title: Probing the substrate specificity of the ergothioneine transporter with methimazole, hercynine, and organic cations.
abstract: Recently, we have identified the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases. ETT was originally designated a multispecific novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68micfrol/(minmgprotein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil (K(i)=11micromol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole (K(i)=7.5mmol/l) was 130-fold lower, and transport of hercynine (K(i)=1.4mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.
authors.affiliation: Department of Pharmacology, University of Cologne, Gleueler Strasse 24, 50931 Cologne, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Grigat
authors.forename: Stephanie
authors.initials: S
authors.lastname: Harlfinger
authors.forename: Sonia
authors.initials: S
authors.lastname: Pal
authors.forename: Ralph
authors.initials: R
authors.lastname: Striebinger
authors.forename: Stefan
authors.initials: S
authors.lastname: Golz
authors.forename: Andreas
authors.initials: A
authors.lastname: Geerts
authors.forename: Andreas
authors.initials: A
authors.lastname: Lazar
authors.forename: Edgar
authors.initials: E
authors.lastname: Schömig
authors.forename: Dirk
authors.initials: D
authors.lastname: Gründemann
mesh_terms: D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism; D001692:Biological Transport; D002478:Cells, Cultured; D004880:Ergothioneine / Q000378:metabolism*; D006639:Histidine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008713:Methimazole / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D013379:Substrate Specificity; D027981:Symporters
chemical_list: D027701:Organic Cation Transport Proteins; D012333:RNA, Messenger; C109807:SLC22A4 protein, human; D027981:Symporters; D001622:Betaine; D006639:Histidine; D008713:Methimazole; D004880:Ergothioneine; C007928:hercynine