Oppositely directed H+ gradient functions as a driving

force of rat H +/organic cation antiporter MATE1

Masahiro Tsuda, Tomohiro Terada, Jun-ichi Asaka, Miki Ueba, Toshiya Katsura
and Ken-ichi Inui
Am J Physiol Renal Physiol 292:F593-F598, 2007. First published 17 October 2006;
doi:10.1152/ajprenal.00312.2006

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Oppositely directed H⫹ gradient functions as a driving force of rat
H⫹/organic cation antiporter MATE1
Masahiro Tsuda, Tomohiro Terada, Jun-ichi Asaka, Miki Ueba, Toshiya Katsura, and Ken-ichi Inui
Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto, Japan
Submitted 9 August 2006; accepted in final form 14 October 2006
Tsuda M, Terada T, Asaka J-i, Ueba M, Katsura T, Inui K-i.
Oppositely directed H⫹ gradient functions as a driving force of rat
H⫹/organic cation antiporter MATE1. Am J Physiol Renal Physiol
292: F593–F598, 2007. First published October 17, 2006;
doi:10.1152/ajprenal.00312.2006.—Recently, we have isolated the rat
(r) H⫹/organic cation antiporter multidrug and toxin extrusion 1
(MATE1) and reported its tissue distribution and transport character-
istics. Functional characterization suggested that an oppositely di-
rected 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 [14C]tet-
raethylammonium was stimulated in intracellular acidified HEK-
rMATE1 cells but not mock cells. The transport of [14C]tetraethyl-
ammonium in membrane vesicles from HEK-rMATE1 cells exhibited
the overshoot phenomenon only when there was an outwardly di-
rected 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 [14C]tetraethylammonium uptake by
an H⫹ gradient [intravesicular H⫹ concentration ([H⫹]in) ⬎ extrave-
sicular H⫹ concentration ([H⫹]out)] was significantly reduced in the
presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphe-
nylhydrazone (FCCP). [14C]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.
multidrug and toxin extrusion 1; transporter; tetraethylammonium;
renal secretion; membrane vesicles
THE SECRETION OF DRUGS AND xenobiotics is an important phys-
iological function of the renal proximal tubules. Cationic drugs
are secreted from blood to urine by cooperative functions of
two distinct classes of organic cation transporters: one driven
by the transmembrane potential difference in the basolateral
membranes and the other driven by the transmembrane H⫹
gradient in the brush-border membranes (7, 16). So far, several
membrane potential-dependent organic cation transporters
(OCT1–3) have been identified, and their physiological and
pharmacokinetic roles have been evaluated (2, 5, 10). How-
ever, the molecular nature of H⫹/organic cation antiport sys-
tems has remained to be elucidated.
Address for reprint requests and other correspondence: K. Inui, Dept. of
Pharmacy, Kyoto Univ. Hospital, Sakyo-ku, Kyoto 606-8507, Japan (e-mail:
inui@kuhp.kyoto-u.ac.jp).
http://www.ajprenal.org 0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society
Am J Physiol Renal Physiol 61: F593–F598, 2007.
First published October 17, 2006; doi:10.1152/ajprenal.00312.2006.
Recently, Moriyama and co-workers (3, 15) have identified
human (h) and mouse MATE1 and MATE2, which are or-
thologs of the multidrug and toxin extrusion (MATE) family of
bacteria. They demonstrated that MATE1 was predominantly
expressed at the luminal membranes of the urinary tubules and
bile canaliculi and transported tetraethylammonium, a proto-
typical organic cation, in a pH-dependent manner (3, 15). We
also isolated cDNAs for rat (r) MATE1 (20) and the human
kidney-specific isoform MATE2-K (13). rMATE1 was signif-
Downloaded from ajprenal.physiology.org on March 30, 2011
icantly expressed in the kidney and placenta, but not in the
liver, and real-time PCR analyses of microdissected nephron
segments showed that rMATE1 was expressed in the proximal
convoluted and straight tubules (20). On the other hand,
hMATE2-K was only expressed in the kidney and was located
at the brush-border membranes of renal proximal tubular cells
(13). By conducting functional analyses, we showed that
rMATE1 and hMATE2-K can transport a wide variety of
organic cations including tetraethylammonium, N1-methylni-
cotinamide, and metformin (13, 20). These characteristics of
MATE1 are similar to those of the H⫹/organic cation antiport
system revealed by renal brush-border membrane vesicle stud-
ies (4, 14, 18, 19, 23).
MATE1 exhibited pH-dependent transport of tetraethylam-
monium in cellular uptake and efflux studies, and intracellular
acidification by NH4Cl pretreatment stimulated tetraethylam-
monium transport (3, 13, 15, 20), suggesting that MATE1
utilized an oppositely directed H⫹ gradient as a driving force.
However, these analyses are not enough to prove the H⫹/
tetraethylammonium antiport mechanism of MATE1, because
it is possible that the pH-dependent transport of tetraethylam-
monium by MATE1 is regulated not by an H⫹ gradient but by
pH itself. Accordingly, in addition to the data obtained using
the cell culture model, we need more direct evidence that an
H⫹ gradient is the driving force for MATE1.
In the present study, we developed HEK293 cells stably
expressing rMATE1 (HEK-rMATE1 cells) and elucidated the
driving force of rMATE1 by uptake studies using plasma
membrane vesicles from HEK-rMATE1 cells for the first time.
MATERIALS AND METHODS
Materials. [14C]levofloxacin (1.07 GBq/mmol) was kindly pro-
vided by Daiichi Pharmaceutical (Tokyo, Japan). [14C]tetraethylam-
monium bromide (2.035 GBq/mmol), [14C]creatinine (2.035 GBq/
mmol), [14C]procainamide (2.035 GBq/mmol), [3H]quinidine (740
GBq/mmol), [3H]quinine (740 GBq/mmol), L-[N-methyl-3H]carnitine
(3.145 TBq/mmol), and [N-methyl-14C]nicotine (2.035 GBq/mmol)
were obtained from American Radiolabeled Chemicals (St. Louis,
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
F593
F594 DRIVING FORCE OF TETRAETHYLAMMONIUM TRANSPORT BY rMATE1
MO). [14C]metformin (962 MBq/mmol), [14C]guanidine hydro-
chloride (1.961 GBq/mmol), [8-3H]acyclovir (110 GBq/mmol),
and [8-3H]ganciclovir (370 GBq/mmol) were purchased from
Moravec Biochemicals (Brea, CA). [3H]1-methyl-4-phenylpyri-
dinium acetate (2.7 TBq/mmol), [3H]estrone sulfate ammonium
salt (2.1 TBq/mmol), and [14C]p-aminohippurate (1.9 GBq/mmol)
were purchased PerkinElmer Life Analytical Sciences (Boton,
MA). [N-methyl-3H]cimetidine (451 GBq/mmol) was obtained
from Amersham Biosciences (Uppsala, Sweden). All other chem-
icals used were of the highest purity available.
Cell culture and transfection. HEK293 cells (American Type Culture
Collection CRL-1573) were cultured in complete medium consisting of
Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum in an
atmosphere of 5% CO2-95% air at 37°C. pcDNA 3.1 (⫹) containing
cDNA encoding rMATE1 or empty vector was transfected into HEK293
cells using LipofectAMINE 2000 Reagent (Invitrogen) according to the
manufacturer’s instructions. At 48 h after transfection, the cells were split
in complete medium containing G418 (0.5 mg/ml, Nacalai Tesque,
Kyoto, Japan) at a dilution of 1:200. Fifteen days after transfection,
single colonies were picked out. Cells expressing rMATE1 (HEK-
rMATE1 cells) were selected by measuring [14C]tetraethylammo-
nium uptake. Cells transfected with empty vector (HEK-pcDNA
cells) were used as controls. These transfectants were maintained
in complete medium with G418 (0.5 mg/ml).
Uptake experiments by HEK-rMATE1 cells. The cellular uptake of
[14C]tetraethylammonium was measured by using monolayers grown
on poly-D-lysine-coated 24-well plates as reported previously with
some modifications (13, 20, 22). Briefly, the cells were preincubated
with 0.2 ml of incubation medium, pH 7.4 (in mM: 145 NaCl, 3 KCl,
1 CaCl2, 0.5 MgCl2, 5 D-glucose, and 5 HEPES) containing 30 mM
NH4Cl for 20 min at 37°C. The medium was then removed, and 0.2
ml of incubation medium (pH 7.4) containing each radiolabeled
compound was added. After an appropriate period of incubation, the
medium was aspirated, and the monolayers were rapidly washed twice
with 1 ml of ice-cold incubation medium (pH 7.4). The cells were
solubilized in 0.5 ml of 0.5 N NaOH, and then the radioactivity in
aliquots was determined by liquid scintillation counting. The protein
content of the solubilized cells was determined by the method of
Bradford (1) using a Bio-Rad Protein Assay Kit (Bio-Rad Laborato-
ries, Hercules, CA) with bovine ␥-globulin as a standard.
Preparation of membrane vesicles from HEK-rMATE1 cells.
Plasma membrane vesicles were prepared according to previous reports
(6, 9). HEK-rMATE1 or HEK-pcDNA cells were seeded on 100-mm
plastic dishes (4 ⫻ 106 cells/dish), and 20 or 40 dishes were used to
prepare membrane vesicles in a single preparation. All procedures were
performed at 4°C. At the third day after seeding, HEK-rMATE1 or
HEK-pcDNA cells were washed with PBS and scraped with a rubber
policeman into PBS. The cell suspension was centrifuged at 200 g for 10
min, suspended in 20 ml of PBS, and recentrifuged at 200 g for 10 min.
The packed cell pellet was resuspended in 20 vol of 250 mM mannitol/10
mM HEPES-Tris (pH 7.5)/0.5 mM MgCl2 (buffer A), and the cells were
gently suspended with five strokes of a loose-fitting Dounce homoge-
nizer. The washed cell suspension was placed in a nitrogen cavitation
bomb (Parr Instrument) at 700 lb/in.2 for 15 min. After the homogenate
was collected, K2EDTA (pH 7.5) was added to a final concentration of 1
mM. The homogenate was centrifuged at 750 g for 15 min, and the
supernatant was centrifuged at 20,000 g for 15 min. The supernatant was
centrifuged at 100,000 g for 60 min. The pellet was resuspended in 100
mM mannitol/10 mM MES-KOH (pH 6.0; experimental buffer) or 100
mM mannitol/10 mM HEPES-KOH (pH 7.5; experimental buffer)
and centrifuged again at 100,000 g for 60 min. The pellet was
suspended in the same experimental buffer (pH 6.0 or 7.5) by
sucking the suspension 10 times through a fine needle (⬃4 –10 mg
protein/ml). KCl (pH 6.0 or 7.5) was added to a final concentration
of 100 mM.
Transport experiments by membrane vesicles. The uptake of
[14C]tetraethylammonium by membrane vesicles was measured by a
AJP-Renal Physiol • VOL
rapid filtration technique with a slight modification (8, 19). In the
regular assays, the reaction was initiated rapidly by adding 80 ␮l of
buffer, containing 31.25 ␮M [14C]tetraethylammonium, to 20 ␮l of
membrane vesicle suspension at 25°C. After specified periods, the
incubation was terminated by diluting the reaction mixture with 1 ml
of ice-cold stop solution containing (in mM) 150 KCl, 20 HEPES-Tris
(pH 7.5), 0.1 HgCl2, and 1 tetraethylammonium. The mixture was
poured immediately onto Millipore filters (HAWP, 0.45 ␮m, 2.5 cm
in diameter), and the filters were washed with 5 ml of ice-cold stop
solution. The radioactivity of [14C]tetraethylammonium trapped in
membrane vesicles was determined using an ACS II (Amersham
Biosciences) by liquid scintillation counting. The protein content
was determined by the method of Bradford (1) using a Bio-Rad
Protein Assay Kit (Bio-Rad Laboratories) with bovine ␥-globulin
as a standard.
Western blot analysis. Polyclonal antibody was raised against a
synthetic peptide corresponding to the intracellular domain of
rMATE1 (CQQAQVHANLKVN, no. 465– 477) (13). Brush-border
membrane vesicles from rat kidney cortex were prepared as described
previously (12). Membrane fractions were separated by SDS-PAGE
and analyzed by Western blotting as described previously (17, 21).
Downloaded from ajprenal.physiology.org on March 30, 2011
Data analysis. Data were analyzed statistically with a one-way
analysis of variance followed by Scheffé’s test and are expressed as
means ⫾ SE.
RESULTS
Generation of HEK-rMATE1 cells. First, we generated and
characterized HEK293 cells stably expressing rMATE1. As
shown in Fig. 1, an immunoreactive protein with a molecular
weight of ⬃70 kDa was detected in HEK-rMATE1 cells and rat
renal brush-border membranes but not in HEK-pcDNA cells. The
functional expression of rMATE1 was assessed by measuring the
Fig. 1. Western blot analysis of rat renal brush-border membranes and plasma
membranes obtained from HEK-rat multidrug and toxin extrusion 1( rMATE1)
and HEK-pcDNA cells. Renal brush-border membranes (20 ␮g) and plasma
membranes (5 or 20 ␮g) obtained from HEK-rMATE1 and HEK-pcDNA cells
were separated by SDS-PAGE (10%) and blotted onto polyvinylidene difluo-
ride membranes. The antiserum for rMATE1 (1:1,000) was used as a primary
antibody. A horseradish peroxidase-conjugated anti-rabbit IgG antibody was
used for detection of bound antibodies, and the strips of blots were visualized
by chemiluminescence on X-ray film. The arrowhead indicates the position of
rMATE1. Lanes were as follows: lane 1, rat renal brush-border membranes;
lane 2, HEK-rMATE1 (5 ␮g); lane 3, HEK-rMATE1 (20 ␮g); lane 4,
HEK-pcDNA (5 ␮g); and lane 5, HEK-pcDNA (20 ␮g).
61 • FEBRUARY 2007 • www.ajprenal.org
 DRIVING FORCE OF TETRAETHYLAMMONIUM TRANSPORT BY rMATE1 F595
uptake of [14C]tetraethylammonium in the HEK-rMATE1 cells
under the intracellular acidified conditions caused by NH4Cl
pretreatment. A time- and concentration-dependent uptake of
[14C]tetraethylammonium by HEK-rMATE1 cells was observed
(Fig. 2, A and B). [14C]tetraethylammonium uptake by HEK-
rMATE1 cells exhibited saturable kinetics, and an apparent Km
value of 304 ⫾ 80 ␮M was calculated from three separate
experiments. When the extracellular pH was changed from 6.0 to
8.5, a bell-shaped pH profile of [14C]tetraethylammonium uptake
via rMATE1 was observed, and the uptake was greatest at pH 7.5
and lowest at pH 6.0 (Fig. 2C).
Uptake of various compounds by HEK-rMATE1 cells. We
then examined the substrate specificity of rMATE1. As shown in
Fig. 3, rMATE1 mediated the transport of various organic cations
with different chemical structures such as [14C]tetraethylammo-
nium, [3H]1-methyl-4-phenylpyridinium acetate, [3H]cimetidine,
and [14C]metformin. The transport of other organic cations such
as [14C]procainamide, [14C]creatinine, and [14C]guanidine was
greater in HEK-rMATE1 cells than in HEK-pcDNA cells, al-

Downloaded from ajprenal.physiology.org on March 30, 2011

though the stimulation was not remarkable.
Characteristics of [14C]tetraethylammonium transport by
membrane vesicles from HEK-rMATE1 cells. Next, we per-
formed transport experiments using plasma membrane vesicles
isolated from HEK-rMATE1 cells and HEK-pcDNA cells. In the
presence of an H⫹ gradient [intravesicular H⫹ concentration
([H⫹]in) ⬎ extravesicular H⫹ concentration ([H⫹]out)], a marked

Fig. 2. Transport of [14C]tetraethylammonium (TEA) by HEK-rMATE1

cells. A: time course of [14C]TEA uptake by HEK-rMATE1 and HEK-
pcDNA cells. HEK-rMATE1 cells (F) and HEK-pcDNA cells (E) were
preincubated with 30 mM NH4Cl (pH 7.4) for 20 min. Then, the preincu-
bation medium was removed, and the cells were incubated with 5 ␮M of
[14C]TEA (pH 7.4) for indicated time at 37°C. Each point represents the
mean ⫾ SE of 3 monolayers. This figure is representative of 3 separate
experiments. B: concentration dependence of [14C]TEA uptake by HEK-
rMATE1 cells. HEK-rMATE1 cells were preincubated with 30 mM NH4Cl
Fig. 3. Uptake of various compounds by HEK-rMATE1 cells. HEK-pcDNA
(pH 7.4) for 20 min. Then, the preincubation medium was removed, and the
cells (open bars) and HEK-rMATE1 cells (filled bars) were preincubated with
cells were incubated with various concentration of [14C]TEA (pH 7.4) in
30 mM NH4Cl (pH 7.4) for 20 min. Then, the preincubation medium was
the absence (F) or presence (E) of 5 mM TEA for 30 s at 37°C. Each point
removed, and the cells were incubated with [14C]TEA (5 ␮M), [3H]1-methyl-
represents the mean ⫾ SE of 3 monolayers. C: effect of extracellular pH on
4-phenylpyridinium acetate (3.8 nM), [3H]cimetidine (11.1 nM), [14C]met-
[14C]TEA uptake by HEK-rMATE1 and HEK-pcDNA cells. HEK-rMATE1
formin (10 ␮M), [14C]creatinine (5 ␮M), [14C]guanidine hydrochloride (5
cells (F) and HEK-pcDNA cells (E) were preincubated with 30 mM NH4Cl
␮M), [14C]procainamide (5 ␮M), [3H]quinidine (13.9 nM), [3H]quinine (13.9
(pH 7.4) for 20 min. Then, the preincubation medium was removed, and the
nM), [3H]carnitine (3.3 nM), [14C]nicotine (5 ␮M), [14C]levofloxacin (14
cells were incubated with 5 ␮M of [14C]TEA (indicated pH) for 30 s at
␮M), [3H]acyclovir (92 nM), [3H]ganciclovir (28 nM), [3H]estrone sulfate
37°C. Each point represents the mean ⫾ SE of 3 monolayers. The figure is
(4.86 nM), or [14C]p-aminohippurate (5 ␮M) for 30 s at 37°C. Each bar
representative of 2 separate experiments.
represents the mean ⫾ SE of 3 monolayers. The figure is representative of 2
separate experiments. *P ⬍ 0.05 significantly different from HEK-pcDNA
cells.

AJP-Renal Physiol • VOL 61 • FEBRUARY 2007 • www.ajprenal.org

F596 DRIVING FORCE OF TETRAETHYLAMMONIUM TRANSPORT BY rMATE1
Fig. 4. Time course of [14C]TEA uptake by membrane vesicles from HEK-
pcDNA and HEK-rMATE1 cells. The uptake of [14C]TEA by membrane
vesicles from HEK-pcDNA cells (E, ‚) and HEK-rMATE1 cells (F, Œ) was
examined in the absence (E, F) or presence (‚, Œ) of 10 mM TEA. Membrane
vesicles were prepared in the experimental buffer at pH 6.0. The uptake of
[14C]TEA was examined in the experimental buffer containing 31.25 ␮M
[14C]TEA and 100 mM KCl at pH 7.5 in the absence or presence of 10 mM
TEA. Each point represents the mean ⫾ SE of 3 determinations.
stimulation of [14C]tetraethylammonium uptake (overshoot phe-
nomenon) was observed in membrane vesicles from HEK-
rMATE1 cells, but not in those from HEK-pcDNA cells (Fig. 4).
The overshoot phenomenon disappeared in the presence of an
excess of cold tetraethylammonium.
Driving force for [14C]tetraethylammonium transport by
membrane vesicles from HEK-rMATE1 cells. To elucidate the
driving force of tetraethylammonium transport by rMATE1, we
Fig. 5. Effect of H⫹ gradient on [14C]TEA uptake by membrane vesicles from
HEK-rMATE1 cells. Membrane vesicles were prepared in the experimental
buffer at pH 6.0 (E, ‚) or 7.5 (F, Œ). The uptake of [14C]TEA was examined
in the experimental buffer containing 31.25 ␮M [14C]TEA and 100 mM KCl
at pH 6.0 (‚, Œ) or 7.5 (E, F). Each point represents the mean ⫾ SE of 3
determinations. The figure is representative of 2 separate experiments. pHin,
intravesicular pH; pHout, extravesicular pH.
AJP-Renal Physiol • VOL 61 • FEBRUARY 2007 •
performed [14C]tetraethylammonium transport experiments using
membrane vesicles from HEK-rMATE1 cells. As shown in Fig. 5,
the presence of an H⫹ gradient ([H⫹]in ⬎ [H⫹]out) induced a
marked stimulation of [14C]tetraethylammonium uptake against
the concentration gradient. On the other hand, no stimulation of
[14C]tetraethylammonium uptake was observed in the absence of
the gradient or in the presence of the reverse gradient ([H⫹]in ⬍
[H⫹]out). The final amount of [14C]tetraethylammonium taken up
in the presence of the H⫹ gradient ([H⫹]in ⬎ [H⫹]out) was not so
different from that attained in the absence of the gradient or in the
presence of the reverse gradient ([H⫹]in ⬍ [H⫹]out).
To further evaluate the effect of an outwardly directed H⫹
gradient on [14C]tetraethylammonium uptake, the influence of
a protonophore, FCCP, was examined. As shown in Fig. 6A,
the initial rate of [14C]tetraethylammonium uptake in the pres-
ence of an H⫹ gradient ([H⫹]in ⬎ [H⫹]out) was markedly
Downloaded from ajprenal.physiology.org on March 30, 2011
Fig. 6. Effect of FCCP (A) and valinomycin (B) on [14C]TEA uptake in the
presence of an outwardly directed H⫹ gradient by membrane vesicles from
HEK-rMATE1 cells. A: membrane vesicles were prepared in the experimental
buffer at pH 6.0. The uptake of [14C]TEA was examined in the experimental
buffer containing 31.25 ␮M [14C]TEA and 100 mM KCl at pH 7.5 in the
absence (E) or presence (F) of 40 ␮M FCCP. Each point represents the mean ⫾
SE of 3 determinations. The figure is a representative of 2 separate experi-
ments. B: membrane vesicles were prepared in the experimental buffer at pH
6.0. The uptake of [14C]TEA was examined in the experimental buffer
containing 31.25 ␮M [14C]TEA and 100 mM CsCl at pH 7.5 in the absence (E)
or presence (F) of 8 ␮M valinomycin. Each point represents the mean ⫾ SE
of 3 determinations. The figure is representative of 2 separate experiments.
www.ajprenal.org
 DRIVING FORCE OF TETRAETHYLAMMONIUM TRANSPORT BY rMATE1
reduced by FCCP, although the values at 30 min were similar
in the absence or presence of FCCP.
To determine whether [14C]tetraethylammonium uptake de-
pends on membrane potential, the effect of a K⫹ diffusion
potential generated by valinomycin on [14C]tetraethylammo-
nium uptake was examined. As shown in Fig. 6B, the H⫹
gradient-stimulated [14C]tetraethylammonium uptake was not
altered by the presence of valinomycin. Furthermore, we also
examined the effect of a K⫹ diffusion potential generated by
valinomycin on rMATE1-mediated [14C]tetraethylammonium
uptake in the absence of H⫹ gradient ([H⫹]in ⫽ [H⫹]out, pH
7.5). [14C]tetraethylammonium uptake was not significantly
changed with or without valinomycin at 30 s (with valinomy-
cin, 1.77 ⫾ 0.21; without valinomycin, 1.84 ⫾ 0.14 pmol䡠mg
protein⫺1䡠30 s⫺1; n ⫽ 3) and at 1 min (with valinomycin,
3.10 ⫾ 0.18; without valinomycin, 3.48 ⫾ 0.68 pmol䡠mg
protein⫺1䡠min⫺1; n ⫽ 3). These results indicate that the inside-
negative membrane potential does not affect [14C]tetraethyl-
ammonium uptake by rMATE1, suggesting the electroneutral
antiport of H⫹ and [14C]tetraethylammonium.
DISCUSSION
Transport studies in brush-border and basolateral membrane
vesicles from renal epithelial cells have been successfully
utilized to characterize a number of transport systems under
well-defined in vitro conditions. The membrane vesicle studies
are particularly useful for identifying the driving forces of
secondary active transport systems, compared with other anal-
yses. This is because the ionic composition inside or outside
membrane vesicles is easily manipulated, and ion gradients and
membrane potential can be provided artificially. In fact, it was
clearly demonstrated that organic cation transport systems at
the renal brush-border membranes are driven by an outwardly
directed H⫹ gradient (4, 19, 23). Recent cloning and functional
studies of MATE1 from various species have suggested that an
oppositely directed H⫹ gradient was a driving force of tetra-
ethylammonium transport by MATE1 (3, 13, 15, 20), but there
had been no evidence of a direct coupling of organic cation
transport to H⫹.
In the present study, by using membrane vesicles from
HEK-rMATE1 cells, we provide the first direct evidence that
MATE1 mediates the H⫹-coupled uphill transport of [14C]tet-
raethylammonium. Furthermore, this stimulation disappeared
in the presence of a protonophore, FCCP, indicating that
MATE1 functions as the H⫹/organic cation antiporter. The K⫹
diffusion potential generated by valinomycin had no effect on
[14C]tetraethylammonium uptake by membrane vesicles from
HEK-rMATE1 cells with or without an H⫹ gradient. This is
consistent with a report that the tetraethylammonium uptake by
brush-border membrane vesicles was not enhanced by in-
side-negative membrane potential (19). Taken together, it is
suggested that the antiport of H⫹ and tetraethylammonium
via rMATE1 is electroneutral and that the stoichiometry
might be 1:1.
In our previous study (20), using rMATE1-transiently ex-
pressing cells without NH4Cl pretreatment, we assessed the
time course of [14C]tetraethylammonium uptake (pH 8.4), pH
profile of [14C]tetraethylammonium uptake, and substrate spec-
ificity at the pH 8.4. In the present study, using HEK-rMATE1
cells with NH4Cl pretreatment, the transport characteristics for
AJP-Renal Physiol • VOL
F597
rMATE1 were analyzed by [14C]tetraethylammonium (pH 7.4)
or various compounds (pH 7.4). It was reported that the
intracellular pH of HEK293 cells is ⬃7.2 and transiently
acidified to 6.0 – 6.5 by NH4Cl pretreatment (11). These dis-
tinct experimental conditions may have affected the different
transport characteristics of rMATE1. For example, we previ-
ously reported that the intracellular accumulation of [14C]tet-
raethylammonium via rMATE1 showed a time-dependent in-
crease. In the present study, [14C]tetraethylammonium intra-
cellular accumulation by rMATE1 peaked at 30 – 60 s and then
gradually decreased. This may be due to the consumption of
the outward H⫹ gradient within 30 – 60 s and subsequent back
flux of [14C]tetraethylammonium via MATE1. In addition,
[3H]1-methyl-4-phenylpyridinium acetate and [14C]procain-
amide were transported by rMATE1 in the present study, but
not in the previous study. This may be due to the lack of a
strong enough driving force to transport [3H]1-methyl-4-phe-
nylpyridinium acetate and [14C]procainamide in the previous
conditions.
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In conclusion, we generated HEK293 cells stably expressing
rMATE1, and clearly demonstrated that the driving force of
tetraethylammonium transport by rMATE1 is an oppositely
directed H⫹ gradient using membrane vesicles from this stable
transfectant. These findings can provide important information
about the renal tubular secretion of organic cations, and these
experimental strategies may be useful for elucidating the mech-
anisms of action used by single transporters in heterologous
expression systems.
GRANTS
This work was supported by the 21st Century COE Program “Knowledge
Information Infrastructure for Genome Science,” a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan, and a Grant-in-Aid for Research on Advanced Medical
Technology from the Ministry of Health, Labor, and Welfare of Japan. J.
Asaka is supported as a research assistant by the 21st Century COE program
“Knowledge Information Infrastructure for Genome Science.”
REFERENCES
1. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72: 248 –254, 1976.
2. Burckhardt BC, Burckhardt G. Transport of organic anions across the
basolateral membrane of proximal tubule cells. Rev Physiol Biochem
Pharmacol 146: 95–158, 2003.
3. Hiasa M, Matsumoto T, Komatsu T, Moriyama Y. Wide variety of
locations for rodent MATE1, a transporter protein that mediates the final
excretion step for toxic organic cations. Am J Physiol Cell Physiol 291:
C678 –C686, 2006.
4. Holohan PD, Ross CR. Mechanisms of organic cation transport in kidney
plasma membrane vesicles: 2. ⌬pH studies. J Pharmacol Exp Ther 216:
294 –298, 1981.
5. Inui K, Masuda S, Saito H. Cellular and molecular aspects of drug
transport in the kidney. Kidney Int 58: 944 –958, 2000.
6. Inui K, Moller DE, Tillotson LG, Isselbacher KJ. Stereospecific hexose
transport by membrane vesicles from mouse fibroblasts: membrane vesi-
cles retain increased hexose transport associated with viral transformation.
Proc Natl Acad Sci USA 76: 3972–3976, 1979.
7. Inui K, Okuda M. Cellular and molecular mechanisms of renal tubular
secretion of organic anions and cations. Clin Exp Nephrol 2: 100 –108,
1998.
8. Inui K, Saito H, Hori R. H⫹-gradient-dependent active transport of
tetraethylammonium cation in apical-membrane vesicles isolated from
kidney epithelial cell line LLC-PK1. Biochem J 227: 199 –203, 1985.
9. Inui K, Tillotson LG, Isselbacher KJ. Hexose and amino acid transport
by chicken embryo fibroblasts infected with temperature-sensitive mutant
61 • FEBRUARY 2007 • www.ajprenal.org
F598 DRIVING FORCE OF TETRAETHYLAMMONIUM TRANSPORT BY rMATE1
of Rous sarcoma virus. Comparison of transport properties of whole cells
and membrane vesicles. Biochim Biophys Acta 598: 616 – 627, 1980.
10. Koepsell H, Endou H. The SLC22 drug transporter family. Pflügers Arch
447: 666 – 676, 2004.
11. Lang K, Wagner C, Haddad G, Burnekova O, Geibel J. Intracellular
pH activates membrane-bound Na⫹/H⫹ exchanger and vacuolar H⫹-
ATPase in human embryonic kidney (HEK) cells. Cell Physiol Biochem
13: 257–262, 2003.
12. Maeda S, Takano M, Okano T, Ohoka K, Inui K, Hori R. Transport of
organic cation in renal brush-border membrane from rats with renal
ischemic injury. Biochim Biophys Acta 1150: 103–110, 1993.
13. Masuda S, Terada T, Yonezawa A, Tanihara Y, Kishimoto K, Ka-
tsura T, Ogawa O, Inui K. Identification and functional characterization
of a new human kidney-specific H⫹/organic cation antiporter, kidney-
specific multidrug and toxin extrusion 2. J Am Soc Nephrol 17: 2127–
2135, 2006.
14. McKinney TD, Kunnemann ME. Cimetidine transport in rabbit renal
cortical brush-border membrane vesicles. Am J Physiol Renal Fluid
Electrolyte Physiol 252: F525–F535, 1987.
15. Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H,
Moriyama Y. A human transporter protein that mediates the final excre-
tion step for toxic organic cations. Proc Natl Acad Sci USA 102: 17923–
17928, 2005.
16. Pritchard JB, Miller DS. Mechanisms mediating renal secretion of
organic anions and cations. Physiol Rev 73: 765–796, 1993.
AJP-Renal Physiol • VOL 61 • FEBRUARY 2007 •
17. Saito H, Okuda M, Terada T, Sasaki S, Inui K. Cloning and charac-
terization of a rat H⫹/peptide cotransporter mediating absorption of
␤-lactam antibiotics in the intestine and kidney. J Pharmacol Exp Ther
275: 1631–1637, 1995.
18. Takano M, Inui K, Okano T, Hori R. Cimetidine transport in rat renal
brush border and basolateral membrane vesicles. Life Sci 37: 1579 –1585,
1985.
19. Takano M, Inui K, Okano T, Saito H, Hori R. Carrier-mediated
transport systems of tetraethylammonium in rat renal brush-border and
basolateral membrane vesicles. Biochim Biophys Acta 773: 113–124,
1984.
20. Terada T, Masuda S, Asaka J, Tsuda M, Katsura T, Inui K. Molecular
cloning, functional characterization and tissue distribution of rat H⫹/
organic cation antiporter MATE1. Pharm Res 23: 1696 –1701, 2006.
21. Terada T, Saito H, Mukai M, Inui K. Identification of the histidine
residues involved in substrate recognition by a rat H⫹/peptide cotrans-
porter, PEPT1. FEBS Lett 394: 196 –200, 1996.
22. Urakami Y, Akazawa M, Saito H, Okuda M, Inui K. cDNA cloning,
functional characterization, and tissue distribution of an alternatively
spliced variant of organic cation transporter hOCT2 predominantly ex-
pressed in the human kidney. J Am Soc Nephrol 13: 1703–1710, 2002.
23. Wright SH, Wunz TM. Transport of tetraethylammonium by rabbit renal
Downloaded from ajprenal.physiology.org on March 30, 2011
brush-border and basolateral membrane vesicles. Am J Physiol Renal
Fluid Electrolyte Physiol 253: F1040 –F1050, 1987.
www.ajprenal.org