
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism* / Q000493:pharmacokinetics; D050483:Dopamine Plasma Membrane Transport Proteins; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; 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 / Q000494:pharmacology*; D013395:Sucrose / Q000494:pharmacology
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D013395:Sucrose; D008536:Meglumine; D012964:Sodium; D003042:Cocaine; D011188:Potassium; D004298:Dopamine