THE JOURNAL OF BIOLOGICAL CHEMISTRY
2773–2779, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Stoichiometry and Kinetics of the High-affinity H1-coupled Peptide
Transporter PepT2*
(Received for publication, July 20, 1998, and in revised form, November 13, 1998)
Xing-Zhen Chen‡, Tong Zhu§, David E. Smith§, and Matthias A. Hediger‡¶
From the ‡Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 and
the §College of Pharmacy and Upjohn Center for Clinical Pharmacology, The University of Michigan, Ann
Arbor, Michigan 48109
Proton-coupled peptide transporters mediate the ab-
sorption of a large variety of di- and tripeptides as well
as peptide-like pharmacologically active compounds.
We report a kinetic analysis of the rat kidney high-
affinity peptide transporter PepT2 expressed in Xeno-
pus oocytes. By use of simultaneous radioactive uptake
and current measurements under voltage-clamp condi-
tion, the charge to substrate uptake ratio was found to
be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indi-
cating that the H1:substrate stoichiometry is 2:1 and 3:1
for neutral and anionic dipeptides, respectively. The
higher stoichiometry for anionic peptides suggests that
they are transported in the protonated form. For D-Phe-
L-Lys, the charge:uptake ratio averaged 2.4 from pooled
experiments, suggesting that Phe-Lys crosses the mem-
brane via PepT2 either in its deprotonated (neutral) or
its positively charged form, averaging a H1:Phe-Lys stoi-
chiometry of 1.4:1. These findings led to the overall con-
clusion that PepT2 couples transport of one peptide mol-
ecule to two H1. This is in contrast to the low-affinity
transporter PepT1 that couples transport of one peptide
to one H1. Quinapril inhibited PepT2-mediated currents
in presence or in absence of external substrates. Oocytes
expressing PepT2 exhibited quinapril-sensitive out-
ward currents. In the absence of external substrate, a
quinapril-sensitive proton inward current (proton leak)
was also observed which, together with the observed
pH-dependent PepT2-specific presteady-state currents
(Ipss), indicates that at least one H1 binds to the trans-
porter prior to substrate. PepT2 exhibited Ipss in re-
sponse to hyperpolarization at pH 6.5– 8.0. However,
contrary to previous observations on various transport-
ers, 1) no significant currents were observed corre-
sponding to voltage jumps returning from hyperpolar-
ization, and 2) at reduced extracellular pH, no
significant Ipss were observed in either direction. To-
gether with observed lower substrate affinities and de-
creased PepT2-mediated currents at hyperpolarized Vm,
our data are consistent with the concept that hyperpo-
larization exerts inactivation effects on the transporter
which are enhanced by low pH. Our studies revealed
distinct properties of PepT2, compared with PepT1 and
other ion-coupled transporters.
* This work was supported by the International Human Frontier
Science Program, Long Term Fellowship (to X.-Z.C.) and by National
Institutes of Health Grants GM35498 (to D. E. S.) and DK43171 (to
M.A.H.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ To whom correspondence should be addressed: Renal Division,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
02115. E-mail: mhediger@rics.bwh.harvard.edu.
This paper is available on line at http://www.jbc.org
Vol. 274, No. 5, Issue of January 29, pp.
Printed in U.S.A.
In kidney and intestine, enzymatic degradation of proteins
and peptides produces oligopeptides that are absorbed across
the brush-border membrane of epithelial cells, followed by
breakdown into free amino acids (1–3). The absorption is car-
ried out by proton-driven cotransport systems, as demon-
Downloaded from http://www.jbc.org/ at University of Manitoba Libraries on June 21, 2015
strated by studies using brush-border membrane vesicles, ep-
ithelial cells in culture, intact epithelial tubules, and
recombined peptide transporters (4 –10). A large variety of di-
and tripeptides, as well as pharmacologically active peptide-
like compounds such as b-lactam antibiotics and angiotensin-
converting enzyme inhibitors, are transported (11–13). Peptide
transporters have been cloned since 1994 (3, 14 –19) and were
shown to accept many oligopeptides and peptide-derived com-
pounds as substrates, highlighting the physiological signifi-
cance of these transporters in nutrient and drug absorption.
Functional studies of these membrane proteins allow a better
understanding of the mechanism of substrate-transporter in-
teraction and may help establishing therapeutic strategies in-
volving peptide-based drugs.
An electrophysiological study of PepT2 has been carried out
recently on a rabbit homologue (10). However, the H1:peptide
stoichiometry is still controversial (6, 10, 13, 20). In the present
study, we report novel characteristics of the rat PepT2 demon-
strated by neutral, anionic, and cationic substrates under var-
ious conditions, using the two-microelectrode voltage-clamp
technique. We simultaneously measured the radiolabeled pep-
tide uptakes and the PepT2-mediated currents under voltage-
clamp conditions in order to determine the H1:substrate stoi-
chiometry. We also demonstrated quinapril-sensitive PepT2-
mediated outward currents, a proton leak, and pH-dependent
presteady-state currents (Ipss).
EXPERIMENTAL PROCEDURES
Isolation of PepT2 Clone and Oocyte Preparation—A kidney cortex
lgt10 cDNA library was screened using a 0.52-kb PepT2 probe gener-
ated by specific PCR primers. The complete PepT2 sequence was iso-
lated and subcloned into PTLN2 plasmid. Sequence analysis of both
strands confirmed a 100% identity to the published PepT2 sequence
(GenBankTM accession number D63149, Ref. 21). Xenopus laevis oo-
cytes were prepared as described previously (22) except that oocytes
were defolliculated by incubating them in the calcium-free Barth’s
solution containing collagenase (3 mg/ml) at 18 °C for 3–3.5 h. Capped
cRNA of rat PepT2 was synthesized by in vitro transcription from
cDNAs in PTLN2 and injected (with 15–25 ng) into oocytes. The same
volumes of H2O were injected as the control.
Electrophysiology—The two-microelectrode voltage-clamp experi-
ments were performed using a commercial amplifier (Clampator One,
model CA-1B, Dagan Co., Minneapolis, MN) and the pCLAMP software
(Version 6, Axon Instruments, Inc., Foster City, CA). In experiments
involving voltage jumps, currents or membrane potentials were re-
corded by digitizing at 150 ms/sample and by the Bessel filtering at 10
kHz. When recording currents at a holding potential, digitization at 0.5
s/sample and filtering at 20 Hz were used. Control solutions used for
extracellular perfusion contained (in mM): NaCl, 100; HEPES, 10;
2773
2774 Kinetics of PepT2
1
MES, 2.5; Tris base 2.5; KCl, 2; CaCl2, 1; MgCl2, 1; pH 5.0 – 8.0 by
N-methyl-D-glucamine or HEPES. After 3;5 min of membrane poten-
tial stabilization following microelectrode impalements, the oocyte was
clamped to the holding potential (Vh) of 250 mV. 100-ms voltage pulses
of between 2160 and 160 mV, in increments of 120 mV, were then
applied, and steady-state currents were obtained as the average values
in the interval from 80 to 95 ms after the initiation of the voltage pulses.
Voltage-clamped Tracer Measurements—Substrate-evoked currents
and uptake of [4-3H-D-Phe]-Ala, [4-3H-D-Phe]-Glu, or [4-3H-D-Phe]-Lys
were simultaneously measured under voltage-clamp conditions, accord-
ing to a method similar to the one described previously (23). The
radioactive phenylalanyl-dipeptides were synthesized by Zeneca Cam-
bridge Research Biochemicals (Northwich, Cheshire, United Kingdom)
and dissolved in 10% aqueous ethanol. The specific radioactivity is 12
Ci/mmol, and the concentration is 1 mCi/ml (or 83.3 mM) for all three
peptides. The unlabeled Phe-Glu and Phe-Lys were synthesized as
described previously (24), and other peptides were purchased from
Sigma. The control solution or HEPES-rich solution (in mM: NaCl, 70;
HEPES, 60; MES, 2.5; KCl, 2; CaCl2, 1; MgCl2, 1; pH 5.5– 6.5 by
N-methyl-D-glucamine) was used for external perfusion. The uptake
solution consisted of the control or HEPES-rich solution plus cold (0.5,

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0.5, or 1 mM of Phe-Ala, Phe-Glu, or Phe-Lys, respectively) and hot
substrate (1.33, 1.33, or 2.67 ml of radiolabeled Phe-Ala, Phe-Glu, or
Phe-Lys, respectively, in 200 ml of solution). The hot substrates con-
tained in the uptake solutions represented only 0.11% of the total
substrates. Before starting tracer uptakes, oocyte was clamped at 280
mV and perfused with substrate-free solution. Then the perfusion was
stopped, and the uptake solution (200 ml) was added manually using a
pipettor, which washed out the substrate-free solution. The uptake
lasted 5 min in the chamber whose volume is about 200 ml and termi-
nated by perfusing (washing) the oocyte with the substrate-free solution.
Statistics and Data Analysis—Experimental results were expressed
in the form of mean 6 S.E. (n), where n indicates the number of oocytes
obtained from at least two different donors. The curve-fitting proce-
dures were performed using the SigmaPlot program (Version 4, Jandel
Scientific Software, San Rafael, CA), and each fitted parameter is
expressed in the form parameter 6 S.E., where S.E. represents the
standard error in the fitting estimates.

RESULTS
Substrate and Proton Affinities—PepT2 mediates the high-
affinity transport of most di- and tripeptides at physiological
membrane potentials (Vm) but not at hyperpolarized Vm (Fig. 1,
A and B). Apparent affinities for both glycyl-dipeptides (Gly-
Glu, Gly-Leu, Gly-Lys) and D-phenylalanyl-dipeptides (Phe-
Glu, Phe-Ala, Phe-Lys) were strongly voltage-dependent and
decreased about 10-fold when hyperpolarizing from 250 to
2160 mV (Table I and Fig. 1B). Although affinities differ
largely according to the charge status of glycyl-dipeptides, the
maximal currents for glycyl-dipeptides remain approximately
the same in the whole voltage range (Fig. 1C). High concentra-
tions of glycyl-dipeptides evoked currents that did not saturate
by hyperpolarization, while modest or low concentrations of
these substrates did saturate (Fig. 2A). In contrast, saturation
at hyperpolarization was observed for phenylalanyl-dipeptides,
FIG. 1. Affinity constants and maximal currents for glycyl-
dipeptides at pH 6.0 and various membrane potentials. A, dose
response of averaged H1-Gly-Glu cotransport currents at membrane
potentials 250 mV () and 2160 mV (ƒ). B, apparent affinity constants
(Km) for Gly-Lys (●), Gly-Leu (E), and Gly-Glu () were determined
when currents elicited by substrates at various concentrations were
measured and fitted to the Hill Equation. Inset, Km for Gly-Leu (E)
shown in an expanded scale. C, maximal currents (Imax) for substrates.
Data are averages derived from four to six oocytes.
TABLE I
Apparent affinity constants for a variety of dipeptides
Currents due to addition of various concentrations of substrates were
measured in oocytes expressing the rat PepT2 and at pH 6.0. Km values
were obtained by fitting the data to the Hill equation using the Sig-
maPlot 4 Program.
a a
Substrate K250
m K2160
m K2160
m /K250
m

even at high concentrations (Fig. 2B), presumably due to rela- mM

tively low substrate affinity (Table I). Depending on pH, sub- D-Phe-L-Glu 48 6 4 520 6 60 11
strate type, and substrate concentration, PepT2 may exhibit D-Phe-L-Ala 75 6 6 570 6 70 7.6
decreases in current with hyperpolarization (Fig. 2) due to low D-Phe-L-Lys 135 6 6 890 6 230 6.6
affinities for substrates. The affinities for cationic peptides are L-Gly-L-Glu 16 6 1 490 6 80 30
L-Gly-L-Leu 4.4 6 0.5 30 6 2 6.9
relatively low compared with those for anionic and neutral L-Gly-L-Lys 51 6 3 560 6 10 11
peptides, in analogy to PepT1 (25, 26).
The proton affinities were determined at various membrane
a
K250
m 5 Km at 250 mV; K2160
m 5 Km at 2160 mV.
potentials using glycyl-dipeptide concentrations approximately
affinities for proton at Vm 5 250 mV were determined when
equal to five times that of their Km values. Substrate-evoked
data were fitted to the Hill Equation. The apparent affinity
currents generally increase with an increase in [H1], which is
constants were 2.8 6 0.5 (n 5 6), 0.7 6 0.1 (n 5 3), and 0.5 6
consistent with a proton-driven transport. However, as alluded
0.1 mM (n 5 5) (corresponding to pH 5.5, 6.1, and 6.3) for
to above, currents evoked by low substrate concentrations (es-
Gly-Glu, Gly-Leu, and Gly-Lys, respectively. The Hill coeffi-
pecially those evoked by cationic substrates) decreased with
cients were 1.06 6 0.03, 1.27 6 0.09, and 1.44 6 0.06, corre-
increasing [H1] and hyperpolarization (Fig. 2, D and E). The
spondingly. This appears to suggest the coupling of a single
proton ion to Gly-Glu and more than one proton ion to Gly-Leu
1
The abbreviations used is: MES, morpholineethanesulfonic acid. and Gly-Lys. However, Hill coefficients obtained at other Vm
 Kinetics of PepT2
FIG. 2. Comparison of voltage de-
pendence of PepT2-mediated cur-
rents under different pH and sub-
strate concentrations. A, currents
evoked by Gly-Glu ranging from 10 to 500
mM, pH 6.0. B, currents evoked by Phe-Ala
from 10 to 500 M. C, currents elicited by
250 mM Gly-Glu at various pH between
7.5 and 5.0, in increments of 0.5 pH units.
D, currents elicited by 50 mM Gly-Leu at
pH between 8.0 and 5.0. E, currents elic-
ited by 500 mM Gly-Lys at pH between 8.0
and 5.0. F, proton dependence of PepT2-
mediated currents evoked by 50 mM Gly-
Leu at Vm of 250 (●) and 2100 mV (E).
Data shown in this panel are extracted
from D.
were significantly different, indicating that this method is not
accurate for stoichiometry evaluation. In addition, since cur-
rents at low pH experienced decreases at hyperpolarized Vm
(see Fig. 2F, at 2100 mV), they no longer obey the Michaelis-
Menten or the Hill relationships. Thus, it is inaccurate to
evaluate stoichiometric ratios based on Hill coefficients. More
direct approaches are necessary to determine the H1:substrate
stoichiometry.
Determination of Stoichiometry by Tracer Method—The pro-
ton:substrate coupling ratio (stoichiometry) can be accurately
determined when the proton and substrate fluxes mediated by
PepT2 are measured under the same conditions. Simultaneous
monitoring of transporter-specific currents and radioactive
substrate uptakes from the same oocyte under voltage-clamp
conditions is one of the few approaches proven to be accurate.
Voltage-clamp condition is critical to monitor the PepT2-spe-
cific currents, because background currents (before substrate
addition) change due to depolarization that is elicited by sub-
strate addition.
We observed that, during the uptake period, substrate-elic-
ited currents significantly decreased after reaching initial peak
values (Fig. 3, A–C), which poses the question of whether these
decreases interfere with stoichiometry determination. De-
creases were less pronounced when the buffer concentration
was high (60 versus 10 mM HEPES). For a given buffer concen-
tration, decreases in current were less pronounced when
PepT2-specific currents were lower. With similar initial peak
currents, Phe-Glu-evoked currents exhibited more decrease
than Phe-Ala-evoked currents. We also observed a spike cur-
rent upon perfusing an oocyte with substrate-free solution after
substrate applications. The spikes were significantly higher
2775
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when the buffer concentration was lower. In the same oocytes,
decreases in Gly-Leu-evoked current were also present during
continuous perfusion but were accelerated when perfusion was
stopped (not shown). These data indicate that decreases in
current were partly due to decreased proton concentrations at
the immediate proximity of the extracellular membrane. De-
creases in current during perfusion may be due to intracellular
substrate or H1 accumulation (trans-inhibition), in analogy to
observations for other transporters (34, 35). None of these
factors are expected to compromise the accuracy of stoichiom-
etry determination.
No significant difference in the charge:uptake ratios were
observed when using either control (10 mM HEPES) or HEPES-
rich solution (see “Experimental Procedures”). When charge
was converted into picomole, the charge to uptake ratios for
Phe-Glu, Phe-Ala, and Phe-Lys were determined and averaged
1.91 6 0.04 (n 5 11), 2.16 6 0.02 (n 5 8), and 2.42 6 0.03 (n 5
16), respectively (Fig. 3D). Data obtained using either solution
were both plotted in Fig. 3D. These results indicate that the
number of protons cotransported is 3 and 2, respectively, for
each anionic and neutral peptide transported. The cotrans-
ported proton number per Phe-Lys molecule is 1.4, which is
significantly larger than 1, suggesting the possibility that Phe-
Lys is transported either under the deprotonated (neutral)
form (60%) or under the positively charged form (40%).
Quinapril-inhibited PepT2-mediated Currents—Quinapril,
an angiotensin-converting enzyme inhibitor, was found to in-
hibit the PepT2-mediated currents in oocytes expressing PepT2
(Fig. 4, A, B, and D) but had no effects on control oocytes. The
inhibition was demonstrated to be noncompetitive with an IC50
close to 1 mM by an independent study (data from our labora-
2776 Kinetics of PepT2
FIG. 3. Stoichiometry determination by simultaneous meas-
urements of substrate-evoked currents and tracer uptakes un-
der voltage-clamp conditions (Vh 5 280 mV). A, representative
examples of currents generated by 500 mM Phe-Glu (cold 1 hot). Re-
cordings were obtained using solutions containing 10 mM (left panel) or
60 mM HEPES (right panel) (see “Experimental Procedures” for detail).
The charge moved was calculated by integrating the Phe-Glu-evoked
current over the uptake period. B, representative examples using 500
mM Phe-Ala. C, representative examples using 1 mM Phe-Lys (see the
scale difference). D, charge moved was converted to pmol and plotted
against radiolabeled uptake. The slopes of the linear fits corresponding
to Phe-Lys (), Phe-Ala (E), and Phe-Glu (●) are 2.44 6 0.02, 2.15 6
0.02 and 1.88 6 0.03, respectively.
tory).2 Both inward and outward currents were inhibited (Fig.
4, A–C), indicating that there exists a reversed transport mode.
In the absence of external substrates, an inward quinapril-
sensitive proton current (proton leak) was observed in oocytes
expressing the rat PepT2 (Fig. 4, D and E). Similar uncoupled
leak currents exist in several other transporters (see Refs. 23,
34, and 37). Together with the observation that no currents
were elicited by substrate addition at high pH (see Fig. 2, C–E),
our data demonstrates that H1 binds to PepT2 prior to the
substrate. Quinapril-sensitive proton leaks exhibited similar
decreases with hyperpolarization compared with currents
evoked by low external substrate concentrations, indicating
that this is a characteristic property of PepT2.
PepT2 Presteady-state Currents—By applying voltage jumps
from 250 mV to hyperpolarized potentials, PepT2 exhibited
pH-dependent presteady-state currents (Ipss) (Fig. 5). At pH
5.0 – 6.0, no significant or low Ipss were observed in the whole
voltage range tested. The PepT2-specific charge displacement
2
Zhu, T., Chen, X.-Z., Hediger, M. A., and Smith, D. E., manuscript
in preparation.
(Q, obtained by eliminating the capacitative component) at pH
7.0 did not saturate up to 2160 mV (see Fig. 5E). Since Q at pH
6.0 saturated by hyperpolarization, it is obvious from the figure
that the maximal charge displacement (Qmax) at pH 6.0 repre-
sents less than 15% of the value at pH 7.0. Surprisingly, at the
tested pHo range (5.0 – 8.0), no significant Ipss were observed for
the off-responses (returning from test potentials Vm to Vh) (see
examples in Fig. 5, A–C) and Ipss at pH 6.0 recovered by
addition of substrate (Fig. 5D; steady-state currents were sub-
tracted). These abnormal behaviors of PepT2 are not readily
accounted for by previously described kinetic models (see
“Discussion”).
Between 2160 and 280 mV, relaxation time constants (t) of
Ipss for the on-responses ranged from 10 to 40 ms. With exter-
nal alkalization, the Vm corresponding to the maximal t shifted
to more negative values (Fig. 5F), similar to other transporters
such as PepT1 (9) and SGLT1 (27). At more depolarized poten-
tials, t could not be evaluated due to small Ipss. t was only
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slightly affected by intracellular acidification using a previ-
ously reported approach (9) (not shown), in contrast to t of
PepT1 that showed large responses (9).
DISCUSSION
Using biophysical approaches, we have revealed unique ki-
netic properties of rat PepT2 expressed in Xenopus oocytes.
These include the stoichiometries for different types of sub-
strates determined under voltage-clamp conditions, the proton
leak, and the reversed transport mode as well as presteady-
state currents.
Stoichiometry—The number of proton ions (“n”) necessary for
cotransporting one substrate molecule (stoichiometry “n”) de-
termines the concentration of intracellular substrate that cells
can keep at equilibrium. A high H1:substrate ratio allows
maintenance of high intracellular substrate concentrations but
also requires a relatively large amount of H1 electrochemical
energy. Earlier studies on isolated intestinal tissue prepara-
tions and the Caco-2 cell line derived a H1:peptide ratio of
greater than 2:1 based on measurements of short circuit cur-
rents and peptide fluxes (5, 28). In contrast, according to equi-
librium intracellular substrate estimation (14) and Hill plot
analysis (13, 26, 29) using Xenopus oocytes expressing low
affinity PepT1, a 1:1 coupling ratio was deducted for neutral
substrates. More recently, our laboratory used current and
tracer measurements to study the stoichiometry of PepT1 for
neutral and charged peptides (25). These experiments revealed
1:1, 2:1, and 1:1 ratios for neutral, anionic, and cationic pep-
tides, respectively.
Studies of the high-affinity peptide transporter using rat
kidney brush-border membrane vesicles (6) and oocytes ex-
pressing rabbit renal PepT2 (10, 13) revealed a Hill coefficient
(nH) close to 1 for charged and neutral peptides, suggesting a
1:1 stoichiometry. However, using brush-border membrane
vesicles of rat kidney cortex, Temple et al. (20) found a nH close
to 1 and 2 for neutral and anionic dipeptides, respectively, and
suggested a stoichiometry of 1:1 and 2:1, correspondingly.
Since nH value may depend on the substrate concentration and
binding cooperativity of different protons in case of coupling to
multiple protons, nH is usually not equal to the actual stoichi-
ometric ratio. Protons are also known to have multiple effects
on membrane proteins as well as on substrates (30). The eval-
uation of nH necessitates experiments to be performed over a
wide pH range. However, proteins and/or substrates might not
have the same activity in the whole range, which would signif-
icantly influence the profile of current versus proton concentra-
tion ([H1]). Although in epithelial cells PepT2 is in contact with
a luminal unstirred layer, which has a pH ranging between 5.5
 Kinetics of PepT2 2777

FIG. 4. Inhibition of PepT2-specific

currents by quinapril. A, time course of
total currents in the presence of 50 mM
Gly-Leu upon voltage pulses from a hold-
ing potential of 250 mV to final potentials
ranging between 2160 and 160 mV, each
separated by 20 mV. For clarity, only cur-
rents corresponding to Vm of 2160, 2120,
280, 250, 220, 120, and 160 mV are
shown. B, time course of currents in the
presence of both 50 mM Gly-Leu and 1.2
mM quinapril, obtained from the same oo-
cyte as in A. C, quinapril-sensitive cur-
rents in presence 50 mM Gly-Leu as ob-
tained by subtracting the recording in B

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from that in A. The inset is a depiction at
depolarized potentials, highlighting out-
ward currents at depolarized potentials.
D, quinapril inhibition of PepT2-mediated
currents at a holding potential of 250
mV. Solid and hatched bars represent ap-
plications of 25 mM Gly-Leu and 1.2 mM
quinapril, respectively. E, PepT2-specific
quinapril-sensitive currents in the ab-
sence of external substrate (●, also inset)
were compared with currents due to addi-
tion of 50 mM Gly-Leu (E) in the same
oocytes. Represented data were mean val-
ues from five oocytes.

and 6.0 (31), and proton affinity constants of PepT2 are around
pH 6.0 (see “Results”), PepT2-mediated currents decreased when
pHo approached 5.0 – 6.5 (Fig. 2). This means that PepT2 cur-
rents no longer obey the Michaelis-Menten or Hill relationships.
Our results from simultaneous measurements of radiola-
beled peptide uptakes and PepT2-mediated currents under
voltage-clamp condition show that the charge:uptake ratios are
close to 2 for both anionic and neutral substrates. These data
indicate that PepT2 possesses two H1- and one substrate-
binding sites. In the case of anionic substrates, one additional
proton is needed, most likely for substrate protonation before or
during binding. Because of the hydrophobic environment pro-
vided by the membrane and the transporter, the charges that
the loaded transporter is permitted to carry within the mem-
brane should be well controlled and relatively constant (12 for
neutral and anionic dipeptides). In the case of cationic sub-
strates (S1) such as Gly-Lys and Phe-Lys, the charges on the
loaded transporter (2H1 and 1S1) are 13, and the cotransport
might be unfavorable. The lysine residue under physiological
pH range (pH 5.0 – 8.5) predominantly carries one positive
charge. However, PepT2 protein might help in deprotonating
(neutralizing) the lysine residue even at external physiological
pH and, thereby, transport the resulting neutral form of dipep-
tide by coupling to two protons. This process is apparently
equivalent to the stoichiometry of 1H1:1S1 and corresponds to
carried charges of 12. Observed charge:Phe-Lys ratio of 2.4 is
consistent with the interpretation that both cationic and neu-
tral forms of Phe-Lys are transported. The former (2H1:1S1)
accounts for 40% and the latter (1H1:1S1) accounts for 60% of
observed Phy-Lys-evoked currents. However, neither of these
two coupling mechanisms satisfies both the 2:1 H1:substrate
stoichiometry requirement and the 2:1 charge:substrate ratio
requirement, which may explain observed significantly lower
affinities for cationic substrates compared with neutral or an-
ionic substrates.
The characteristics of high stoichiometry and overall high
affinity of PepT2 as compared with PepT1 are consistent with
its S3 localization in the kidney (32), where PepT2 can effi-
ciently reabsorb peptides using higher electrochemical energy
of protons. In contrast, the 1:1 stoichiometry and low affinity of
PepT1 allow economic and efficient substrate absorption in the
intestine and early parts of renal proximal tubules.
The Reversed Transport Mode—As required by the principle
of microscopic reversibility, reversed transport must occur pro-
vided that substrates are available in the intracellular side of
the membrane. Usually, forward and reversed transports are
not symmetrical in terms of substrate/ion affinity. Transport by
SGLT1 exhibits a strong inward rectification in both cotrans-
port and Na-leak modes, as revealed using the cut-open oocyte
technique (33). Reversed transport has been demonstrated by
using transporter-specific inhibitors in oocytes expressing
SGLT1 (by phlorizin, Ref. 33), EAAC1 (by kainate, Ref. 38), and
SDCT1 (by phloretin, Ref. 23), etc. Reversed transport depends
on the availability of sufficient levels of substrates inside the
oocyte. However, no significant levels of dipeptides were ob-
served in Xenopus oocytes (39) as they are efficiently digested
by intracellular peptidases. The observed quinapril-inhibitable
outward currents of PepT2 might originate either from re-
2778 Kinetics of PepT2

FIG. 5. Rat PepT2-specific pre-

steady-state currents (Ipss). The hold-
ing potential was 250 mV. For clarity,
only currents at final potentials of 2160,
2140, 2120, and 140 mV were shown.
Time between two consecutive jumps was
650 ms. Steady-state currents were re-
moved from all recordings by base-line
subtraction using the Pclamp6 Program.
A–C, Ipss were recorded in the absence of
external substrates at pH 7.0, 6.5, and
6.0, respectively. D, Ipss were evoked by
250 mM Gly-Glu at pH 6.0. E, charge dis-

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placements upon voltage jumps from 250
mV to various test potentials were meas-
ured by integrating the currents versus
time and eliminating the capacitative
component that was determined when
data were fitted to the sum of two expo-
nential functions. F, relaxation time con-
stants (t) at various Vm and pH values.

versed cotransport of unknown PepT2 substrates or from pro-

ton-leak current.
Presteady-state Properties of PepT2—Transporters and chan-
nels possess charged residues within the membrane which, in
response to voltage changes (jumps) applied across the mem-
brane, move (or relax) to new equilibrium positions, thus gen-
erating electrical signals (i.e. currents). These currents vanish
when the new equilibrium (steady state) is reached and are
therefore called presteady-state currents (Ipss). Binding/disso-
ciation of coupling ions or substrates are often voltage-depend-
ent and contribute to the observed Ipss. Characterization of
these currents provides unique information on properties of
membrane transporters.
We propose a kinetic model to help understanding how
PepT2 Ipss are associated with conformational changes from
one steady state to another (Fig. 6). For simplicity, the model
was drawn with ordered binding and mirror symmetry, in
analogy to models proposed for PepT1 (9, 29). At low external FIG. 6. Schematic illustration of a kinetic model for PepT2.
driving-ion concentrations and in the absence of external sub- Each configuration represents a state. Outside, from states I to IV, two
strate, the transporter should be predominantly inward facing proton ions and one substrate (S) orderly bind to the protein. Fully
(states I9 to IV9, Fig. 6). Ipss elicited under these (or similar) loaded configuration (state IV) undergoes a conformational change dur-
ing which two H1 and one S are translocated across the membrane.
conditions have been interpreted as being associated with con- Subsequently, the dissociation of these ions and substrate occurs, as
formational changes of the unloaded transporter from the in- represented by reaction steps from state IV9 to I9 (inside). When the free
ward-faced to the outward-faced configurations (27, 36). Con- carrier changes from the inward-faced state (I9) to the outward-faced
versely, when [H1]o is high, previous kinetic models predict state (I), the whole cycle of forward transport is accomplished, which
generates a net inward current. In the presence of intracellular H1 and
that the transporter is predominantly outward facing (states II substrates, the backward (reversed) transport can occur as well, pro-
and III) and that voltage jumps to depolarized Vm generate ducing outward currents. After binding of either one or two H1, the
positive Ipss. However, these predictions were not applicable to translocation across the membrane may occur in the absence of peptide
PepT2 (see below), even though they were extensively verified substrates, generating observed H1 leaks.
in a number of transporters such as rat PepT1 (9), human
PepT1 (29), and SGLT1 (27, 36). The reported models for these vided that the driving ion is still present and 2) the charge
transporters also predict that 1) the maximal charge displace- movement associated with the on-response from Vh to a test
ment (Qmax) is independent of driving-ion concentration pro- potential Vm (Qon) is equal to that corresponding to the off-
 Kinetics of PepT2
response from Vm to Vh (Qoff), consistent with strict charge
conservation.
As alluded to above, these predictions are not applicable to
PepT2. First, Qmax was greatly reduced at low pHo (Fig. 5E).
Second, in contrast to Qon, Qoff (from hyperpolarized Vm to Vh of
250 mV) was negligible at any pHo tested. Third, in contrast to
previous observations that the presence of external substrate
diminishes or eliminates Ipss (see SGLT1, Ref. 36; the Na1-
iodide transporter, Ref. 34; PepT1, Ref. 9), PepT2-specific Ipss
were increased by substrate addition at low pHo (Fig. 5D).
Finally, when the time interval separating two consecutive
voltage jumps (Vh 5 250 mV) was reduced from 650 to 200 or
50 ms, Ipss were largely reduced after the first jump (to 2160
mV), whereas there was no significant reduction in Ipss when
using time intervals of 650 ms or longer (not shown).
These observations indicate that hyperpolarization results in
transporter inactivation, which can be reversed by depolariza-
tion (250 mV) during a 650-ms period. Hyperpolarization ap-
pears to have inactivation effects on steady-state properties as
well. At low pHo, hyperpolarization resulted in decreases in
substrate-evoked current (Fig. 2), substrate affinity (Table I
and Fig. 1B), and proton leak (Fig. 4E). External substrate
appears to prevent the transporter from being inactivated by
hyperpolarization, as we can see from increases in Ipss by
substrate addition (Fig. 5D) and from increases in maximal
steady-state currents evoked by saturating substrate concen-
trations (Fig. 1C). Mechanisms underlying such a hyperpolar-
ization-stimulated inactivation are still unclear.
Although PepT2 possesses considerable sequence homology
to PepT1 (;50% identity), our findings revealed profound dif-
ferences in several aspects such as stoichiometry, substrate
affinity, effects of hyperpolarization, and presteady-state prop-
erties. While PepT1 was shown to exhibit remarkable symmetry
with respect to the effects of intra- and extracellular pH on Ipss,
more studies are needed to elucidate corresponding effects of pH
on PepT2. It remains to be seen whether binding of an additional
proton in PepT2 is the origin of some of these differences.
Acknowledgments—We are grateful to Dr. Donald W. Hilgemann for
valuable discussions and suggestions on the manuscript and to Dr.
Ji-Bin Peng for valuable discussions and technical assistance. Radioac-
tive and cold phenylalanyl-dipeptides were kindly provided by Dr.
C. A. R. Boyd.
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MEMBRANES AND BIOENERGETICS:
Stoichiometry and Kinetics of the
High-affinity H +-coupled Peptide
Transporter PepT2

Xing-Zhen Chen, Tong Zhu, David E. Smith

and Matthias A. Hediger
J. Biol. Chem. 1999, 274:2773-2779.
doi: 10.1074/jbc.274.5.2773

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