<Emphasis Type="Italic">N</Emphasis>-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter |
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Authors: | Birger Wenge Heinz Bönisch |
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Institution: | (1) Institute of Pharmacology and Toxicology, University of Bonn, Reuterstr. 2b, 53113 Bonn, Germany |
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Abstract: | 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 3H]NA uptake and 3H]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. |
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Keywords: | N-Ethylmaleimide Noradrenaline transporter ATP MPP+ Antidepressants |
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