In human proximal tubules, organic cations are taken up from blood into cells by human organic cation transporter 2 [hOCT2/ solute carrier (SLC) 22A2] and then eliminated into the lumen by apical H ϩ /organic cation antiporters, human multidrug and toxin extrusion 1 (hMATE1/SLC47A1) and hMATE2-K (SLC47A2). To evaluate drug interactions of cationic drugs in the secretion process, epithelial cells engineered to express both hOCT2 and hMATE transporters are required to simultaneously evaluate drug interactions with renal basolateral and apical organic cation transporters. In the present study, therefore, we assessed the drug interaction between cimetidine and metformin with double-trans- ]metformin from the basolateral side, suggesting that cimetidine at a low concentration inhibits apical hMATE1, rather than basolateral hOCT2. Actually, in concentration-dependent inhibition studies by a single transporter expression system, such as human embryonic kidney 293 stably expressing hMATE1, hMATE2-K, or hOCT2, cimetidine showed higher affinity for hMATEs than for hOCT2. These results suggest that apical hMATE1 is involved in drug interactions between cimetidine and cationic compounds in the proximal tubular epithelial cells.
Recently, we have isolated the rat (r) H(+)/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H(+) gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [(14)C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [(14)C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H(+) gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [(14)C]tetraethylammonium uptake by an H(+) gradient [intravesicular H(+) concentration ([H(+)](in)) > extravesicular H(+) concentration ([H(+)](out))] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [(14)C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H(+) gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.
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