Interaction of H⁺ with the extracellular and intracellular aspects of hMATE1

Human multidrug and toxin extrusion 1 (hMATE1, SLC47A1) is a major candidate for being the molecular identity of organic cation/proton (OC/H⁺) exchange activity in the luminal membrane of renal proximal tubules. Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake, i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction, i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, we measured the time course of hMATE1- mediated efflux of the prototypic MATE1 substrate, [3H]1-methyl-4- phenylpyridinium, under a variety of intra- and extracellular pH conditions, from Chinese hamster ovary cells that stably expressed the transporter. In this study, we showed that an IC50/Ki for interaction between extracellular H⁺ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] was not significantly different (P = 0.6). Furthermore, kinetics of interaction between intracellular H_ and inward-facing hMATE1 determined using the efflux protocol revealed an IC50 for H⁺ of 11.5 nM (pH 7.91), consistent with symmetrical interactions of H⁺ with the inward-facing and outward-facing aspects of hMATE1.