Electrophysiologic Characterization of an Organic Anion

Transporter Cloned from Winter Flounder Kidney (fROAT)

BIRGITTA CHRISTINA BURCKHARDT, NATASCHA A. WOLFF, and
GERHARD BURCKHARDT
Zentrum Physiologie und Pathophysiologie, Georg-August Universitat Gottingen, Gottingen, Germany.
Abstract. The two-electrode voltage clamp technique was used
to demonstrate translocation of p-aminohippurate (PAH) and
related compounds such as loop diuretics in Xenopus laevis
oocytes expressing the renal organic anion transporter from
winter flounder kidney (fROAT). In fROAT-expressing oo-
cytes, PAH (0.1 mM) induced a depolarization of 4.2 0.4
mV and at a holding potential of 60 mV an inward current of
22.6 3.5 nA. PAH-induced current and the current calcu-
lated from [
3
H]-PAH uptake were of similar magnitude. De-
polarization, inward current, and current-to-uptake relation in-
dicated exchange of the monovalent PAH with a divalent
anion, possibly -ketoglutarate (-KG), causing net efflux of
one negative charge. The kinetic analysis of PAH-induced
currents revealed that translocation is dependent on membrane
potential, saturable with an apparent K
m
of 58 8 M, and
sensitive to probenecid and furosemide. In contrast to probenecid
and furosemide, the loop diuretics bumetanide, ethacrynic acid,
and tienilic acid and the nephrotoxic mycotoxin ochratoxin A
elicited inward currents indicating translocation through
fROAT. Substrate-dependent currents provide a tool to eluci-
date the structure/function relationship of the renal organic
anion transporter.
The renal extraction of organic anions, including xenobiotics,
loop diuretics, and other drugs from the blood, exceeds the
filtered load, indicating that tubule cells take up these anions
from the blood through their basolateral membrane (1). Stop-
flow and microperfusion experiments have proven that the
proximal tubule is the main site of organic anion secretion. The
mechanisms of secretion are delineated mainly from investi-
gations of few transported compounds, considered as represen-
tative of other secreted organic anions, for example, p-amino-
hippurate (PAH). PAH transport by basolateral membranes, as
shown by studies in rat (24), pig (5), bovine (6), and rabbit
(7,8) membrane preparations, occurs through an exchange with
dicarboxylates, which are subsequently returned into the cell
via a Na

-dependent dicarboxylate transporter. The affinity of

the PAH/anion exchanger is high for -ketoglutarate (-KG),
the only natural dicarboxylate accepted by the transporter.
Other dicarboxylates, such as succinate, malonate, and oxalate,
are not substrates for the exchanger. Nonphysiologic dicar-
boxylates such as glutarate, adipate, and suberate can substitute
for -KG (reviewed in reference (9).
The knowledge of the structure of the PAH/-KG exchanger
is necessary to understand how the transporter is able to ac-
commodate a large variety of substrates (reviewed in refer-
ences 9 and 10). Several groups have recently succeeded in
expression- or homology-cloning of cDNAs encoding rat (11
13), mouse (14), human (1518), and winter flounder (19)
organic anion transporters. Common to all of these transporters
is their operation as PAH/-KG exchangers. Demonstration
of transport of other substrates besides PAH thus far is
limited to the studies of Sekine et al., Uwai et al., and Lu et
al. (11,13,17), who studied tracer uptake of methotrexate,
cAMP, cGMP, PGE
2
, urate, -KG, and probenecid in Xe-
nopus oocytes or in HeLa cells expressing the organic anion
transporter.
Most substrate specificity studies are based on the ability of
a substrate to compete for transport with PAH. Such studies
demonstrate an affinity of the carrier system for organic anions
but do not establish transport per se. Electrophysiologic mea-
surements provide the chance to discriminate between mere
binding to the carrier and transport of a substrate. Therefore,
we applied two-electrode voltage clamp studies to elucidate the
effects of several potential substrates, including some clinically
important loop diuretics, on the basolateral PAH/-KG ex-
changer cloned from winter flounder kidney, fROAT (flounder
renal organic anion transporter) (19).
Materials and Methods
Oocyte Preparation and Maintenance
Stage V and VI oocytes from Xenopus laevis (Nasco, Fort Atkin-
son, WI) were manually dissected and stored in control solution (for
composition, see Solutions). They were injected with 30 nl of cRNA
(1 g/l) encoding fROAT 1 d after their isolation and maintained at
16 to 18C in control solution supplemented with 100 kU/L penicillin,
0.1 mg/L streptomycin, and 2.5 mM sodium pyruvate. After 3 to 4 d
of incubation, oocytes were used for the electrophysiologic as well as
for the uptake studies.
Received April 29, 1999. Accepted July 15, 1999.
Correspondence to Dr. Birgitta-Christina Burckhardt, Zentrum Physiologie
und Pathophysiologie, Georg-August Universitat Gottingen, Humboldtallee
23, D-37073 Gottingen, Germany. Phone: 49 551 395880; Fax: 49 551
395883; E-mail: bcburckhardt@veg-physiol.med.uni-goettingen.de
1046-6673/1101-0009
Journal of the American Society of Nephrology
Copyright 2000 by the American Society of Nephrology
J Am Soc Nephrol 11: 917, 2000
Electrophysiology
Oocytes were placed in a chamber that was continuously perfused
at a rate of 4 ml/min with control or different test solutions at room
temperature. The two-electrode voltage clamp method (Warner OC
725 A, B, New Haven, CT) was used either in the current clamp mode
to analyze potential changes upon PAH addition or in the voltage
clamp mode to analyze currents evoked by PAH. The holding poten-
tial of the oocytes was 60 mV, which resembles the cell membrane
potential of a proximal tubule cell in situ. In a series of 10-s pulses, the
membrane potential was clamped at values ranging from 90 to 10
mV to obtain current/voltage (I/V) relations. Upon initiation of the
clamp, there was a capacitative transient and currents reached steady
state within less than 1 s. The reported steady-state currents for I/V
relations were taken from the last 5 s of the voltage step. In general,
the I/V protocol was applied under control conditions and 30 s after
changing the perfusion to the test solution. The substrate-evoked
currents were evaluated as the difference between currents recorded
before and after substrate addition. Experimental results were ex-
pressed as means SEM (n), where n indicates the number of oocytes
investigated from at least two different donors.
Comparison between Current and Tracer PAH Uptake
One approach consisted of measuring PAH-induced current and
[
3
H]-PAH uptake on the same fROAT-expressing oocyte. After de-
termination of the resting membrane potential in the current clamp
mode, the fROAT-expressing oocyte was clamped to this potential
and the current induced by 0.1 mM PAH was recorded. Afterward,
uptake of [
3
H]-PAH (5 Ci/ml) (aminohippuric acid, P-[glycyl-2-
3
H],
1.28 Ci/mmol, New England Nuclear, Cologne, Germany) was as-
sayed for 60 min at room temperature in control solution containing
0.1 mM unlabeled and 0.0039 mM labeled PAH. The uptake was
terminated by aspiration of the incubation medium and 3 3 ml
washes with ice-cold control solution. Each individual oocyte was
then dissolved in 0.1 ml of 1N NaOH and, after neutralization with 0.1
ml of 1N HCl, the [
3
H] content was assayed by liquid scintillation
counting. The [
3
H] uptake (mol/s) was converted to current using
Faradays constant.
Similar to the procedure described for [
3
H]-PAH uptake above,
uptake of [
3
H]-bumetanide (5 Ci/ml) (10 Ci/mmol) (generously
provided by Dr. E. Petzinger, Institute of Pharmacology and Toxicol-
ogy, University of Giessen, Germany) was performed using 0.05 mM
unlabeled and 0.005 labeled bumetanide.
Solutions
The control solution was composed of (in mM): 90 NaCl, 3 KCl, 2
CaCl
2
, 1 MgCl
2
, and 5 Hepes/Tris, pH 7.6. All chemicals including
the phenoxyacetates, sulfamoylbenzoates, and ochratoxin A were of
analytical grade and were purchased from Merck (Darmstadt, Ger-
many) or Sigma (Deisenhofen, Germany).
Results
Basic Characterization of the PAH-Induced Currents in
fROAT-Expressing Oocytes
Under current clamp conditions, oocytes injected with
cRNA encoding the organic anion transporter fROAT exhib-
ited a membrane potential of 36.9 3.2 mV (n 13).
Addition of 0.1 mM PAH resulted in a depolarization by 4.2
0.4 mV. When the same oocytes were investigated under
voltage clamp conditions at a holding potential of 60 mV,
they showed an inward current of 22.6 3.5 nA when PAH
was added. A representative example of such an experiment is
presented in Figure 1A. Depolarization (Figure 1A, top trace)
and inward current (Figure 1A, bottom trace) are compatible
with influx of the monovalent PAH in exchange for a divalent
anion, most likely endogenous -KG, leading to one net neg-
ative charge leaving the oocyte, which causes an inward cur-
rent. Changes of cell membrane potential or inward current
upon PAH were absent in water-injected control oocytes (data
not shown). To determine the coupling ratio of PAH uptake to
charge efflux, we first measured PAH-induced current and then
[
3
H]-PAH uptake on the same individual oocyte. This experi-
mental procedure was possible because PAH (0.1 mM) elicited
only a small depolarization, which does not appreciably change
the electrochemical driving force for PAH. Plotting current
calculated from [
3
H]-PAH uptake versus current measured at
60 mV upon PAH, a linear fit with a slope equal to 1.02 (n
15), was obtained (Figure 1B), supporting our assumption of
influx of monovalent PAH in exchange of a divalent anion. In
Figure 1C, the steady-state currents in the absence (f) and
presence () of PAH are plotted as a function of membrane
potential. Upon addition of 0.1 mM PAH, the reversal poten-
tial, E
rev
, shifted from 39.2 5.2 mV to 33.7 4.8 mV
(n 13) and the slope conductance increased. The resulting
PAH-induced current, I
PAH
(F), i.e., the difference between
the current in the presence () and in the absence (f) of PAH,
is shown in Figure 1D. This PAH-induced current increased
with increasing membrane potential and was only observed in
oocytes injected with cRNA coding for fROAT (not shown).
The PAH-induced currents, I, were then studied as a func-
tion of the external PAH concentration (Figure 2A). At 60
mV, the current increased upon increasing PAH concentrations
and tended to saturate. By Eadie-Hofstee analysis of five
oocytes from three donors, a K
m
of 58 8 M was obtained.
Increasing glutarate concentrations applied simultaneously
with PAH (0.1 mM) inhibited the PAH-induced current (Figure
2B). Half-maximal inhibition was observed at approximately
0.2 mM glutarate (n 6). In the absence of PAH, glutarate
evoked only a small outward current in fROAT-expressing
oocytes. In some oocytes, this current was not distinguishable
from zero. In six paired experiments with oocytes from three
donors at 60 mV, the currents expressed by 0.1 mM PAH and
1 mM glutarate were 18.5 2.3 nA and 2.8 2.1 nA,
respectively.
Influence of Different Inhibitors on the
PAH-Induced Currents
Figure 3 summarizes the I/V relations of substrate-depen-
dent steady-state currents, I (n 8), obtained in fROAT-
injected oocytes by 0.1 mM PAH (Figure 3A), by simultaneous
application of 0.1 mM PAH and 1 mM probenecid (Figure 3B),
and by 1 mM probenecid alone (Figure 3C), which were tested
on the same oocyte in random order. Probenecid (1 mM)
abolished the PAH-induced current shown in Figure 3A and
even turned it into an outward current deflection (Figure 3B) at
potentials more negative than 10 to 20 mV. This outward
current was larger at negative potentials. An outward current
was also observed when probenecid (1 mM) was applied alone
10 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 917, 2000
(Figure 3C). The reversal potential, E
rev
, of all of these currents
was in the range of 10 to 20 mV. To determine whether the
outward current deflection observed in the presence of probe-
necid was due to an inhibition of an inward current, the I/V
relations in the absence and presence of PAH were compared
with those obtained in the absence and presence of probenecid
on the same oocyte (Figure 4, A and B). As can be seen from
the steepness of the I/V relations, the conductance in the
presence of PAH increased, whereas the conductance in the
presence of probenecid decreased, indicating inhibition of a
preexisting current by probenecid.
Substrate Specificity of fROAT-Expressing Oocytes
It was shown in the rat proximal tubule in situ (20) that
diuretics of the sulfamoylbenzoate, as well as of the phenoxy-
acetate-type inhibit basolateral [
3
H]-PAH influx. The differ-
ence currents evoked by some sulfamoylbenzoate and phe-
noxyacetate analogues applied in a concentration of 0.1 mM as
measured on 10 oocytes of three donors in random order are
summarized in Figure 5. At 60 mV, the magnitude of the
inward current decreased in the order ethacrynic acid
PAH tienilic acid bumetanide sulfanilamide acet-
azolamide. Sulfanilamide and acetazolamide showed barely
Figure 1. p-Aminohippurate (PAH)-induced currents in oocytes expressing fROAT (flounder renal organic anion transporter). (A) Depolar-
ization and inward current at a holding potential of 60 mV measured in response to application of 0.1 mM PAH during the period indicated
by the horizontal bars. Baseline potential and current have been suppressed to show PAH-induced changes only. The traces were obtained from
a representative oocyte of a batch of 13 oocytes used in this study. (B) Stoichiometry of [
3
H]-PAH uptake (converted to current) to PAH-evoked
current (measured current). Currents evoked by 0.1 mM PAH when oocytes were clamped to their resting potential were compared with those
calculated from [
3
H]-PAH uptake. Current and tracer uptake were performed on the same oocyte. (C) The steady-state currents obtained at the
last 5 s of the voltage step in the absence (f) and presence () of 0.1 mM PAH are plotted as a function of membrane potential. (D) The
difference between the two current/voltage (I/V) relations presented in Panel C is the PAH-induced current, I
PAH
(F), in fROAT-expressing
oocytes. n denotes the number of oocytes tested.
J Am Soc Nephrol 11: 917, 2000 Electrophysiologic Characterization of fROAT 11
detectable currents, indicating slow translocation by fROAT.
The currents observed in the presence of furosemide were
similar to those obtained for probenecid, e.g., furosemide dis-
played outward current deflections. Again, as in the case of
probenecid, the observed outward currents could be due to an
inhibition of a preexisting inward current. Similar to probene-
cid, furosemide applied at a concentration of 1 mM inhibited
the PAH-induced current completely (Figure 6). Bumetanide
transport via fROAT was also demonstrated by uptake of the
radiolabeled compound. [
3
H]-bumetanide uptake was in con-
trol and fROAT-expressing oocytes 12.1 4.4 pmol/h
oocyte and 43.4 2.8 pmol/h oocyte, respectively, in three
independent experiments each carried out on five to 14 oocytes
per treatment.
It was demonstrated in single rabbit renal proximal tubules
(21,22) as well as in opossum kidney cells (23) that the
mycotoxin ochratoxin A (OTA) interacts with the basolateral
Figure 3. Influence of probenecid on the PAH-induced current,
I
PAH
, in fROAT-expressing oocytes. The potential-dependent in-
ward current induced by 0.1 mM PAH (A) was turned into a potential-
dependent outward current by 1 mM probenecid (B). This potential-
dependent outward current was not dependent on the presence of PAH
(C). I/V relations were obtained as described in Figure 1, C and D. All
three experimental conditions were investigated on individual oocytes
in random order.
Figure 2. Steady-state PAH-induced currents, I
PAH
, at 60 mV as a
function of increasing PAH concentrations (A) and in the presence of
increasing glutarate concentrations at a fixed PAH concentration (0.1
mM) (B). The data summarize the results of five oocytes (A) or six
oocytes (B), where all experimental conditions were tested in random
order. Water-injected, control oocytes (E) showed a PAH-induced
current 5 nA at concentrations 0.5 mM PAH.
12 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 917, 2000
PAH transporter via cis-inhibition. In fROAT-expressing oo-
cytes, OTA (0.01 mM) evoked an inward current reversing into
an outward current at 22.6 4.2 mV (Figure 7) compared to
21.0 2.1 mV for PAH. A comparison of the PAH-induced
(0.1 mM) and the OTA-induced (0.01 mM) currents at 60
mV in four paired experiments revealed that on average OTA
was more potent than PAH in evoking a current.
Discussion
Recently, the renal basolateral organic anion transporters
cloned from various species including winter flounder
(fROAT) (19), rat (OAT1 or ROAT1) (1113), mouse
(mOAT) (14), and human (hROAT1, hOAT1, or hPAHt) (15
18) were expressed in Xenopus laevis oocytes, Hela, or COS 7
cells. Substrate specificity of these transporters was character-
ized mostly by cis-inhibition experiments using radiolabeled
PAH as reporter anion and unlabeled anions as inhibitors.
Similar to experiments on the intact kidney (10), these studies
revealed a broad specificity of the substrate binding site of the
organic anion transporters, but did not prove translocation of
competing anions. Sekine et al. (11), Uwai et al. (13), and Lu
et al. (17), however, demonstrated uptake of radiolabeled sub-
strates other than PAH by the cloned transporters, indicating
that chemically unrelated substrates are being translocated. To
further elucidate the spectrum of transported substrates, a
method is desirable that does not depend on the availability of
radiolabeled compounds, e.g., an electrophysiologic approach.
Here we report the first electrophysiologic characterization
of the PAH transporter from winter flounder kidney (fROAT)
as an alternative method to test translocation of substrates.
Addition of 0.1 mM PAH to fROAT-expressing oocytes re-
sulted in a depolarization, an increase in membrane conduc-
tance, and, at a holding potential of 60 mV, an inward
current. The increase in membrane conductance reflects net
translocation of charges through the expressed fROAT. Depo-
larization and inward current at negative clamp potentials
indicate that either positive charges enter, or negative charges
leave, the oocytes upon addition of PAH. On the basis of
earlier findings on renal membranes (110), the most likely
explanation is an exchange of an extracellular monovalent
PAH molecule for an intracellular divalent -ketoglutarate
molecule (-KG), resulting in a surplus of one negative charge
leaving the oocyte per transport cycle. As obtained on the same
oocyte, the current calculated from the uptake of [
3
H]-PAH
and the current measured in the presence of the same PAH
concentration were similar, which is in agreement with the
movement of one charge per transported PAH molecule.
Although PAH-induced -KG efflux from fROAT-express-
ing oocytes has not yet been demonstrated directly, it is likely
that PAH/-KG exchange is the prevailing mode of PAH
uptake. Trans-stimulation of radiolabeled PAH uptake was
observed earlier by preloading of fROAT-expressing oocytes
with glutarate (19) or by coexpression of a Na

-coupled di-
carboxylate cotransporter with OAT1 or hOAT1 (11,16), sup-
porting the notion that the expressed transporters are operating
in the PAH/dicarboxylate exchange mode. Moreover, increas-
ing concentrations of glutarate, a nonmetabolizable analogue
of -KG, in the bath inhibited PAH-induced currents. Glutarate
alone did not produce a current, because the exchange of an
intracellular -KG for an extracellular glutarate is electroneu-
tral. The inhibition of PAH-induced currents by glutarate in the
bath is most likely due to a competition for a common binding
site at fROAT.
Since charge movement is accompanied by PAH uptake, an
inside negative potential should serve as an additional driving
force for PAH/-KG exchange. Indeed, PAH-induced current
was greatest at 90 mV and declined with more positive
potentials. This is in line with studies using basolateral mem-
Figure 4. Current/voltage (I/V) relations of fROAT-expressing oocytes in the absence and presence of PAH (A) or probenecid (B). Data points
under control conditions, i.e., in the absence of PAH or probenecid, are indicated as f, whereas those in the presence of PAH or probenecid
are indicated as . I/V relations in the presence of PAH (0.1 mM) were steeper than those obtained in the presence of probenecid (1 mM).
J Am Soc Nephrol 11: 917, 2000 Electrophysiologic Characterization of fROAT 13
Figure 5. Effect of sulfamoylbenzoate and phenoxyacetate compounds compared with PAH in fROAT-expressing oocytes. For better
comparison, all compounds were applied at a concentration of 0.1 mM. Data were obtained on the same individual oocyte with the test substrate
applied in random order, and means SEM were computed. For furosemide, the currents were corrected by the furosemide-elicited currents
observed in water-injected control oocytes.
14 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 917, 2000
brane vesicles from rabbit proximal tubule (8), in which a
significantly greater [
3
H]-PAH uptake was observed in the
presence of an inside negative compared with an inside posi-
tive potential. In contrast, in in situ measurements on rat kidney
(24), in studies on bovine basolateral membrane vesicles (25),
as well as on ROAT1-expressing oocytes (12), PAH uptake
was unchanged or only marginally affected by either depolar-
ization induced by high K

media or by imposition of an inside

negative potential (K

i
K

o
). Whether the discrepancies in
potential dependence are due to species differences or due to
different experimental approaches is not known. Alternatively,
changes in membrane potential may be without a measurable
effect on an electrogenic transporter, if the rate-determining
step of the overall translocation process consisting of substrate
binding, occlusion, and translocation is voltage-independent.
The reversal potential, E
rev
, of the PAH-induced current was
between 20 and 10 mV. At membrane potentials more
positive than 10 mV, PAH induced an outward current, as if
PAH would flow out of, and -KG into, the oocytes. Unstirred
layers on both sides of the oocytes membrane may lead to
local concentrations of PAH and -KG that deviate from those
in the bulk fluid. In particular, -KG may accumulate close to
the outer surface of the membrane, and PAH at the inner
surface, causing a reversal of PAH/-KG exchange at positive
membrane potentials. As long as the local concentration of
PAH and -KG are not known, the equilibrium potential of
PAH/-KG exchange cannot be predicted. In addition, we
cannot exclude that other ions are directly or indirectly in-
volved in PAH uptake. The E
rev
of PAH-induced current is
close to the chloride equilibrium potential of Xenopus laevis
oocytes (26). In this regard, it is important to mention that a
replacement of Cl

ions by either gluconate or methanesulfo-

nate inhibited PAH/-KG exchange in rat and bovine renal
basolateral membrane vesicles (27,28) and in oocytes express-
ing hOAT1 (16) or fROAT (29). In contrast, no change in
basolateral PAH uptake upon anion replacement was observed
in the rat kidney in situ (24). How Cl

affects PAH/-KG
exchange is not understood and remains to be determined.
At 60 mV, the inward current was concentration-depen-
dent and saturable with a K
m
value of 58 8 M. The K
m
derived from [
3
H]-PAH uptake in fROAT-expressing oocytes
was 21 4 M, and for the winter flounder renal tubules in
vitro K
m
was 157 M (30), respectively. Different K
m
values
were also observed for PAH/-KG exchange of rat kidney: K
m
values for the cloned rat OAT1 transporter were 14.3 2.9
M (11) and 70 M (12), respectively, whereas for the rat
kidney in situ a K
m
of 80 10 M was detected (25).
Opossum kidney cell preparations (31,32) showed K
m
values
ranging from 34 to 64 M. Smaller K
m
values of 9.3 1 M
(16) and 5 M (17) were reported for the heterologously
expressed human OAT1 and hPAHt. The reason for the dif-
Figure 7. Currents induced by PAH (A) and ochratoxin A (OTA) (B) in fROAT-expressing oocytes. Both currents reversed at approximately
22 mV.
Figure 6. Effect of furosemide on PAH-induced currents in fROAT-
expressing oocytes. Currents were corrected for the furosemide-
evoked currents observed in water-injected control oocytes.
J Am Soc Nephrol 11: 917, 2000 Electrophysiologic Characterization of fROAT 15
ferent K
m
values even within one species may be related to
different technical approaches. Nevertheless, the K
m
of approx-
imately 60 M determined in this study is well within the
range of other published results.
In all species tested thus far, PAH transport is inhibited by
probenecid (9). In fROAT-expressing oocytes, the PAH-in-
duced current was abolished by 1 mM probenecid, but probe-
necid itself was not able to induce a current. This result is in
agreement with data obtained by Uwai et al. (13) showing by
HPLC that probenecid was not transported by rat renal OAT1.
From these and our data, we propose that probenecid binds
tightly and is not, or only slowly, translocated. At negative
clamp potentials, probenecid evoked an outward current re-
gardless of whether PAH was present. The slight decrease in
membrane conductance in the presence of probenecid suggests
that the recorded outward current may be due to the inhibition
of an inward current. In the absence of PAH, this inward
current may reflect operation of fROAT with anions present in
the bath, e.g., Cl

/-KG exchange, which is inhibited by

probenecid. No effect of probenecid was seen with water-
injected control oocytes.
Testing 0.1 mM of the phenoxy compounds ethacrynic and
tienilic acid, we found inward currents comparable to those
induced by PAH. Among the sulfamoyl compounds, sulfanil-
amide and acetazolamide did not evoke currents in fROAT-
expressing oocytes, whereas a similar concentration of bumet-
anide did. Hence, sulfanilamide and acetazolamide are not
measurably translocated at a concentration of 0.1 mM, whereas
bumetanide is. To our surprise, the loop diuretic furosemide
did not evoke an inward current in fROAT-expressing oocytes.
Furosemide inhibited PAH-induced currents and caused an
outward current deflection much as that seen with probenecid.
These results suggest that furosemide acts as an inhibitor of
PAH transport through fROAT, but is itself not, or only slowly,
translocated. Since furosemide is therapeutically applied in
humans, it must be clarified whether flounder and human PAH
transporters differ with respect to the specificity of their trans-
location steps or whether furosemide is secreted in proximal
tubules via another route.
OTA is an organic anion causing severe nephrologic dam-
age, i.e., nephropathy. Previous studies have shown that the
renal accumulation of OTA occurs via the PAH/-KG ex-
change from blood into proximal tubule cells (2123). In these
studies, OTA uptake was cis-inhibited by PAH, probenecid,
and -KG. Accordingly, we demonstrate here a very efficient
translocation of OTA by fROAT. The current evoked by 0.01
mM OTA was as large as the current evoked by 0.1 mM PAH.
Taken together, electrophysiologic measurements on oo-
cytes expressing the renal organic anion transporter fROAT
provide a tool to test the specificity of substrate translocation.
In particular, several potential substrates can be tested on a
single oocyte facilitating a direct comparison. Thus far, the
method is restricted to monovalent test anions, because the
exchange of a divalent test anion with -KG is electroneutral
and not detectable with electrophysiologic techniques. Exper-
iments in progress show that this limitation can be overcome
by PAH-preloading of the oocytes, since these oocytes show an
outward current upon exposure to extracellular -KG. System-
atic investigations on transport specificity will provide infor-
mation on the molecular requirement of substrate translocation.
This information is needed to understand the renal excretion of
endogenous and potentially toxic exogenous compounds.
Acknowledgments
We thank I. Markmann and G. Dallmeyer for excellent technical
assistance in the electrophysiologic and tracer uptake experiments,
respectively, and E. Thelen for the artwork.
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