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
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ABSTRACT |
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Proton-coupled peptide transporters
mediate the absorption 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 Xenopus oocytes. By use of simultaneous
radioactive uptake and current measurements under voltage-clamp
condition, the charge to substrate uptake ratio was found to be close
to 2 for both D-Phe-L-Ala and
D-Phe-L-Glu, indicating that the
H+: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
membrane via PepT2 either in its deprotonated (neutral) or its
positively charged form, averaging a H+:Phe-Lys
stoichiometry of 1.4:1. These findings led to the overall conclusion
that PepT2 couples transport of one peptide molecule to two
H+. This is in contrast to the low-affinity transporter
PepT1 that couples transport of one peptide to one H+.
Quinapril inhibited PepT2-mediated currents in presence or in absence
of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward 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
H+ binds to the transporter prior to substrate. PepT2
exhibited Ipss in response to hyperpolarization
at pH 6.5-8.0. However, contrary to previous observations on various
transporters, 1) no significant currents were observed corresponding to
voltage jumps returning from hyperpolarization, and 2) at reduced
extracellular pH, no significant Ipss were
observed in either direction. Together with observed lower substrate
affinities and decreased PepT2-mediated currents at hyperpolarized
Vm, our data are consistent with the concept that
hyperpolarization 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.
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 carried out by proton-driven
cotransport systems, as demonstrated by studies using brush-border
membrane vesicles, epithelial 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 An electrophysiological study of PepT2 has been carried out recently on
a rabbit homologue (10). However, the H+:peptide
stoichiometry is still controversial (6, 10, 13, 20). In the present
study, we report novel characteristics of the rat PepT2 demonstrated by
neutral, anionic, and cationic substrates under various conditions,
using the two-microelectrode voltage-clamp technique. We simultaneously
measured the radiolabeled peptide uptakes and the PepT2-mediated
currents under voltage-clamp conditions in order to determine the
H+:substrate stoichiometry. We also demonstrated
quinapril-sensitive PepT2-mediated outward currents, a proton leak, and
pH-dependent presteady-state currents
(Ipss).
Isolation of PepT2 Clone and Oocyte Preparation--
A kidney
cortex Electrophysiology--
The two-microelectrode voltage-clamp
experiments 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 recorded
by digitizing at 150 µs/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; MES,1 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 potential stabilization following microelectrode
impalements, the oocyte was clamped to the holding potential
(Vh) of 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, according to a method similar to the
one described previously (23). The radioactive phenylalanyl-dipeptides
were synthesized by Zeneca Cambridge Research Biochemicals (Northwich, Cheshire, United Kingdom) and dissolved in 10% aqueous ethanol. The
specific radioactivity is 12 Ci/mmol, and the concentration is 1 µCi/µl (or 83.3 µM) 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, 0.5, or 1 mM of
Phe-Ala, Phe-Glu, or Phe-Lys, respectively) and hot substrate (1.33, 1.33, or 2.67 µl of radiolabeled Phe-Ala, Phe-Glu, or Phe-Lys, respectively, in 200 µl of solution). The hot substrates contained in
the uptake solutions represented only 0.11% of the total substrates. Before starting tracer uptakes, oocyte was clamped at Statistics and Data Analysis--
Experimental results were
expressed in the form of mean ± S.E. (n), where
n indicates the number of oocytes obtained from at least two
different donors. The curve-fitting procedures were performed using the
SigmaPlot program (Version 4, Jandel Scientific Software, San Rafael,
CA), and each fitted parameter is expressed in the form parameter ± S.E., where S.E. represents the standard error in the fitting estimates.
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
The proton affinities were determined at various membrane potentials
using glycyl-dipeptide concentrations approximately equal to five times
that of their Km values. Substrate-evoked currents
generally increase with an increase in [H+], which is
consistent with a proton-driven transport. However, as alluded to
above, currents evoked by low substrate concentrations (especially
those evoked by cationic substrates) decreased with increasing
[H+] and hyperpolarization (Fig. 2, D and
E). The affinities for proton at Vm = Determination of Stoichiometry by Tracer Method--
The
proton: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-specific currents, because background currents
(before substrate addition) change due to depolarization that is
elicited by substrate addition.
We observed that, during the uptake period, substrate-elicited currents
significantly decreased after reaching initial peak values (Fig.
3, A-C), which poses the
question of whether these decreases interfere with stoichiometry
determination. Decreases were less pronounced when the buffer
concentration was high (60 versus 10 mM HEPES).
For a given buffer concentration, 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 current upon
perfusing an oocyte with substrate-free solution after substrate
applications. The spikes were significantly higher 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. Decreases in current during perfusion may be due to
intracellular substrate or H+ accumulation
(trans-inhibition), in analogy to observations for other transporters
(34, 35). None of these factors are expected to compromise the accuracy
of stoichiometry 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 ± 0.04 (n = 11), 2.16 ± 0.02 (n = 8),
and 2.42 ± 0.03 (n = 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 cotransported 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 inhibit 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
laboratory).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
H+ 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
Between Using biophysical approaches, we have revealed unique kinetic
properties of rat PepT2 expressed in Xenopus oocytes. These include the stoichiometries for different types of substrates 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") determines the concentration of intracellular
substrate that cells can keep at equilibrium. A high
H+:substrate ratio allows maintenance of high intracellular
substrate concentrations but also requires a relatively large amount of H+ electrochemical energy. Earlier studies on isolated
intestinal tissue preparations and the Caco-2 cell line derived a
H+:peptide ratio of greater than 2:1 based on measurements
of short circuit currents and peptide fluxes (5, 28). In contrast, according to equilibrium 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 peptides, respectively.
Studies of the high-affinity peptide transporter using rat kidney
brush-border membrane vesicles (6) and oocytes expressing 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
stoichiometric ratio. Protons are also known to have multiple effects
on membrane proteins as well as on substrates (30). The evaluation 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 significantly influence
the profile of current versus proton concentration
([H+]). Although in epithelial cells PepT2 is in contact
with a luminal unstirred layer, which has a pH ranging between 5.5 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
currents no longer obey the Michaelis-Menten or Hill relationships.
Our results from simultaneous measurements of radiolabeled 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
H+- 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 provided by the membrane and the transporter, the charges
that the loaded transporter is permitted to carry within the membrane should be well controlled and relatively constant (+2 for neutral and
anionic dipeptides). In the case of cationic substrates
(S+) such as Gly-Lys and Phe-Lys, the charges on the loaded
transporter (2H+ and 1S+) are +3, 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 dipeptide by
coupling to two protons. This process is apparently equivalent to the
stoichiometry of 1H+:1S+ and corresponds to
carried charges of +2. Observed charge:Phe-Lys ratio of 2.4 is
consistent with the interpretation that both cationic and neutral forms
of Phe-Lys are transported. The former
(2H+:1S+) accounts for 40% and the latter
(1H+:1S+) accounts for 60% of observed
Phy-Lys-evoked currents. However, neither of these two coupling
mechanisms satisfies both the 2:1 H+: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 anionic 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 efficiently 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 provided 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 cotransport 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 observed in
Xenopus oocytes (39) as they are efficiently digested by
intracellular peptidases. The observed quinapril-inhibitable outward
currents of PepT2 might originate either from reversed cotransport of
unknown PepT2 substrates or from proton-leak current.
Presteady-state Properties of PepT2--
Transporters and channels
possess charged residues within the membrane which, in response to
voltage changes (jumps) applied across the membrane, move (or relax) to
new equilibrium positions, thus generating 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/dissociation of coupling ions or substrates are often
voltage-dependent 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 driving-ion concentrations and in
the absence of external substrate, the transporter should be
predominantly inward facing (states I' to IV',
Fig. 6). Ipss elicited under these (or similar)
conditions have been interpreted as being associated with
conformational changes of the unloaded transporter from the
inward-faced to the outward-faced configurations (27, 36). Conversely,
when [H+]o is high, previous kinetic models
predict that the transporter is predominantly outward facing (states II
and III) and that voltage jumps to depolarized Vm
generate positive Ipss. However, these
predictions were not applicable to PepT2 (see below), even though they
were extensively verified in a number of transporters such as rat PepT1
(9), human PepT1 (29), and SGLT1 (27, 36). The reported models for
these transporters also predict that 1) the maximal charge displacement
(Qmax) is independent of driving-ion
concentration provided that the driving ion is still present and 2) the
charge movement associated with the on-response from
Vh to a test potential Vm
(Qon) is equal to that corresponding to the
off-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
These observations indicate that hyperpolarization results in
transporter inactivation, which can be reversed by depolarization (
Although PepT2 possesses considerable sequence homology to PepT1
(~50% identity), our findings revealed profound differences in
several aspects such as stoichiometry, substrate affinity, effects of
hyperpolarization, and presteady-state properties. 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.
INTRODUCTION
Top
Abstract
Introduction
References
-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 compounds as
substrates, highlighting the physiological significance of these
transporters in nutrient and drug absorption. Functional studies of
these membrane proteins allow a better understanding of the mechanism
of substrate-transporter interaction and may help establishing
therapeutic strategies involving peptide-based drugs.
EXPERIMENTAL PROCEDURES
gt10 cDNA library was screened using a 0.52-kb PepT2
probe generated by specific PCR primers. The complete PepT2 sequence
was isolated 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 oocytes 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.
50 mV. 100-ms voltage pulses of
between
160 and +60 mV, in increments of +20 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.
80 mV and perfused with substrate-free solution. Then the perfusion was stopped,
and the uptake solution (200 µl) 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 µl and terminated by
perfusing (washing) the oocyte with the substrate-free solution.
RESULTS
50 to
160 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 concentrations 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, even at
high concentrations (Fig. 2B), presumably due to relatively low substrate affinity (Table I). Depending on pH, substrate type, and
substrate concentration, PepT2 may exhibit decreases in current with
hyperpolarization (Fig. 2) due to low affinities for substrates. The
affinities for cationic peptides are relatively low compared with those
for anionic and neutral peptides, in analogy to PepT1 (25, 26).
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Fig. 1.
Affinity constants and maximal currents for
glycyl-dipeptides at pH 6.0 and various membrane potentials.
A, dose response of averaged H+-Gly-Glu
cotransport currents at membrane potentials 50 mV (
) and
160 mV
(
). B, apparent affinity constants
(Km) for Gly-Lys (
), Gly-Leu (
), 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 (
) shown in an
expanded scale. C, maximal currents
(Imax) for substrates. Data are averages derived
from four to six oocytes.
Apparent affinity constants for a variety of dipeptides
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Fig. 2.
Comparison of voltage dependence of
PepT2-mediated currents under different pH and substrate
concentrations. A, currents evoked by Gly-Glu ranging
from 10 to 500 µM, pH 6.0. B, currents evoked
by Phe-Ala from 10 to 500 M. C, currents
elicited by 250 µM Gly-Glu at various pH between 7.5 and
5.0, in increments of 0.5 pH units. D, currents elicited by
50 µM Gly-Leu at pH between 8.0 and 5.0. E,
currents elicited by 500 µM Gly-Lys at pH between 8.0 and
5.0. F, proton dependence of PepT2-mediated currents evoked
by 50 µM Gly-Leu at Vm of
50 (
) and
100 mV (
). Data shown in this panel are extracted
from D.
50 mV were determined when data were fitted to the Hill Equation. The
apparent affinity constants were 2.8 ± 0.5 (n = 6), 0.7 ± 0.1 (n = 3), and 0.5 ± 0.1 µM (n = 5) (corresponding to pH 5.5, 6.1, and 6.3) for Gly-Glu, Gly-Leu, and Gly-Lys, respectively. The Hill
coefficients were 1.06 ± 0.03, 1.27 ± 0.09, and 1.44 ± 0.06, correspondingly. This appears to suggest the coupling of a
single proton ion to Gly-Glu and more than one proton ion to Gly-Leu
and Gly-Lys. However, Hill coefficients obtained at other
Vm were significantly different, indicating that
this method is not accurate for stoichiometry evaluation. In addition,
since currents at low pH experienced decreases at hyperpolarized
Vm (see Fig. 2F, at
100 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
H+:substrate stoichiometry.
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Fig. 3.
Stoichiometry determination by simultaneous
measurements of substrate-evoked currents and tracer uptakes under
voltage-clamp conditions (Vh = 80
mV). A, representative examples of currents generated
by 500 µM Phe-Glu (cold + hot). Recordings 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 µM
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 (
), and
Phe-Glu (
) are 2.44 ± 0.02, 2.15 ± 0.02 and 1.88 ± 0.03, respectively.
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Fig. 4.
Inhibition of PepT2-specific currents by
quinapril. A, time course of total currents in the
presence of 50 µM Gly-Leu upon voltage pulses from a
holding potential of 50 mV to final potentials ranging between
160
and +60 mV, each separated by 20 mV. For clarity, only currents
corresponding to Vm of
160,
120,
80,
50,
20, +20, and +60 mV are shown. B, time course of currents
in the presence of both 50 µM Gly-Leu and 1.2 mM quinapril, obtained from the same oocyte as in
A. C, quinapril-sensitive currents in
presence 50 µM Gly-Leu as obtained by subtracting the
recording in B from that in A. The
inset is a depiction at depolarized potentials, highlighting
outward currents at depolarized potentials. D, quinapril
inhibition of PepT2-mediated currents at a holding potential of
50
mV. Solid and hatched bars represent applications
of 25 µM Gly-Leu and 1.2 mM quinapril,
respectively. E, PepT2-specific quinapril-sensitive currents
in the absence of external substrate (
, also inset) were
compared with currents due to addition of 50 µM Gly-Leu
(
) in the same oocytes. Represented data were mean values from five
oocytes.
50 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 (Q, obtained by eliminating the capacitative component) at pH 7.0 did not saturate up to
160 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 represents
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 subtracted).
These abnormal behaviors of PepT2 are not readily accounted for by
previously described kinetic models (see "Discussion").
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Fig. 5.
Rat PepT2-specific presteady-state
currents (Ipss). The holding
potential was 50 mV. For clarity, only currents at final potentials
of
160,
140,
120, and +40 mV were shown. Time between two
consecutive jumps was 650 ms. Steady-state currents were removed 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 µM Gly-Glu at pH 6.0. E, charge displacements
upon voltage jumps from
50 mV to various test potentials were
measured by integrating the currents versus time and
eliminating the capacitative component that was determined when data
were fitted to the sum of two exponential functions. F,
relaxation time constants (
) at various Vm and pH
values.
160 and
80 mV, relaxation time constants (
) of
Ipss for the on-responses ranged from 10 to 40 ms. With external alkalization, the Vm corresponding
to the maximal
shifted to more negative values (Fig.
5F), similar to other transporters such as PepT1 (9) and
SGLT1 (27). At more depolarized potentials,
could not be evaluated
due to small Ipss.
was only slightly affected by intracellular acidification using a previously reported approach (9) (not shown), in contrast to
of PepT1 that showed large
responses (9).
DISCUSSION
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Fig. 6.
Schematic illustration of a kinetic model for
PepT2. Each configuration represents a state. Outside, from
states I to IV, two proton ions and one substrate
(S) orderly bind to the protein. Fully loaded configuration
(state IV) undergoes a conformational change during which
two H+ and one S are translocated across the membrane.
Subsequently, the dissociation of these ions and substrate occurs, as
represented by reaction steps from state IV' to
I' (inside). When the free carrier changes from the
inward-faced state (I') to the outward-faced state
(I), the whole cycle of forward transport is accomplished,
which generates a net inward current. In the presence of intracellular
H+ and substrates, the backward (reversed) transport can
occur as well, producing outward currents. After binding of either one
or two H+, the translocation across the membrane may occur
in the absence of peptide substrates, generating observed
H+ leaks.
50 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 Na+-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 =
50 mV) was reduced from 650 to 200 or 50 ms, Ipss were largely reduced after the first
jump (to
160 mV), whereas there was no significant reduction in
Ipss when using time intervals of 650 ms or
longer (not shown).
50
mV) during a 650-ms period. Hyperpolarization appears 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 concentrations (Fig. 1C). Mechanisms
underlying such a hyperpolarization-stimulated inactivation are still unclear.
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ACKNOWLEDGEMENTS |
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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. Radioactive and cold phenylalanyl-dipeptides were kindly provided by Dr. C. A. R. Boyd.
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FOOTNOTES |
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* 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. The 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{at}rics.bwh.harvard.edu.
The abbreviation used is: MES, morpholineethanesulfonic acid.
2 Zhu, T., Chen, X.-Z., Hediger, M. A., and Smith, D. E., manuscript in preparation.
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REFERENCES |
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