title: Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1.
abstract: 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.
authors.affiliation: Dept. of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tsuda
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Terada
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Asaka
authors.forename: Miki
authors.initials: M
authors.lastname: Ueba
authors.forename: Toshiya
authors.initials: T
authors.lastname: Katsura
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Inui
mesh_terms: D000818:Animals; D017920:Antiporters / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000502:physiology*; D027701:Organic Cation Transport Proteins / Q000502:physiology*; D051381:Rats; D013757:Tetraethylammonium Compounds / Q000378:metabolism
chemical_list: D017920:Antiporters; D027701:Organic Cation Transport Proteins; C515734:Slc47a1 protein, rat; D013757:Tetraethylammonium Compounds; D006859:Hydrogen