Human multidrug and toxin extrusion 1 (hMATE1) 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 (RPT). 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, I measured the time course of hMATE1-mediated efflux of the prototypic MATE1-substrate, [3H]1-methyl-4-phenylpyridinium ([3H]MPP), under a variety of conditions, including different values for intra- and extracellular pH, from CHO cells that stably expressed hMATE1. I 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] were not significantly different (P = 0.6). To test a hypothesis that H+ interacts symmetrically with each face of hMATE1, kinetics of interaction between intracellular H+ and inward-facing hMATE1 were determined using the efflux protocol. The IC50 for interaction with H+ was 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1. The efflux protocols demonstrated in this study are a potential means to examine kinetics at cytoplasmic face of hMATE1 and also a practical tool to screen uptake of substrates at extracellular face of hMATE1.

O rim é tanto um órgão-alvo como a principal fonte de produção de ET-1, peptídio que regula a excreção de Na+ e água por este órgão que expressa os seus respectivos receptores, ETA e ETB, além dos trocadores NHE1 e NHE3 que são essenciais para o equilíbrio ácido base e hidroeletrolítico das células. Assim, o objetivo deste estudo foi investigar, em células IRPTC, o papel de ET-1 na atividade dos trocadores NHE1 e NHE3. Nossos resultados indicam que o tratamento agudo com ET-1 (10-9 M) aumenta a velocidade de recuperação do pHi (dpHi/dt) nos dois primeiros minutos após o pulso ácido, sugerindo aumento na atividade dos trocadores NHE1 e NHE3, que ocorre via ativação dos receptores ETA e ETB e parece ser secundária à atividade da p90RSk e p38MAPK. O tratamento crônico com ET-1 (10-9 M) reduz a dpHi/dt nos dois primeiros minutos após o pulso ácido, o que sugere redução na atividade dos trocadores NHE1 e NHE3, que pode ser secundária à inibição da Na+, K+-ATPase por ET-1. / The kidney is both a target organ and the main source of ET-1 production, a peptide that regulates the excretion of Na+ and water by this organ that expresses its respective receptors, ETA and ETB, in addition to the NHE1 and NHE3 exchanger which are Essential for the basic acid and electrolyte balance of cells. Thus, the objective of this study was to investigate, in IRPTC cells, the role of ET-1 in the activity of the NHE1 and NHE3 exchanger. Our results indicate that the acute treatment with ET-1 (10-9 M) increases the rate of recovery of pHi (dpHi/dt) in the first two minutes after the acid pulse, suggesting an increase in the activity of the NHE1 and NHE3 exchanger, which occurs via activation Of ETA and ETB receptors and appears to be secondary to the activity of p90RSk and p38MAPK. Chronic treatment with ET-1 (10-9 M) reduces dpHi/dt in the first two minutes after the acid pulse, suggesting a reduction in NHE1 and NHE3 exchanger activity, which may be secondary to inhibition of Na+, K+- ATPase by ET-1.

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