Subtype-Specific Affinity for Corticosterone of Rat Organic Cation Transporters rOCT1 and rOCT2 Depends on Three Amino Acids within the Substrate Binding Region

Abstract

The affinity of corticosterone to organic cation transporters (OCTs) is subtype-and species-dependent. For example, the IC 50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are ϳ150 and ϳ4 M, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane ␣ helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K m values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC 50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC 50 value for the inhibition of . This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.

Substrate-Dependent Inhibition of Human MATE1 by Cationic Ionic Liquids

“…Similarly, Zolk et al (2009) reported that a set of structurally diverse cationic drugs were markedly more effective inhibitors of OCT2-mediated metformin transport than of MPP transport. These observations with OCTs, and those presented here for the interaction of selected ILs with the MATEs, support the view that ligand interactions with multidrug transporters can include close-order (competitive) interactions and distant (allosteric) interactions (Gorboulev et al, 2005). We suggest that rather than having a single unique binding site, the MATEs have a binding surface that permits spatially distinct interactions with structurally distinct ligands.…”

“…MPP is a comparatively amphiphilic, planar, heterocyclic ring compound. Given the characteristic multiselectivity of MATEs (Tanihara et al, 2007) and the potential of xenobiotic transporters to display kinetically complex interactions with substrates and inhibitory ligands (e.g., Gorboulev et al, 2005;Harper and Wright, 2013), we elected to establish the kinetics of MATE1-mediated transport of two structurally dissimilar OCs, namely, the tetra-alkylammonium compound TEMA and the fluorescent substrate NBD-MTMA (Fig. 1).…”

“…This substrate-dependent inhibitor interaction is inconsistent with a purely competitive model of substrate-inhibitor interaction (Segel, 1975). Instead, this behavior was reminiscent of corticosterone's inhibition of transport mediated by selected site-directed mutants of rOct1, which displayed IC 50 values for MPP versus TEA that differed by 5-fold (Gorboulev et al, 2005). Similarly, Zolk et al (2009) reported that a set of structurally diverse cationic drugs were markedly more effective inhibitors of OCT2-mediated metformin transport than of MPP transport.…”

“…We suggest that rather than having a single unique binding site, the MATEs have a binding surface that permits spatially distinct interactions with structurally distinct ligands. Similar ideas have been invoked to explain kinetically complex substrate-inhibitor interactions in other efflux transporters (e.g., the prokaryotic multidrug transporter, BmrR) (Vazquez-Laslop et al, 2000) and with the organic cation transporters (OCTs) (Gorboulev et al, 2005), providing a potential mechanistic basis for the polyselectivity of xenobiotic transporters. With respect to these latter ideas, it is interesting to note that the central mass of NBuPy (and MPP) is a six-membered ring (i.e., pyridinium), whereas Bmim and BmPy include smaller, five-membered rings (imidazolium and pyrrolidinium, respectively).…”

Charge-to-substrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine

“…It has been shown first that OCTs are able to mediate sodiumindependent electrogenic transport of organic cations in both directions across the plasma membrane (3, 17); second, that a nontransported competitive inhibitor can inhibit rat OCT2 (rOCT2) from either side of the plasma membrane, suggesting inwardly and outwardly facing orientations of the substrate binding site (31); third, that rOCT1 contains high-and lowaffinity binding sites for individual organic cations (11); and fourth, that rOCT2 mediates electrogenic uptake of the inorganic cation Cs ϩ with high selectivity over Na ϩ and K ϩ (24). Mutagenesis identified seven amino acids in rOCT1 that are involved in substrate binding (9,10,21). In a three-dimensional model of rOCT1 based on the structure of the inwardfaced conformation of LacY permease (1) these amino acids could be located within a large inward-open cleft (21).…”