Stoichiometry of organic anion/dicarboxylate exchange in membrane

vesicles from rat renal cortex and hOAT1-expressing cells

Amy Aslamkhan,
1
Yong-Hae Han,
1
Ramsey Walden,
1
Douglas H. Sweet,
2
and John B. Pritchard
1
1
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
National Institutes of Health, Research Triangle Park, North Carolina 27709; and
2
Department of
Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425
Submitted 8 April 2003; accepted in nal form 26 June 2003
Aslamkhan, Amy, Yong-Hae Han, Ramsey Walden,
Douglas H. Sweet, and John B. Pritchard. Stoichiometry
of organic anion/dicarboxylate exchange in membrane vesi-
cles from rat renal cortex and hOAT1-expressing cells. Am J
Physiol Renal Physiol 285: F775F783, 2003. First published
July 1, 2003; 10.1152/ajprenal.00140.2003.Although mem-
brane vesicle studies have established the driving forces that
mediate renal organic anion secretion and the organic anion
transporter Oat1 has now been cloned in several species, its
stoichiometry has remained uncertain. In this study, we used
electrophysiology, kinetic measurements, and static head
experiments to determine the coupling ratio for Oat1-medi-
ated organic anion/dicarboxylate exchange. Initial experi-
ments demonstrated that uptake of PAH by voltage-clamped
Xenopus laevis oocytes expressing rOat1 led to net entry of
positive charge, suggesting that coupling was one-to-one.
This conclusion was conrmed by kinetic analysis of PAH
and glutarate uxes in native basolateral membrane vesicles
from the rat renal cortex, which showed a Hill coefcient of 1.
Similarly, static head experiments on the rat vesicles also
showed a 1:1 coupling ratio. To conrm these conclusions in
a system expressing a single cloned transporter, Madin-
Darby canine kidney cells were stably transfected with the
human exchanger hOAT1. The hOAT1-expressing cell line
showed extensive PAH transport, which was very similar in
all respects to transport expressed by hOAT1 in Xenopus
oocytes. Its K
m
for PAH was 8 M and glutarate effectively
trans-stimulated PAH transport. When stoichiometry was
assessed using plasma membranes isolated from the hOAT1-
expressing cells, both kinetic and static head data indicated
that hOAT1 also demonstrated a 1:1 coupling between or-
ganic anion and dicarboxylate.
p-aminohippurate; hOAT1; organic anion transport; anion
exchange; Madin-Darby canine kidney cells
A CRITICAL FUNCTION of the kidney is elimination of po-
tentially toxic chemicals, both foreign and endogenous,
from the body (17). The primary effector for the excre-
tion of negatively charged chemicals and metabolites is
the classical organic anion secretory system that trans-
ports PAH and other small anions (500 Da). A good
substrate such as PAH may be completely cleared from
the renal plasma in a single pass through the kidney.
In the 1980s, membrane vesicle studies led to the
elucidation of the mechanisms and driving forces that
enable this system to function so effectively (14, 16,
23). The critical uphill step in the secretory process was
shown to be basolateral exchange of the anionic drug or
xenobiotic for intracellular -ketoglutarate (-KG)
(15). Two organic anion transporters, Oat1 and Oat3,
have been localized to the basolateral membrane
(BLM) (5, 7, 8, 25, 2729). Of these, Oat1 was rst to be
cloned and demonstrated to be an organic anion/-KG
exchanger, initially in rats (22, 28) and later in a
number of other species, including humans (6, 8, 11,
19). Recently, we showed that rat Oat3 is also an
organic anion/-KG exchanger (24).
Surprisingly, given all of this attention, the stoichi-
ometry of Oat1 has remained uncertain. The original
experiments conducted using membrane vesicles iso-
lated from rat kidney showed no change in PAH uptake
when membrane potential was altered by changing
potassium gradients in the presence of the potassium
ionophore valinomycin (14). These data suggested that
exchange was electroneutral, i.e., one divalent -KG
molecule being exchanged for two monovalent PAH
molecules. Similar data were obtained in bovine vesi-
cles (21), and a comparable conclusion was reached
based on observations in the intact tubule (30). Even in
Xenopus laevis oocytes expressing the cloned rat trans-
porter rOat1, no effect on PAH transport was observed
when the oocytes were depolarized (28). Because of the
consistency of these ndings from different laborato-
ries, the stoichiometry was not determined directly.
However, at least one study using rabbit renal BLM
vesicles did show increased PAH uptake when mem-
brane potential was inside negative and decreased
uptake when it was inside positive (12). Furthermore,
when Burckhardt et al. (3) examined the electrophys-
iology of organic anion exchange in oocytes expressing
winter ounder Oat1 under voltage-clamped condi-
tions, entry of organic anions was associated with net
entry of positive charge or exit of negative charge. This
Address for reprint requests and other correspondence: J. B. Prit-
chard, Laboratory of Pharmacology and Chemistry, National Insti-
tute of Environmental Health Sciences, National Institutes of
Health, Research Triangle Park, NC 27709 (E-mail: pritcha3
@niehs.nih.gov).
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.
Am J Physiol Renal Physiol 285: F775F783, 2003.
First published July 1, 2003; 10.1152/ajprenal.00140.2003.
http://www.ajprenal.org F775
result is most simply explained by a 1:1 exchange of
organic anion for dicarboxylate. This issue, which has
major implications in terms of transport mechanism at
the molecular level, clearly requires resolution.
In the studies described below, we reexamined the
stoichiometry of Oat1/OAT1 through 1) electrophysio-
logical measurements in voltage-clamped rOat1-ex-
pressing Xenopus oocytes and 2) kinetic measurements
and static head experiments in native BLMs isolated
from rat kidney and in membranes isolated from Ma-
din-Darby canine kidney (MDCK) cells stably express-
ing the human ortholog of the PAH/-KG exchanger,
hOAT1. In agreement with the electrophysiology data,
the vesicle data show that the coupling ratio of PAH to
-KG is 1:1. Specically, in both membrane prepara-
tions, the initial rate of PAH uptake was a hyperbolic
function of the intravesicular concentration of counter-
ion (Hill coefcient 1) and simultaneous 10-fold gra-
dients for PAH and dicarboxylate balanced one an-
other, yielding no net ux in static head experiments.
METHODS AND MATERIALS
Chemicals
[
3
H]PAH (1.28 Ci/mmol) and [
3
H]taurocholic acid (2.10
Ci/mmol) were purchased from New England Nuclear (Bos-
ton, MA). [
14
C]urate (55 mCi/mmol) and [
14
C]glutarate (15.6
mCi/mmol) were obtained from American Radiolabeled
Chemicals (St. Louis, MO) and ICN (Irvine, CA), respec-
tively. Unlabeled PAH and quinine were obtained from
Sigma (St. Louis, MO). All other chemicals were obtained
from commercial sources and were of the highest grade
available.
Animals
Rats. Kidneys were obtained from 300-g male Sprague-
Dawley rats purchased from Taconic Farms (Germantown,
NY) and maintained in the animal quarters at National
Institute of Environmental Health Sciences (NIEHS) for 1
wk or less. For tissue harvest, animals were euthanized with
100% CO
2
and the kidneys were immediately removed to
ice-cold, oxygenated saline for preparation of isolated BLM.
Frogs. Female X. laevis were purchased from Xenopus One
(Ann Arbor, MI). The animals were anesthetized with 0.3%
tricaine (Sigma), decapitated, pithed, and the ovaries were
removed. Stage V and VI oocytes were isolated by collagenase
digestion as previously described (6, 13, 26). All animal
procedures were carried out in accord with protocols ap-
proved by the NIEHS Animal Care and Use Committee.
Electrophysiology
Three days after cRNA injection, oocyte membrane cur-
rents were measured using a conventional two-electrode (3 M
KCl; resistance of 1 m) voltage-clamp technique (Ge-
neclamp 500B, Axon Instruments, Foster City, CA). The
oocytes were continuously bathed (4 ml/min) with OR-2
buffer (in mM: 82.5 NaCl, 2.5 KCl, 1 Na
2
HPO
4
, 3 NaOH, 1
CaCl
2
, 1 MgCl
2
, 1 Na-pyruvate, 5 HEPES, pH 7.6), and the
oocyte membrane potential was held at 60 mV. Buffer
containing 400 M PAH, with or without 0.5 mM probenecid,
was applied to the oocytes in a pulsed manner via a solenoid-
controlled valve. Recordings were sampled at 100 Hz with a
50-Hz lter frequency. After being sampled, current signals
were processed with a Gaussian low band pass (2 Hz cut-off)
lter. The current voltage (I-V) protocol was initiated from a
50-mV hold. Each holding potential was applied for 100 ms,
and potentials were cycled from 150 to 50 mV in 20-mV
increments, after which the clamping potential was returned
to 50 mV. Steady-state currents were measured at 95 ms
during each voltage pulse. I-V protocol recordings were sam-
pled at 5 kHz with a 500-Hz lter frequency.
Cell Culture
MDCK type II cells (a low transepithelial resistance clone,
originally derived from distal renal tubules of an adult fe-
male cocker spaniel) were provided by Dr. D. Balkovetz
(University of Alabama at Birmingham) and were originally
subcloned in the laboratory of Dr. K. Simons (EMBL, Heidel-
berg, Germany). They were negative for mycoplasma upon
receipt. All MDCK lines were retested before publication and
found to be negative for mycoplasma. Cells were maintained
in Eagles Modied Essential Medium (EMEM; Invitrogen,
Carlsbad, CA) supplemented with 10% fetal bovine serum in
a humidied incubator at 37C with 5% CO
2
. Cultures were
split 1:20 every 34 days.
Transfection
For mammalian cell transfection, the fragment containing
the full-length hOAT1 cDNA was removed from the isolated
library clone, pSPORT1/hOAT1 (6), with the restriction en-
zymes BamH 1 and Kpn 1. The fragment was gel-isolated
and ligated into pcDNA3.1 (Invitrogen, Carlsbad, CA) cut
with BamH 1 and Kpn 1, resulting in the plasmid pcDNA3.1/
hOAT1. Activity of the new construct was conrmed by X.
laevis oocyte expression assay before transfection. One day
before transfection, 2 10
5
MDCK cells were plated into
individual wells of a six-well culture plate (9.1 cm
2
). Cells
were transfected with 10 g pcDNA3.1/hOAT1 plasmid DNA
for 3 h at 37C using SuperFect Reagent (5 l SuperFect/g
DNA; Qiagen, Chatsworth, CA). Transfected cells were
washed with PBS, given fresh medium, and maintained at
37C with 5% CO
2
. Two days after transfection, cells were
lifted, diluted to 1 cell/ml, dispensed (1 ml/well) into 24-well
culture plates, and cultured in the presence of 1 mg/ml G418
(Invitrogen). Surviving cell clones were maintained with 200
g/ml G418 and tested for organic anion transport activity.
Membrane Vesicles
Rat kidney BLM vesicles. Renal cortex was dissected from
the kidneys of 15 rats, and BLM vesicles were isolated by
differential and density (11%Percoll, AmershamBiosciences,
Piscataway, NJ) gradient centrifugation as previously de-
scribed (18). The nal membrane pellet was suspended in
KCl vesicle buffer [in mM: 100 mannitol, 100 KCl, 20
HEPES/Tris (hydroxymethyl) aminoethane (Tris), 1 MgSO
4
,
at pH 7.5] and stored in liquid nitrogen until use. Upon
thawing, vesicles were washed by centrifugation and resus-
pended in fresh buffer with additions as noted in the gure
legends.
Plasma membranes from cultured MDCK cells. Cells were
harvested by scraping and homogenized in buffer (in mM:
300 mannitol, 12 HEPES/Tris, and 0.1 phenylmethylsulpho-
nyl uoride) with 20 strokes using a pestle and glass homog-
enizer. The homogenate was centrifuged at 250 g for 15 min,
and the pellet was discarded. The supernatant was centri-
fuged at 20,500 g for 20 min to collect the plasma mem-
branes. The plasma membrane pellet was washed twice by
resuspension and centrifugation at 20,500 g, and the nal
F776 STOICHIOMETRY OF ORGANIC ANION/DICARBOXYLATE EXCHANGE
AJP-Renal Physiol VOL 285 OCTOBER 2003 www.ajprenal.org
pellet was suspended in vesicle buffer and stored in liquid
nitrogen. Marker enzyme analysis showed four- to sixfold
enrichment of both brush border (alkaline phosphatase) and
BLM (Na-K-ATPase) markers.
Vesicle Transport Measurement
Upon thawing, rat BLM and MDCK plasma membrane
vesicles were centrifuged and resuspended in fresh vesicle
buffer. Vesicles were allowed to equilibrate for 60 min at
2224C. Transport was measured using the rapid ltration
method and Millipore HAWP (0.45 m) membranes as pre-
viously described (12). Briey, 10 l of vesicles were incu-
bated with 190 l of vesicle buffer with additions as indicated
in the gure legends. All vesicle experiments were conducted
under short-circuiting conditions (100 mM KCl in out plus
10 M valinomycin). Uptake was terminated by addition of 1
ml of stop solution (in mM: 300 mannitol, 12 HEPES/Tris,
and 0.1 HgCl
2
, at pH 7.5). PAH uptake values for all vesicle
experiments (kinetic and static head experiments) are re-
ported as probenecid-sensitive uptake. Radioactivity was de-
termined using an LKB model 1216 liquid scintillation
counter with external standard quench correction.
MDCK Cell Transport Assay
For transepithelial ux experiments, 1 10
6
cells were
plated onto 24-mm (4.7 cm
2
) Transwell-Clear microporous
polyester membranes (Costar, Bedford, MA). Cell monolayers
were cultured for 3 days in EMEM supplemented with 10%
fetal bovine serum without G418 (2 ml on each side of the
monolayer) in a humidied incubator at 37C with 5% CO
2
.
The culture medium was changed daily. Before transport
experiments, the culture medium was removed, the monolay-
ers were washed twice with 2 ml of Hanks balanced salt
solution (Sigma) buffered with 10 mM HEPES (transport
buffer), and 2 ml of transport buffer (pH 7.4) were added to
both apical and basolateral compartments. Transport mea-
surement was initiated by replacement of basolateral, apical,
or both media with buffer containing 2 M [
3
H]PAH (2
Ci/ml). After incubation at 37C, the medium was removed
from both sides of the monolayer and the cells were rapidly
rinsed three times with ice-cold 0.1 M MgCl
2
. The cells were
dissolved in 2 ml 1 N NaOH and neutralized with 2 ml 1 N
HCl. Aliquots were removed for protein assay (2) using a
Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules,
CA) with bovine serum albumin used as a standard and for
liquid scintillation counting using 15 ml of Ecolume (ICN
Biomedical, Cleveland, OH). [
3
H]PAH uptake was normal-
ized to cellular protein. For solid support experiments, 1
10
6
cells were plated into individual wells (3.5 cm
2
) of a
12-well tissue culture plate. Cells were handled exactly as
described above for the experiments done on lter inserts,
except that they were cultured for only 2 days after plating.
Estimation of Kinetic Parameters
The kinetic parameters for PAH uptake by hOAT1/MDCK
cells were calculated by tting the data to the following
equation
V
V
max
S
K
m
S
P
diff
S
where V is the uptake rate of PAH (pmol min
1
mg pro-
tein
1
), S is the PAH concentration in the medium (M), K
m
is the apparent Michaelis Menten constant (M), V
max
is the
maximum uptake rate (pmol min
1
mg protein
1
), and P
diff
is the diffusion constant (l min
1
mg protein
1
). Curve
tting was performed by an iterative nonlinear least-squares
method using a MULTI program (31). The input data were
weighed as the reciprocal of the observed values, and the
Damping Gauss Newton Method was used as the tting
algorithm.
Statistics
Data are presented as means SE, and differences were
considered to be signicant when P 0.05. Data between two
Fig. 1. Electrophysiological recording of PAH-induced current (I) in
a rat organic anion transporter (rOat)1-expressing oocyte. A: inward
current (60-mV clamp) observed in response to 400 M PAH with
and without 500 M probenecid (Prob). B: rOat1 PAH (400 M)-
mediated current response to voltage steps. Responses are represen-
tative of results observed in at least 5 oocytes isolated from 3
different animals.
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AJP-Renal Physiol VOL 285 OCTOBER 2003 www.ajprenal.org
experimental groups were compared using the unpaired Stu-
dents t-test.
RESULTS
Electrophysiology
As shown in Fig. 1A, when rOat1-expressing X. lae-
vis oocytes clamped at 60 mV were exposed to 400
M PAH, net entry of positive charge was seen, rang-
ing from 6 to 8 nA in multiple trials. This current was
abolished by 0.5 mM probenecid. Reapplication of PAH
after treatment with probenecid yielded a blunted re-
sponse, probably because of lingering probenecid in the
transporter binding sites. Control oocytes injected with
water rather than rOat1 RNA showed no PAH or
probenecid-induced current (data not shown). These
results indicate that entry of PAH is accompanied by
net entry of positive charge or loss of negative charge.
Because rOat1 is known to mediate PAH/dicarboxylate
exchange, the simplest interpretation consistent with
this observation would be 1:1 exchange, i.e., of one
monovalent organic anion for one divalent dicarboxy-
late anion. As shown in Fig. 1B, the current response of
rOat1-expressing oocytes to 400 M PAH was voltage
sensitive. Inward positive current increased as the
voltage was varied from10 to 150 mV. Current ow
reversed at 10 mV, and outward positive current was
seen at voltages from 10 to 50 mV.
Rat BLM Vesicles
To directly test whether exchange was 1:1, kinetic
(Hill analysis) and static head experiments were con-
ducted in BLM vesicles isolated from rat renal cortex.
As shown in Fig. 2, the basic experimental protocol was
to measure the probenecid-sensitive uptake of [
3
H]-
PAH by vesicles preloaded with 1 mM glutarate and
diluted 20-fold with transport buffer, i.e., an initial
gradient of 1 mM glutarate inside vs. 50 M outside.
Under these conditions, a substantial glutarate-depen-
dent overshoot was readily demonstrated.
Kinetic Analysis
To determine the coupling ratio, the initial rate of
PAH (50 M) uptake was estimated at 15 s. Intrave-
sicular glutarate was varied from 10 M to 2 mM. As
shown in Fig. 3A, probenecid-sensitive PAH uptake
was a hyperbolic function of glutarate concentration
and upon transformation according to the Hill equation
(Fig. 3B), these data yielded a straight line with a slope
(coupling coefcient) of 0.97 (K
m
94 M; V
max
90
pmol mg protein
1
15 s
1
).
Static Head
A second means of assessing the coupling between
two transported species is to determine the gradients
that balance one another and yield no net ux of
substrate. This was done in short-circuited vesicles
(100 mM KCl in out, plus 10 M valinomycin). For
these experiments, the glutarate gradient was xed at
Fig. 2. Time course of probenecid-sensitive [
3
H]PAH uptake in con-
trol (E) and glutarate-preloaded (F) rat renal cortex basolateral
membrane vesicles (BLMV). BLMV were preloaded with 1 mM
glutarate by incubation for 1 h. BLMV (10 l) were then mixed with
190 l vesicle buffer containing 50 M [
3
H]PAH and 10 M valino-
mycin in the presence and absence of 5 mM probenecid. Values are
the means SE (n 3 vesicle preparations analyzed in triplicate).
*Signicantly different from control, P 0.05 by unpaired Students
t-test.
Fig. 3. Kinetics of probenecid-sensitive glutarate exchange for [
3
H]-
PAHin rat renal cortex BLMV. BLMV were preloaded with glutarate
(10 M to 2 mM) as described in Fig. 2. BLMV were then diluted 1:20
with vesicle buffer containing 50 M [
3
H]PAH and 10 M valinomy-
cin in the presence and absence of 5 mM probenecid. Uptake was
measured at 15-s time points. A: probenecid-sensitive uptake of PAH
as a function of the glutarate concentration. B: Hill transformation of
these data. Hill coefcient 0.97. Values are means SE (n 3
vesicle preparations analyzed in triplicate). GA, glutamate.
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10:1 inout and, while maintaining a constant specic
activity, in:out
3
[H]PAH gradients of 1:1, 5:1, 10:1,
50:1, and 100:1 were tested. A coupling ratio of 1
PAH:1 glutarate would yield no net ux at equal gra-
dients. As shown in Fig. 4, no net ux was observed
when the gradients were 10:1 for both ions, indicating
a 1:1 coupling ratio.
hOAT1-Transfected MDCK Cells
To determine whether membranes expressing a sin-
gle cloned Oat would behave similarly to the native
BLM in which multiple paralogs (e.g., Oat1 and Oat3)
might be expressed, we generated an MDCK line sta-
bly transfected with hOAT1. As shown in Fig. 5, apical
and basal uptake of PAH in hOAT1-expressing cells
(hOAT1/MDCK) cultured on permeable lters in-
creased as a function of time. Uptake of PAH by the
parental, nontransfected MDCK cells was minimal and
unaffected by other agents (not shown). When both
faces of the epithelium were exposed simultaneously,
uptake was larger than from either basal or apical
sides alone and approximated their sum at all the time
points. Clearly, hOAT1 expression in this clonal cell
line is evident over the entire plasma membrane.
Therefore, all subsequent characterization experi-
ments were conducted using cells grown on solid sup-
port, i.e., assessing apically expressed hOAT1.
PAH uptake over 1 min by hOAT1/MDCK cells was
saturable over the range of 0.5200 M (Fig. 6). The
apparent K
m
in these hOAT1-expressing cells for PAH
was 8.5 1.3 M with a V
max
166 21 pmol
min
1
mg protein
1
and a nonmediated component,
P
diff
, of 0.47 0.29 l min
1
mg protein
1
(Fig. 6).
Uptake of 2 M [
3
H]PAH by these cells was markedly
inhibited by unlabeled PAH, glutarate, -KG, probene-
cid, and uric acid (Fig. 7) in a manner very similar to
our previous data on hOAT1 expressed in X. laevis
oocytes (6). Fluorescein and the anionic herbicide,
2,4-D, also strongly inhibited PAH uptake. On the
other hand, several larger organic anions including
benzyl-penicillin, taurocholate, methotrexate, and leu-
kotriene-C
4
did not inhibit PAH uptake by the hOAT1/
MDCK cells. Similarly, the p-glycoprotein substrate
cyclosporin A and the organic cations tetraethylammo-
nium and N
1
-methylnicotinamide did not signicantly
inhibit PAH uptake in the transfected cell line (Fig. 7).
Substrate specicity of the hOAT1/MDCK cells was
also evaluated directly by assessing uptake of radiola-
Fig. 4. Static head analysis of PAH/glutarate exchange in rat renal
cortex BLMV. BLMV were preloaded for 30 min in vesicle buffer (100
mM mannitol, 100 mM KCl, 20 mM Tris/HEPES at pH 7.5) contain-
ing 500 M [
3
H]PAH and 500 M glutarate. Vesicles were then
diluted into vesicle buffer containing 10 M valinomycin to achieve
a 10-fold gradient for glutarate (50 M outside; 500 M inside),
whereas the [
3
H]PAH gradient (with 500 M inside) was varied from
1- to 100-fold (i.e., 500 M to 5 M outside). A constant specic
activity was maintained throughout. The probenecid-sensitive [
3
H]-
PAH loss (efux) or gain (inux) was then plotted as a function of
[
3
H]PAH dilution. Values are means SE (n 3 vesicle prepara-
tions analyzed in triplicate).
Fig. 5. Accumulation of 2 M [
3
H]PAH by stably transfected human
(h)OAT1-expressing Madin-Darby canine kidney (MDCK) cells.
Open bars indicate uptake from the basolateral face of the mono-
layer; gray bars show apical uptake; and black bars indicate uptake
at both surfaces. Values are means SE (n 3 experiments
conducted in triplicate).
Fig. 6. Concentration dependence of [
3
H]PAH(0.5 to 200 M) uptake
in stably transfected hOAT1 MDCK cells. Samples were taken at 1
min to approximate the initial rate of uptake. Values are means
SE (n 3 experiments).
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beled glutarate, uric acid, and taurocholate. Figure 8A
shows that glutarate (5 M) is preferentially trans-
ported into hOAT1/MDCK cells compared with un-
transfected MDCK cell controls. In contrast, uptakes of
uric acid (2 M) and taurocholate (10 M) were smaller
than for glutarate and were not notably different be-
tween the transfected and untransfected lines (Fig. 8,
B and C). Finally, both uptake (not shown) and efux of
PAH were trans-stimulated by 250 M unlabeled PAH,
glutarate, and -KG, but not by TEA, at the same
concentration, just as expected of an organic anion/
dicarboxylate exchanger such as hOAT1 (Fig. 9).
Plasma Vesicles from hOAT1/MDCK Cells
Kinetic analysis. The preceding studies indicate
clearly that hOAT1 expressed in this MDCK cell line
has the properties expected of this transporter (Figs.
59). Thus plasma membrane vesicles prepared from
these cells should show hOAT1 activity. When tested,
vesicles preloaded with 1 mM glutarate and diluted
20-fold into medium containing 50 M [
3
H]PAH exhib-
ited a marked stimulation of PAH uptake with a tran-
sient overshoot of vefold (data not shown), much the
same as that depicted in Fig. 2 for native rat BLM
vesicles. As shown in Fig. 10, the Hill plot for PAH/
glutarate exchange conducted in the same manner as
with rat renal cortex BLM vesicles yielded a slope of
0.97, indicating a coupling ratio of 1:1 for the hOAT1-
expressing plasma membrane vesicles.
Static head. hOAT1/MDCK cell plasma membrane
vesicles were preloaded with glutarate and diluted
10-fold to create a 10:1 inout gradient, and the net
ux of [
3
H]PAH was determined at gradients from 1:1
to 100:1 inout. Once again, as for the native mem-
branes from rat kidney (Fig. 3), the cloned hOAT1-
expressing membranes showed no net ux when both
glutarate and PAH gradients were 10:1 (Fig. 11). Thus
both kinetic and static head analysis of the expressed
hOAT1 demonstrated a 1:1 coupling ratio.
Fig. 7. Inhibition of 2 M [
3
H]PAH uptake (1 min) by stably trans-
fected hOAT1-expressing MDCK cells. All compounds were tested at
200 M except cyclosporin A (CSA; 10 M) and leukotriene C4
(LTC4; 1 M). Values are means SE (n 2 experiments conducted
in triplicate). *Signicantly different from control, P 0.05 by
unpaired Students t-test. -KG, -ketoglutarate; 2,4-D, 2-4-dichlo-
rophenoxyacetic acid; TEA, tetraethylammonium; MTX, methotrex-
ate; NMN, N
1
-methylnicotinamide.
Fig. 8. Time course of accumulation (pmol/mg
protein) of 5 M [
14
C]glutarate (A), 2 M [
3
H]-
taurocholate (B), and 10 M [
14
C]urate (C) in
nontransfected (lled symbols) and stably trans-
fected hOAT1 MDCK cells (open symbols). Val-
ues are means SE (n 2 experiments con-
ducted in triplicate). *Signicantly different from
nontransfected, P 0.05 by unpaired Students
t-test.
Fig. 9. Effect of external PAH, glutarate, -KG, or TEA on [
3
H]PAH
efux from stably transfected hOAT1 MDCK cells. Cells were incu-
bated with 2 M [
3
H]PAH for 30 min at room temperature. The
PAH-containing transport buffer was then removed, and the cells
were rapidly rinsed twice with transport buffer. Cells were then
incubated at 37C with 2 ml transport buffer 250 M organic anion
or cation. Total [
3
H]PAH cell content was calculated from the sum of
total radioactivity recovered in the medium and that remaining in
the cells at the conclusion of the 10-min efux period. Values are
means SE (n 2 experiments conducted in triplicate). *Signi-
cantly different from control, P 0.05 by unpaired Students t-test.
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DISCUSSION
Stoichiometry of Oat1
As shown in Fig. 1, accumulation of PAH by rOat1-
expressing oocytes is accompanied by net entry of pos-
itive charge (or loss of negative charge). This result is
comparable to that obtained by Burckhardt et al. (3) for
winter ounder Oat1. Because basolateral organic an-
ion transporters mediate exchange of singly charged
anions such as PAH for doubly charged dicarboxylates
(6, 22, 28), this result is most simply explained by a 1:1
coupling of organic anion (singly charged) and dicar-
boxylate (doubly charged) with the resulting net loss of
one negative charge per transporter cycle. This conclu-
sion was conrmed in native rat BLM vesicles and in
plasma membranes isolated from MDCK cells express-
ing the human OAT1 ortholog, based on both Hill
analysis (Figs. 3 and 10) and static head experiments
(Figs. 4 and 11).
The similarity of ndings for native rat BLMs and
membranes expressing a single OAT paralog has par-
ticularly interesting implications. Clearly, based on
Figs. 10 and 11, when hOAT1 was expressed in the
absence of other Oats, it mediated 1:1 exchange of PAH
for dicarboxylate. However, native rat BLMs express
multiple Oat paralogs; the most prominent, in addition
to Oat1, is Oat3 (9). Indeed, in the rat, Oat1 and Oat3
have very similar afnities for PAH [K
m
of 70 M (28)
and K
m
of 65 M (10), respectively] and both should
contribute to BLM vesicle PAH uptake. Certainly, re-
cent ndings in an Oat3 knockout mouse (27) indicate
that this is the case for the mouse kidney, where Oat3
accounted for slightly more than half of the PAH up-
take by renal slices. Thus our analysis of the rat mem-
branes should have been inuenced by both Oat para-
logs, i.e., both would have contributed to the probene-
cid-sensitive uptake of PAH. In addition, we recently
showed that Oat3 is, similar to Oat1, driven by organic
anion/dicarboxylate exchange (24), meaning that a por-
tion of the glutarate-driven, probenecid-sensitive PAH
uptake observed in the rat BLM vesicles must have
been driven by rOat3. Nevertheless, both Hill analysis
and static head determination (Figs. 3 and 4) demon-
strate that total probenecid-sensitive PAH uptake by
the rat kidney BLM shows 1:1 coupling between PAH
and glutarate. Thus, by implication, it appears that
both Oat1 and Oat3 mediate PAH transport by 1:1
exchange for glutarate.
The electrophysiological results, the Hill analysis,
and the static head experiments all provide a consis-
tent answer to the question of coupling ratio. It is
exchange of one PAH

for one dicarboxylate

2
. Thus
the overall exchange process is electrogenic with the
net loss of one negative charge per transporter cycle.
How then may the previous results showing a lack of
sensitivity of PAH uptake to manipulation of mem-
brane potential in vesicles (14, 20), intact tubules (30),
and the cloned carrier (28) be explained? There are
several possibilities, but at the moment, there is little
evidence to support any of them. For example, a
charged species such as chloride or proton might bind
to and/or be transported during the carrier cycle. In-
deed, our vesicle studies demonstrated a strong chlo-
ride dependence for PAH transport by rat basolateral
vesicles (14). Also, an electrophysiological study in
ounder Oat1-expressing oocytes revealed that a 10-
fold reduction in bath chloride abolished PAH-medi-
ated current and reduced PAH transport, strongly sug-
gesting a role of chloride in Oat transport (4). However,
the detailed studies of Schmitt and Burckhardt (20) in
vesicles indicated that chloride was not cotransported
with PAH and it was concluded that the effect of
chloride was allosteric in nature. Another possible ex-
planation may lie in the experimental approach of the
earlier studies. In each case noted above (14, 20, 28,
30), the earlier studies examined the response of PAH
transport following changes in membrane potential.
Thus it is possible that what they show is that the
rate-limiting step in the transport of PAH is not inu-
enced by membrane potential. Thus, if the transport
event is electrogenic, as shown here and in Burck-
hardts studies of the ounder Oat1, it may be that the
exchange of PAH for glutarate is not the rate-limiting
Fig. 11. Static head analysis of PAH/glutarate exchange in plasma
membrane vesicles prepared from stably transfected hOAT1 MDCK
cells. See Fig. 4 legend for details. Values are means SE (n 3
vesicle preparations analyzed in triplicate).
Fig. 10. Hill-transformed kinetics of probenecid-sensitive [
3
H]PAH
uptake in glutarate-preloaded plasma membrane vesicles prepared
from stably transfected hOAT1/MDCK cells. Vesicles were preloaded
with glutarate (10 M to 2 mM) as described in Fig. 2. They were
then diluted 20-fold in buffer containing 50 M [
3
H]PAH and 10 M
valinomycin (5 mM probenecid). Uptake was determined at 15-s
time points. Hill 0.97. Values are means SE (n 3 vesicle
preparations analyzed in triplicate).
F781 STOICHIOMETRY OF ORGANIC ANION/DICARBOXYLATE EXCHANGE
AJP-Renal Physiol VOL 285 OCTOBER 2003 www.ajprenal.org
step in the overall process. Other candidates for this
role might be the on- or off-rate for substrate or
counter-ion or perhaps an allosteric change in trans-
porter structure allowing it to reorient in the mem-
brane or to accept new substrate or counter-ion. Fur-
thermore, it is also possible that differences in the
degree of phosphorylation may alter transport proper-
ties. Resolution of these possibilities must await fur-
ther study.
Transfected MDCK Cell Model
The hOAT1-expressing cell line used here shows
saturable PAH uptake (Fig. 6), an inhibition pattern
identical to that previously shown for this construct
expressed in X. laevis oocytes (6) (Fig. 7), mediates
glutarate uptake (Fig. 8), and demonstrates PAH/glut-
arate exchange (Fig. 9); all properties to be expected of
an OAT1-expressing cell line. Furthermore, as shown
in Fig. 5, hOAT1 is well expressed at the apical face of
these cells. This is a signicant technical advantage in
that it allows uptake and efux experiments to be
conducted in cells grown on solid support. In addition,
because the parent MDCK cell line shows virtually no
probenecid-sensitive uptake of PAH (1) or glutarate
(Fig. 8A), this model has very low background uptake
of organic anions. Thus the hOAT1-expressing cell line
used in these studies is well suited to mechanistic
studies and for screening of toxicity related to OAT1-
mediated transport (1).
Conclusions
The electrophysiological, kinetic, and static head ex-
periments reported above all indicate that the stoichi-
ometry of organic anion/dicarboxylate exchange by
both rat and human OAT1 orthologs is 1:1 and that,
contrary to earlier conclusions based on experimental
manipulation of membrane potential, organic anion
transport is an electrogenic process. In addition, these
data highlight the utility of this MDCK cell line stably
expressing hOAT1 in the absence of other OATs for
studies characterizing the nature of hOAT1-mediated
transport and/or its role in xenobiotic and drug toxic-
ity.
Present address of Y.-H. Han: Dept. of Metabolism and Pharma-
cokinetics, Bristol-Meyers Squibb Pharmaceutical Research Insti-
tute, Princeton, NJ 08543-4000.
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