Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence.
We have constructed stable cell lines expressing transporters for dopamine (DA), norepinephrine (NE), and serotonin (5-HT) by transfection with cloned cDNAs. The parental LLC-PK1 cell does not express any of these neurotransmitter transporters. Therefore, monoamine transport activities in each of these cell lines are due to the transfected DNA only, allowing comparison in the same background. Drug inhibition profiles for each cell line are distinct and as expected for each transporter. LLC-NET and LLC-DAT cells transported both NE and DA and both cell types exhibited a lower KM for DA transport than for NE transport. Analysis of Vmax data for LLC-NET cells suggests that substrate is bound to the NE transporter during the rate-limiting step(s) in transport. The cocaine analog 2-beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binds to each cell type, and is displaced by transport substrate in each case. Binding and transport measurements on parallel cell cultures allowed estimation of turnover numbers for norepinephrine, dopamine, and serotonin transporters. All three transporters require external Na+ and Cl-. The Na+ concentration dependence suggests that a single Na+ ion is involved in transport catalyzed by norepinephrine and serotonin transporters while more than one Na+ ion participate in transport mediated by the dopamine transporter.
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Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Wall
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Rudnick
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D000818:Animals; D001665:Binding Sites; D001679:Biogenic Amines / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D027981:Symporters; D014162:Transfection
D001679:Biogenic Amines; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011994:Recombinant Proteins; C493227:SLC6A2 protein, human; C493214:SLC6A3 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493226:Slc6a2 protein, rat; C493216:Slc6a3 protein, rat; C493232:Slc6a4 protein, rat; D027981:Symporters