Electrophysiological characteristics of the proton-coupled
peptide transporter PEPT2 cloned from rat brain
Hong
Wang,
You-Jun
Fei,
Vadivel
Ganapathy, and
Frederick H.
Leibach
Department of Biochemistry and Molecular Biology, Medical College of
Georgia, Augusta, Georgia 30912-2100
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ABSTRACT |
We have cloned a peptide transporter from rat brain and found it
to be identical to rat kidney PEPT2. In the present study we
characterize the transport function of the rat brain PEPT2, with
special emphasis on electrophysiological properties and interaction with
N-acetyl-L-aspartyl-L-glutamate
(NAAG). When heterologously expressed in HeLa cells and in SK-N-SH
cells, PEPT2 transports several dipeptides but not free amino acids in
the presence of a proton gradient. NAAG competes with other peptides
for the PEPT2-mediated transport process. When PEPT2 is expressed in
Xenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides of
differing charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAG
is a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of the
transport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively charged
peptides is 1, the transport nonetheless is associated with transfer of
positive charge into the oocyte, as indicated by peptide-induced inward
currents.
N-acetyl-L-aspartyl-L-glutamate; electrophysiology; charged peptides
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INTRODUCTION |
PROTON-COUPLED PEPTIDE transporters belong to a unique
gene family of transport proteins whose members are found not only in
the animal kingdom but also in bacteria and plants (12). These transporters mediate the transport of small peptides consisting of two, three, or four amino acids, and the transport process is
active, energized by a transmembrane electrochemical proton gradient
(11, 19, 26). In animals, peptide transport has been functionally
described primarily in the small intestine and the kidney, where the
process functions in the assimilation of dietary protein digestion
products and in the reclamation of peptide-bound amino nitrogen from
the glomerular filtrate, respectively (1, 16-18). Recently, two
distinct peptide transporters have been cloned, one from the intestine
and the other from the kidney (12, 20, 21). The intestinal peptide
transporter, designated PEPT1, is a low-affinity transporter, and its
primary structure has been deduced by expression and molecular cloning
studies in three different animal species, namely rabbit (8, 13), rat
(27, 33), and human (22). The renal peptide transporter, designated
PEPT2, is a high-affinity transporter, and its primary structure has also been deduced by molecular cloning studies in rabbit (7), rat (34),
and human (23). PEPT1 and PEPT2 are products of different genes, the
PEPT1 gene located on human chromosome 13q24-q33 (22) and the PEPT2
gene located on human chromosome 3q13-q21 (31). More recently, a third
peptide transporter, designated PHT1, has been cloned from rat brain
(42). Interestingly, PHT1 shows very little homology to PEPT1 and
PEPT2, but it does transport small peptides in a proton
gradient-dependent manner. PHT1 is expressed in the brain and in the
eye but not in the intestine or in the kidney. Among the
above-mentioned three peptide transporters, only PEPT1 has been
characterized functionally by detailed electrophysiological approaches
(2, 8, 13, 24, 25, 30, 35).
Northern blot hybridization with PEPT2 cDNA as the probe has indicated
that mRNA species hybridizing to the probe are abundantly expressed in
the brain (7, 34). However, it is not clear whether these hybridizing
transcripts represent PEPT2 mRNA or structurally related but hitherto
unidentified transcripts. Therefore, the present study was undertaken
to establish the identity of the transcripts in the brain that are
recognized by the PEPT2 cDNA probe in Northern blot analysis. To this
end, we screened a rat brain cDNA library using the human PEPT2 cDNA as
the probe and isolated a positive full-length cDNA clone. Sequence
analysis of the cDNA has revealed that it is identical to the already
cloned PEPT2. We report here the electrophysiological characteristics of the rat brain PEPT2 and the interaction of this transporter with the
neuropeptide
N-acetyl-L-aspartyl-L-glutamate
(NAAG) and other charged peptides. Furthermore, the rat brain PEPT2
does not interact with histidine or any other amino acid, a
characteristic that is in contrast to the rat brain PHT1, which
interacts with histidine in addition to peptides.
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MATERIALS AND METHODS |
Materials.
[2-14C]glycyl-[1-14C]sarcosine
(Gly-Sar; specific radioactivity, 109 mCi/mmol) was custom synthesized
by Cambridge Research Chemicals (Billingham, Cleveland, UK).
[1-14C]glycyl-L-proline
(6.4 mCi/mmol),
glycyl-L-[U-14C]phenylalanine
(25 mCi/mmol), and
[1-14C]glycylglycine
(9.1 mCi/mmol) were purchased from Amersham Radiochemical Center
(Arlington Heights, IL).
L-[3-3H]threonine
(15.2 Ci/mmol) was also from Amersham Radiochemical Center.
[3,4-3H]glutamine (59 Ci/mmol),
[1-3H]glycine (35 Ci/mmol),
[4,5-3H]leucine (60 Ci/mmol), and
[ring-2,5-3H]histidine
(57 Ci/mmol) were purchased from DuPont-NEN (Boston, MA). NAAG was
obtained from Research Biochemicals (Natick, MA), and all other
peptides were obtained from Sigma (St. Louis, MO).
Isolation of a peptide transporter cDNA clone from a rat brain cDNA
library.
A cDNA library was constructed using
poly(A)+ RNA isolated from rat
brain. The SuperScript plasmid system (Life Technologies, Gaithersburg,
MD) was employed for this purpose. This system was chosen because the
cDNA inserts can be directionally cloned into the vector pSPORT so that
the inserts are under the control of the T7 promoter in the vector.
This feature is essential for functional expression of the cloned cDNAs
using the Xenopus laevis expression technique as well as the vaccinia virus expression technique. Double-stranded cDNA obtained by reverse transcription of the poly(A)+ RNA was modified at both
termini by adapter sequences containing an
Not I site at one terminus and an
Sal I site at the other terminus to
introduce directionality. The resultant cDNAs were size-fractionated by
gel filtration chromatography (Sephacryl S-500 HR) to eliminate small
cDNA fragments shorter than 0.5 kb. The cDNAs were then ligated to
Not
I/Sal I-digested pSPORT vector. The
transformation of ligated cDNAs into Escherichia
coli was done by electroporation using Electro MAX
DH10B competent cells as host cells.
Screening of the cDNA library was done by colony hybridization of the
plasmid cDNA library grown on nylon transfer membranes (MicroSeparation
Systems, Westboro, MA). The probe was a 1.7-kb fragment released from
the coding region of human PEPT2 cDNA (23) by
EcoR I digestion. The
probe was labeled with
[
-32P]dCTP by
random priming using a commercially available oligolabeling kit
(Pharmacia). Hybridization was carried out at 65°C in a solution containing 5× SSPE (1× SSPE is 0.15 M NaCl, 10 mM
NaH2PO4,
and 1 mM EDTA), 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA for 24 h. Washing was done under
low-stringency conditions that involved washing three times, each time
for 30 min, at room temperature in a solution containing 2× SSC
(1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, and 0.5% SDS).
Positive clones were identified, and the colonies were purified by
secondary and tertiary screenings. Sequencing of the isolated cDNA was
done by the dideoxy chain termination method using the Sequenase 2.0 kit (United States Biochemicals, Cleveland, OH).
Functional expression in HeLa cells and in SK-N-SH cells.
Functional expression was achieved using the vaccinia virus expression
technique (5). Subconfluent HeLa cells and SK-N-SH cells (American Type
Culture Collection, Manassas, VA) were first infected with a
recombinant vaccinia virus (VTF 7-3) encoding T7 RNA polymerase and
then transfected with either the vector carrying the full-length cDNA
or the vector alone (control) in the presence of Lipofectin (Life
Technologies). Peptide or amino acid transport activity was assayed
10-12 h after transfection by measuring the uptake of radiolabeled
peptides or amino acids. The uptake medium for peptide or amino acid
uptake measurements was 25 mM MES-Tris (pH 6.0) containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, and 5 glucose. The
incubation time for uptake measurements was 5 min. HeLa cells and
SK-N-SH cells do not possess endogenous peptide transport activity and
hence are highly suitable for heterologous expression analysis of
putative peptide transporter cDNAs.
Functional expression in X. laevis oocytes and electrophysiological
studies.
Oocytes isolated from X. laevis
(Nasco, Fort Atkinson, WI) were partially digested with collagenase A
in a Ca2+-free medium and then
manually defolliculated. Mature (stage V-VI), defolliculated
oocytes were used for injection with cRNA preparations. The
plasmid-cDNA was linearized by Not I
digestion, and the cDNA insert was transcribed by using bacteriophage
T7 RNA polymerase. The Ambion MEGAscript kit was used for this purpose.
RNAase inhibitor and mRNA cap analog [7-methyl diguanosine
triphosphate, mG(5)ppp(5)G] were included. Final
concentration of the capped cRNA was adjusted to 1 µg/µl. Oocytes
were injected with 50 ng cRNA or 50 nl of water. Electrophysiological
measurements in cRNA- or water-injected oocytes were carried out
4-6 days after cRNA injection.
Electrophysiological characteristics of the cDNA-induced transport
activity in the oocytes were studied using the two-microelectrode voltage-clamp technique. In this technique, the membrane potential was
clamped at
50 mV. One microelectrode was used to monitor the
actual membrane potential and the second microelectrode was used to
pass currents into the oocyte in such a way that the membrane potential
remained clamped at
50 mV. The current required to be passed
through the second electrode is the measured parameter of the
cRNA-induced transporter activity. Oocytes were superfused at room
temperature with the medium containing (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 3 HEPES-MES-Tris in the
pH range 5-8. All substrate solutions were made in these buffers.
The substrate-induced currents were averaged over three sweeps. The
oocytes were thoroughly washed before exposure to the next testing
substrate, and several consecutive currents were measured using the
same oocyte. The experiments were repeated in at least three
independent oocytes for data analysis. The data were fitted to the
equation I = ImaxSnH/(K1/2nH + SnH),
where I is the substrate-evoked
current (i.e., the difference in the steady-state current measured in the presence and absence of the substrate),
Imax is the
maximal current, S is the substrate concentration,
nH is the Hill
coefficient, and
K1/2 is the
substrate concentration at which the substrate-evoked current is
one-half of Imax
(Michaelis-Menten constant). The dependence of the substrate-evoked
current on the peptide concentration as well as on the proton
concentration was investigated. To study the relationship between
membrane potential and substrate-evoked current, step changes in
membrane potential (called testing membrane potential) were applied,
each for a duration of 100 ms, in 20-mV increments over the range +50
mV to
150 mV. The voltage-jumping protocol was first applied in
the absence of substrate and then in the presence of substrate. The
substrate-specific current at each testing membrane potential was
determined as the difference between the currents recorded in the
presence and in the absence of substrate. Kinetic analysis was
performed using a commercially available computer program (SigmaPlot,
Jandel Scientific, San Rafael, CA).
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RESULTS |
Isolation and structural characterization of rat brain PEPT2 cDNA.
Screening of the rat brain cDNA library (~6 × 106 colonies) using a fragment of
human PEPT2 cDNA as the probe resulted in the identification of 22 positive colonies. Twelve of these colonies were purified by secondary
and tertiary screening, and the cDNA inserts of the plasmids were
characterized by size and sequence analyses. All 12 cDNA inserts were
found to be identical except for size differences resulting from
truncations of variable length at the 5' end. The largest of
these cDNAs was 3.9 kb long, and this cDNA was subjected to complete
sequence analysis. It was found to be identical to the PEPT2 cDNA
cloned by Saito et al. (34) from a rat kidney cDNA library. The only
difference was that the rat brain PEPT2 cDNA was shorter by 146 bp in
the 5' untranslated region.
Functional characterization of the rat brain PEPT2 cDNA.
To demonstrate that the rat brain PEPT2 cDNA codes for a functional
peptide transporter, the cDNA was expressed in HeLa cells using the
vaccinia virus expression technique. Transport of four dipeptides
(Gly-Sar, Gly-Gly, Gly-Phe, and Gly-Pro) and five amino acids (Gly,
Thr, Leu, Gln, and His) was measured in HeLa cells that were
transfected with either the pSPORT-cDNA construct or the pSPORT plasmid
alone (Table 1). It was found that the cDNA induced the transport of all four peptides severalfold but that the
transport of the free amino acids was not affected. Transport measurements in these experiments were made at an extracellular pH of
6.0, a condition that creates an inwardly directed proton gradient
across the HeLa cell plasma membrane. It was clearly evident from these
results that the PEPT2 cDNA isolated from rat brain encodes a
functional proton-coupled peptide transporter.
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Table 1.
Uptake of peptides and amino acids in HeLa cells transfected with
either empty vector or rat brain PEPT2 cDNA
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Successful isolation of the PEPT2 cDNA from the brain tissue reported
here raises interesting questions as to the possible function of this
peptide transporter in the brain. NAAG, a dipeptide derivative, is
present in high concentrations in mammalian brain. This peptide has
been suggested to function as a neurotransmitter (28, 38). Available
evidence suggests that NAAG is a selective agonist of specific subtypes
of glutamate receptors (37, 40, 41). One of the mechanisms of
inactivation of this putative neurotransmitter is hydrolysis by a
peptidase (4, 9, 32) that is associated with neuronal and glial plasma
membranes (6, 10). In addition to this extracellular degradative
pathway, NAAG is also known to be taken up into neuronal cells by a
transport pathway whose identity remains unknown (39). Because NAAG is a dipeptide derivative and the brain tissue expresses PEPT2, we tested
the possibility that NAAG may be a substrate for PEPT2. We first
studied the ability of NAAG to inhibit the transport of radiolabeled
Gly-Sar in HeLa cells that functionally express PEPT2. Figure
1A
shows that NAAG is an inhibitor of PEPT2-mediated Gly-Sar transport.
Significant inhibition was observed at a concentration of 1 mM. The
IC50 value (i.e., the
concentration of NAAG necessary to inhibit 50% of Gly-Sar transport)
for the inhibition was ~3 mM. Kinetic analysis revealed that NAAG is
a competitive inhibitor of PEPT2-mediated Gly-Sar transport (Fig.
1B). In the absence of NAAG, Gly-Sar
transport in PEPT2-expressing HeLa cells occurred via a single,
saturable mechanism with a
K1/2 of 35 ± 6 µM and a maximal velocity of 1.07 ± 0.07 nmol · 106
cells
1 · 5 min
1. In the presence of 4 mM NAAG, the K1/2
for Gly-Sar increased to 265 ± 48 µM, with no significant change
in the maximal velocity (1.26 ± 0.17 nmol · 106
cells
1 · 5 min
1).

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Fig. 1.
A: influence of increasing
concentrations of
N-acetyl-L-aspartyl-L-glutamate
(NAAG) on Gly-Sar transport in PEPT2-expressing HeLa cells. Transport
of 10 µM
[14C]Gly-Sar (5-min
incubation) was measured in HeLa cells transfected with pSPORT-rat
brain PEPT2 cDNA construct at pH 6.0 in presence of 0-10 mM NAAG.
B: kinetics of inhibition of
PEPT2-mediated Gly-Sar transport by NAAG in HeLa cells. Transport of
Gly-Sar was measured in PEPT2-expressing HeLa cells in either absence
( ) or presence ( ) of 4 mM NAAG. Concentration range for Gly-Sar
was 25-400 µM, and incubation time was 5 min. Transport measured
in HeLa cells transfected with vector alone was subtracted to account
for nonspecific transport. Results are given as Eadie-Hofstee plots
(V vs.
V/S, where
V is Gly-Sar transport in
pmol · 106
cells 1 · 5 min 1 and S is Gly-Sar
concentration in µM).
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HeLa is a human cervical epithelial cell line, and it has been
extensively used in the functional characterization of a variety of
cloned transporters after heterologous expression. To rule out the
possibility that the observed functional features of the brain PEPT2
expressed in HeLa cells may be unique to the cell type, we studied the
function of the brain PEPT2 after heterologously expressing the
transporter in SK-N-SH cells. SK-N-SH is a human neuroblastoma cell
line, and we thought that it would be desirable to establish the key
functional features of the brain PEPT2 in this brain-derived cell line.
We studied the ion dependence, substrate specificity, and saturation
kinetics of the transporter in SK-N-SH cells. As is the case with HeLa
cells, SK-N-SH cells do not possess detectable endogenous peptide
transport activity. The rat brain PEPT2 cDNA could be functionally
expressed in these cells by the vaccinia virus expression technique, as
assessed by the cDNA-induced transport of Gly-Sar. This transport was
Na+ and
Cl
independent and was
energized by an inwardly directed proton gradient (Fig.
2A).
Competition studies showed that the cDNA-induced Gly-Sar transport was
inhibitable by differently charged (neutral, anionic, and cationic)
dipeptides (Ala-Val, Ala-Glu, and Ala-Lys) and by NAAG (Fig.
2B). Kinetic analysis indicated that
the cDNA-induced Gly-Sar transport was saturable with a
K1/2 of 84 ± 8 µM (Fig. 3).

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Fig. 2.
A: influence of
Na+,
Cl , and protons on Gly-Sar
transport in PEPT2-expressing SK-N-SH cells. Transport of 20 µM
[14C]Gly-Sar (5-min
incubation) was measured in SK-N-SH cells transfected with pSPORT-rat
brain PEPT2 cDNA construct using a buffer of pH 6.0 or 8.0. Ionic
composition of buffer was changed in such a way that it contained NaCl,
was Na+ free, or was
Cl free. In
Na+-free buffer, NaCl was
isosmotically replaced by
N-methyl-D-glucamine
chloride. In Cl -free
buffer, all Cl salts (NaCl,
KCl, and CaCl2) were
isosmotically replaced by corresponding gluconate salts.
B: substrate specificity of rat brain
PEPT2 expressed in SK-N-SH cells. Transport (5 min) of 30 µM
[14C]Gly-Sar was
measured at pH 6.0 in presence or absence of Ala-Val (0.5 mM), Ala-Lys
(0.5 mM), Ala-Glu (0.5 mM), or NAAG (4 mM).
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Fig. 3.
Saturation kinetics of Gly-Sar transport mediated by rat brain PEPT2
expressed in SK-N-SH cells. Transport (5 min) of Gly-Sar was measured
in PEPT2-expressing cells over a Gly-Sar concentration range of
25-400 µM. Transport measured in cells transfected with vector
alone was subtracted to account for nonspecific transport.
Inset: Eadie-Hofstee plot
(V/S vs.
V).
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Electrophysiological characteristics of PEPT2-mediated transport of
differently charged peptides.
We investigated the function of PEPT2 expressed in X. laevis oocytes using electrophysiological approaches.
For this investigation, we chose three dipeptides of differing charge
as representative substrates for PEPT2: Ala-Val (a neutral dipeptide),
Ala-Glu (an anionic dipeptide), and Ala-Lys (a cationic dipeptide).
Figure 4 describes the activation of
PEPT2-mediated transport of all three peptides by protons in the
extracellular medium. Although the three peptide substrates are
differently charged under the experimental conditions, the activation
of transport by protons was hyperbolic in all three cases, indicating a
proton-to-peptide coupling ratio of 1. The Hill coefficient for
protons, which is indicative of the coupling ratio, was 1.0 ± 0.4 for Ala-Glu, 1.0 ± 0.1 for Ala-Lys, and 1.3 ± 0.2 for Ala-Val.
There was very little influence of membrane potential on the Hill
coefficient. Thus it appears that each of the three peptides is
cotransported with one proton. The
K1/2 for the
activation of the transport process by protons was in the range
0.75-2.25 µM.

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Fig. 4.
Top: influence of external proton
concentration on peptide-induced currents
(I) in PEPT2-expressing
X. laevis oocytes for Ala-Glu,
Ala-Lys, and Ala-Val. Concentration of peptides was 75 µM, and range
of proton concentrations tested was 31.6 nM to 10 µM (i.e., pH
5.0-7.5). Bottom: Hill
coefficient values
(nH) for proton at
different testing membrane potentials
(Vtest) for the
3 dipeptides.
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Further kinetic studies on the proton and membrane potential dependence
of the transport of the three peptides revealed significant similarities (Fig. 5). The
K1/2 values for
all three peptides were influenced markedly by the external proton
concentration as well as by the membrane potential (Fig. 5,
A-C).
Although the K1/2
did not appear to be affected significantly by external pH in the
physiological range of membrane potential (
50 to
70 mV),
appreciable changes in the
K1/2 were seen
depending on the external pH under hyperpolarizing membrane potentials. Interestingly, in the case of the charged peptides Ala-Glu and Ala-Lys,
there was no change in the
K1/2 when
external pH was switched from 6.0 to 5.5, but further reduction in the
external pH to 5.0 greatly increased the
K1/2. In
contrast, in the case of the neutral peptide Ala-Val, increases in
K1/2 were seen
when the external pH was switched from 6.0 to 5.5 and also when it was
switched from 5.5 to 5.0.

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Fig. 5.
A-C:
influence of external pH and
Vtest on
Michaelis-Menten constant
(K1/2) for the
3 dipeptides Ala-Glu (A), Ala-Lys
(B), and Ala-Val
(C).
D-F:
influence of
Vtest on
peptide-induced currents
(Imax) at
different pH for Ala-Glu (D),
Ala-Lys (E), and Ala-Val
(F). For each pH,
Imax at a
Vtest of
150 mV was taken as 1 for comparison of influence of
Vtest on
magnitude of Imax
at all 3 pH values.
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Despite the differences in the net charge of the three peptide
substrates, the current-membrane potential relationship was similar in
all three cases (Fig. 5,
D-F).
The currents increased as the testing membrane potential was
hyperpolarized from
50 mV and decreased as the testing membrane
potential was depolarized from
50 mV. The currents in all three
cases approached zero when the testing membrane potential was in the
range between 0 and +50 mV.
Electrophysiological characteristics of PEPT2-mediated transport of
NAAG.
The results obtained with PEPT2-expressing HeLa cells show that NAAG
competes with Gly-Sar for interaction with PEPT2. This suggests but
does not prove that NAAG is a transportable substrate for PEPT2. To
demonstrate directly that PEPT2 does indeed transport NAAG, we employed
the X. laevis oocyte expression system
and analyzed the transport-associated currents. Figure
6A shows
the transport-associated currents when PEPT2-expressing oocytes were
superfused with NAAG (5 mM) at pH 7.5 or 5.5. The presence of NAAG in
the perifusion medium at pH 5.5 induced marked inward currents. The
same concentration of NAAG, however, induced much less inward current
when the pH of the perifusion medium was 7.5 instead of 5.5. In
contrast, NAAG failed to induce detectable currents at pH 7.5 or at pH
5.5 in water-injected oocytes (Fig.
6B). These data show that PEPT2 mediates the transport of NAAG in a proton-dependent manner and that
the transport process results in the transfer of positive charge into
the oocytes. We also analyzed the influence of various proton
concentrations in the external medium on the magnitude of 5 mM
NAAG-induced currents (Fig. 6C). The
magnitude of the currents was found to be hyperbolically related to
external proton concentrations (pH range 5.0-7.5). The Hill
coefficient for protons, which is a measure of the proton-to-NAAG
stoichiometry, was 0.8 ± 0.1. Thus the number of protons that is
cotransported with NAAG appeared to be 1.

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Fig. 6.
Influence of external proton concentration on 5 mM NAAG-induced
I in oocytes injected with either
PEPT2 cRNA or water. A: tracings of 5 mM NAAG-induced I at pH 7.5 or 5.5 in
PEPT2-expressing oocytes. ND96 buffer consists of (in mM) 96 NaCl, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, and 5.0 HEPES (pH adjusted
to 7.4 with Tris). B: tracings of
I in water-injected oocytes under same
experimental conditions described in
A. C:
dependence of 5 mM NAAG-induced inward
I on proton concentration in external
medium. Proton concentration was varied in range between 31.6 nM (pH
7.5) and 10 µM (pH 5.0).
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Figure 7A
shows that superfusion of PEPT2-expressing oocytes with increasing
concentrations of NAAG at pH 5.5 induced inward currents of increasing
magnitude. Figure 7B describes the
relationship of current and testing membrane potential for NAAG
transport. The currents were found to increase with increasing
concentrations of NAAG and also with increasingly hyperpolarizing
membrane potential. Similar relationships between the magnitude of the
currents and NAAG concentration were evident at all testing membrane
potentials studied. With a given concentration of NAAG, the currents
increased in magnitude when the testing membrane potential was
increasingly hyperpolarized. These results show that hyperpolarization
dramatically enhances NAAG transport mediated by PEPT2.

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Fig. 7.
A: influence of increasing
concentrations of NAAG on NAAG-induced
I in PEPT2-expressing oocytes at pH
5.5. B:
I-Vtest
relationship at pH 5.5 for various concentrations of NAAG in
PEPT2-expressing oocytes. C:
saturation kinetics of NAAG-induced I
at pH 5.5 in PEPT2-expressing oocytes with different
Vtest.
D: influence of
Vtest on
K1/2 for NAAG.
K1/2 values were
calculated by measuring I at different
Vtest at pH 5.5 in presence of various concentrations of NAAG (0.1-5 mM).
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The transport of NAAG by PEPT2 was found to be saturable at all testing
membrane potentials studied (Fig.
7C). The relationship between NAAG
concentration and the magnitude of the currents was hyperbolic, and the
data were found to fit best to a transport model involving a single
saturable system. We also determined the
K1/2 for NAAG.
The K1/2 was
found to be dependent on the testing membrane potential to a
significant extent (Fig. 7D).
It must be mentioned here that there was significant variation in the
magnitude of peptide-induced currents among different PEPT2-expressing
oocytes. This difference was most likely due to variation in the
expression levels of the transporter protein in different oocytes.
However, there was no qualitative difference in the functional features
of PEPT2 studied in different oocytes. All of the key features,
including the proton dependence, proton-to-peptide stoichiometry,
current-membrane potential relationship, substrate affinities, and
dependence of
K1/2 for peptides
on membrane potential, were reproducible in each oocyte expressing
PEPT2.
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DISCUSSION |
Although Northern blot analysis has indicated the presence of
transcripts hybridizing to PEPT2 cDNA probes in rat brain, the exact
identity of these transcripts has never been established. Furthermore,
peptide transport activity has not been demonstrated in the brain
tissue by any experimental approach. Therefore, the present results
constitute the first evidence for the expression of functionally
competent PEPT2 transcripts in the brain. Yamashita et al. (42)
recently isolated from a rat brain cDNA library a cDNA clone that codes
for a transporter capable of mediating the uptake of small peptides as
well as of the free amino acid histidine. There is, however, very
little homology at the structural level between this peptide/histidine
transporter (PHT1) and PEPT2. Furthermore, although both PHT1 and PEPT2
mediate proton-coupled transport of peptides, PEPT2 does not interact
with histidine as PHT1 does. To date, PEPT2 has been cloned only from
the renal tissue, where it is believed to function in the reabsorption
of peptides from the glomerular filtrate.
Here we report on the functional characteristics of PEPT2
heterologously expressed in two different mammalian cell lines as well
as in X. laevis oocytes. The two
mammalian cell lines are HeLa (a human cervical epithelial cell line)
and SK-N-SH (a human neuroblastoma cell line). The key functional
features, such as the ionic dependence, substrate specificity, and
saturation kinetics, were found to be similar for PEPT2 expressed in
both cell types. This indicates that the functional characteristics of
PEPT2 are not influenced by the cell type in which it is heterologously expressed. Electrophysiological characterization of PEPT2 function was
carried out in X. laevis oocytes.
Because the function of PEPT2 was assessed for the present study using
intact cells, the observed characteristics are attributed to PEPT2
expressed in the plasma membrane of these cells. This is also evident
from the studies with X. laevis
oocytes, in which the PEPT2 function was monitored using
peptide-induced changes in membrane potential across the oocyte plasma
membrane.
The results presented here clearly demonstrate that the neuropeptide
NAAG is a transportable substrate for the proton-coupled peptide
transporter PEPT2, which is expressed in the brain. PEPT2 exhibits much
higher affinity for its substrates than does PEPT1 (31). The
K1/2 for the
interaction of NAAG with PEPT2 is, however, in the low millimolar
range, which suggests that the blocking of the
-amino group by
acetylation greatly reduces the affinity. Nonetheless, the observed
values may be physiologically relevant. NAAG is present in the
mammalian nervous system at very high concentrations (15). The
concentrations of NAAG have been shown to be in the range 0.2-4.5
mM in different regions of the nervous system. However, it is premature
to speculate on the physiological role of PEPT2 in the disposition of
NAAG in the brain. The precise localization of PEPT2 in the brain and
whether this localization coincides with the regions of high NAAG
concentrations remain still to be determined. Furthermore, there is no
information available on the magnitude of the proton gradients across
the plasma membranes of any of the cell types in the nervous system.
Because PEPT2 is expected to function to a significant extent even in
the absence of a transmembrane proton gradient, it is possible that it
plays a role in the transport of NAAG. Whether such a putative function is involved in the uptake or release of NAAG will depend on relative transmembrane electrochemical gradients for NAAG and protons. A
preliminary report by Nickolaus et al. (29) shows that PEPT2 expression
in the brain is primarily restricted to neuronal cells in the
hippocampal region and to epithelial cells of the choroid plexus. This
new information is likely to fuel further research to identify the
precise function of PEPT2 in the brain.
The most interesting finding with respect to the interaction of NAAG
(net charge,
3) and the other three peptides (net charges,
1, +1, and 0) with PEPT2 is that the transport process induced inside-negative currents irrespective of the net charge of the peptide.
The proton-to-peptide stoichiometry, however, appeared to be 1:1 in all
cases. Similar findings have been reported recently by Amasheh et al.
(3) for PEPT2 cloned from rabbit kidney. A simple transport model
assigning the charge translocation during the transport cycle solely to
the movement of the peptide substrate and proton does not therefore
explain the observed findings. The transport protein itself is likely
to be charged, and the interaction of differently charged peptide
substrates might occur preferentially with differently charged states
of the transport protein. The observations with respect to the
electrophysiology of the PEPT2-mediated transport of differently
charged peptides bear striking similarities to the observations made by
three independent groups of investigators in the case of PEPT1-mediated
transport of differently charged peptides (2, 24, 35). PEPT1 is known
to contain essential histidyl residues (14, 36) that can exist either
in a protonated form or in a nonprotonated form in the pH range
5.0-7.0, depending on the microenvironment of the involved
histidyl residue. Similarly, PEPT2 also possesses essential histidyl
residues that are obligatory for the transport function (14). Recently,
Nussberger et al. (30) have proposed a model for the operational
mechanism of PEPT1 with respect to the translocation of proton and
peptide and for the association of the transport process with inward
negative currents irrespective of the net charge of the translocated
peptide substrate. This model implicates the involvement of differently charged amino acids in the substrate-binding pocket of the PEPT1 protein. A similar situation might exist in the case of PEPT2-mediated translocation of differently charged peptides.
 |
ACKNOWLEDGEMENTS |
We thank Sarah A. Taylor and Ida O. Walker for expert secretarial
assistance.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-28389.
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. §1734 solely to indicate this fact.
Address reprint requests to F. H. Leibach.
Received 3 March 1998; accepted in final form 18 June 1998.
 |
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