title: Substrate- and cell contact-dependent inhibitor affinity of human organic cation transporter 2: studies with two classical organic cation substrates and the novel substrate cd2+.
abstract: Polyspecific organic cation transporter Oct2 from rat (gene Slc22A2) has been previously shown to transport Cs(+). Here we report that human OCT2 (hOCT2) is able to transport Cd(2+) showing substrate saturation with a Michaelis-Menten constant (Km) of 54 ± 5.8 μM. Uptake of Cd(2+) by hOCT2 was inhibited by typical hOCT2 ligands (unlabeled substrates and inhibitors), and the rate of uptake was decreased by a point mutation in a substrate binding domain of hOCT2. Incubation of hOCT2 overexpressing human embryonic kidney 293 cells (HEK-hOCT2-C) or rat renal proximal tubule cells expressing rOct2 (NRK-52E-C) with Cd(2+) resulted in an increased level of apoptosis that was reduced by OCT2 inhibitory ligand cimetidine(+). HEK-hOCT2-C exhibited different functional properties when they were confluent or had been dissociated by removal of Ca(2+) and Mg(2+). Only confluent HEK-hOCT2-C transported Cd(2+), and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP(+)) by Cd(2+), MPP(+), tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd(2+) uptake, 4-[4-(dimethylamino)styryl]-N-methylpyridinium(+) (ASP(+)) uptake, and MPP(+) uptake using substrate concentrations far below the respective Km values. Employing a point mutation in the previously identified substrate binding site of rat Oct1 produced evidence that short distance allosteric effects between binding sites for substrates and inhibitors are involved in substrate-dependent inhibitor potency. Substrate-dependent inhibitor affinity is probably a common property of OCTs. To predict interactions between drugs that are transported by OCTs and inhibitory drugs, it is necessary to employ the specific transported drug rather than a model substrate for in vitro measurements.
authors.affiliation: Institute of Physiology and Pathophysiology, ZBAF, University of Witten/Herdecke , Witten, Germany. frank.thevenod@uni-wh.de
authors.forename: Frank
authors.initials: F
authors.lastname: Thévenod
authors.forename: Giuliano
authors.initials: G
authors.lastname: Ciarimboli
authors.forename: Marcus
authors.initials: M
authors.lastname: Leistner
authors.forename: Natascha A
authors.initials: NA
authors.lastname: Wolff
authors.forename: Wing-Kee
authors.initials: WK
authors.lastname: Lee
authors.forename: Irina
authors.initials: I
authors.lastname: Schatz
authors.forename: Thorsten
authors.initials: T
authors.lastname: Keller
authors.forename: Rouvier
authors.initials: R
authors.lastname: Al-Monajjed
authors.forename: Valentin
authors.initials: V
authors.lastname: Gorboulev
authors.forename: Hermann
authors.initials: H
authors.lastname: Koepsell
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002104:Cadmium / Q000378:metabolism* / Q000494:pharmacology; D050482:Catecholamine Plasma Membrane Transport Proteins / Q000378:metabolism; D002460:Cell Line; D002927:Cimetidine / Q000494:pharmacology; D003345:Corticosterone / Q000494:pharmacology; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000075102:Organic Cation Transporter 2; D051381:Rats; D019789:Tetraethylammonium / Q000494:pharmacology
chemical_list: D050482:Catecholamine Plasma Membrane Transport Proteins; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; C491013:SLC22A2 protein, human; C493212:Slc22a1 protein, rat; D002104:Cadmium; D019789:Tetraethylammonium; D002927:Cimetidine; D003345:Corticosterone