N-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter.
Using transfected HEK293 cells that express the human (h) noradrenaline transporter (hNAT), we show differential inhibitory effects of the thiol reagent N-ethylmaleimide (NEM) on [(3)H]NA uptake and [(3)H]nisoxetine binding. Irreversible inhibition of uptake by NEM was complete, faster, and occurred at lower concentrations. Furthermore, hNAT ligands (substrates and inhibitors) prevented NEM-induced inhibition of binding but not that of uptake, indicating different underlying mechanisms of inhibition. NEM-induced uptake inhibition was not primarily due to inhibition of the Na(+)/K(+)-ATPase since ouabain caused only partial inhibition. For the first time, we show that NEM at low concentrations causes a rapid and complete depletion of cellular adenosine triphosphate (ATP) not only in HEK293 cells but also in several other eukaryotic cell lines. Thus, while high NEM concentrations alkylate the NAT protein in a ligand-protectable manner, low concentrations inhibit substrate uptake through a loss of the Na(+) and K(+) gradient as a driving force by depleting cellular ATP.
Wenge
Birger
B
Institute of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, 53113 Bonn, Germany.
Bönisch
Heinz
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005033:Ethylmaleimide / Q000008:administration & dosage / Q000494:pharmacology*; D005473:Fluoxetine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000187:drug effects* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000008:administration & dosage / Q000494:pharmacology*; D014162:Transfection
D008024:Ligands; D050484:Norepinephrine Plasma Membrane Transport Proteins; D013439:Sulfhydryl Reagents; D005473:Fluoxetine; C011386:nisoxetine; D000255:Adenosine Triphosphate; D012964:Sodium; D005033:Ethylmaleimide; D011188:Potassium; D009638:Norepinephrine