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+

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 Cd2+ showing substrate saturation with a Michaelis-Menten constant (K-m) of 54 +/- 5.8 mu M. Uptake of Cd2+ 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 Cd2+ 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 Ca2+ and Me. Only confluent HEK-hOCT2-C transported Cd2+, and confluent and dissociated cells exhibited different potencies for inhibition of uptake of 1-methyl-4-phenylpyridinium(+) (MPP+) by Cd2+, MPP+, tetraethylammonium(+), cimetidine(+), and corticosterone. In confluent HEK-hOCT2-C, largely different inhibitor potencies were obtained upon comparison of inhibition of Cd2+ uptake, 4-[4-(dimethylamino)styryl]N-methylpyridiniurn(+) (ASP(+)) uptake, and MPP+ uptake using substrate concentrations far below the respective K-m values. Employing a point mutation in the previously identified substrate binding site of rat Octl 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.