(Received for publication, October 3, 1995; and in revised form, December 12, 1995)
From the
The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei,
Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L.,
Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol.
Chem. 270, 6456-6463) encodes a
H/oligopeptide cotransporter. Using two-microelectrode
voltage-clamp in Xenopus oocytes expressing hPEPT1, we have
investigated the transport mechanisms of hPEPT1 with regard to voltage
dependence, steady-state kinetics, and transient charge movements. The
currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0
were dependent upon membrane potential (V
) between -150 mV and +50
mV. Gly-Sar-evoked currents increased hyperbolically with increasing
extracellular [H
], with Hill coefficient
1, and the apparent affinity constant (K
) for H
was in
the range of 0.05-1 µM. K
for
Gly-Sar (K
) was dependent upon V
and pH; at -50 mV, K
was minimal (
0.7 mM)
at pH 6.0. Following step-changes in V
,
in the absence of Gly-Sar, hPEPT1 exhibited
H
-dependent transient currents with characteristics
similar to those of Na
-coupled transporters. These
charge movements (which relaxed with time constants of 2-10 ms)
were fitted to Boltzmann relations with maximal charge (Q
) of up to 12 nC; the apparent valence was
determined to be
1. Q
is an index of the
level of transporter expression which for hPEPT1 was in the order of
10
/oocyte. In general our data are consistent with an
ordered, simultaneous transport model for hPEPT1 in which H
binds first.
In the mammalian intestine, absorption of oligopeptides is
mediated by one or more proton-coupled membrane transporters (reviewed
in (1) and (2) ). The sequential actions of the
Na/K
-ATPase pump and the
Na
/H
exchanger in the small intestine
create an acidic extracellular microclimate (pH
6.0(3) )
generating an inward H
electrochemical gradient
sufficient to drive the tertiary active transport of oligopeptides.
Liang et al.(4) cloned and expressed a human
intestinal cDNA encoding a H-dependent oligopeptide
transporter (hPEPT1). (
)hPEPT1 accepts a broad range of
substrates including dipeptides and tripeptides, but not free amino
acids; hPEPT1 also accepts as substrates certain pharmacologically
active compounds such as the tripeptide-like
-lactam antibiotics
and the antineoplastic agent bestatin. hPEPT1 is homologous (81% amino
acid sequence identity(4) ) with the rabbit
H
/oligopeptide cotransporter (rPEPT1): transport is
electrogenic, coupled to an influx of H
, and
independent of Na
, K
, and
Cl
(5, 6) .
We have investigated
the mechanisms of H/oligopeptide cotransport mediated
by the human intestinal PEPT1 transporter and describe the biophysical
and kinetic characterization of hPEPT1 using two-microelectrode
voltage-clamp in cRNA-injected oocytes, with the non-hydrolyzable
dipeptide glycyl-sarcosine (Gly-Sar) as the characterizing substrate.
Presteady-state currents were integrated with time (see legend
to Fig. 5) and charge movements (Q) fitted to the
Boltzmann relation () for which Q = Q
- Q
(where Q
and Q
represent Q at depolarizing and hyperpolarizing limits), z is the apparent valence of the movable charge, V
is the potential for 50% charge translocation, F is Faraday's constant, R is the universal gas
constant, and T is the absolute temperature.
Figure 5:
Charge
translocation associated with hPEPT1 expressed in oocytes. All data
described are for a single hPEPT1 cRNA-injected oocyte at 20-22
°C in the absence of substrate, and were replicated in a second
oocyte (not shown) with Q of up to 12 nC. A, representative carrier-mediated transient currents at pH
5.0: these were obtained from the total currents by subtraction of the
currents due to membrane capacitance (with a decay constant of
0.7
ms) and the steady-state currents, using the fitted method previously
described (9) . Currents (filtered at 100 Hz for display) are
shown from 3 ms after the voltage steps indicated in the top
panel. B, kinetics of transient current relaxation (for
on currents), were described by a single time constant
(
, filled symbols) between 3 ms and upon
reaching steady-state at pH 5.0, 5.5, 6.0, and 6.5 (solid lines are fitted Gaussian relations). The time constants for the off
currents (
is shown for pH 5.0 at V
= +30 mV,
) did not
vary with either V
or pH. C,
charge-voltage relationships for hPEPT1 at pH 6.5, 6.0, 5.5, and 5.0
obtained using the fitted method (9) (filled symbols);
at each pH, the data were fitted to the Boltzmann equation () (solid lines). Q/V
data were normalized such that the depolarizing limits of Q were equalized for each pH
value.
Figure 1:
Voltage dependence of
the Gly-Sar-evoked current for hPEPT1 and rPEPT1. A, currents
evoked by 0.2, 1, 5, and 20 mM Gly-Sar in a single oocyte
expressing the human intestinal oligopeptide transporter hPEPT1; the
oocyte was superfused with 100 mM Na buffer
at pH 5.0, and at 20-22 °C. B, currents evoked by 20
mM (saturating) Gly-Sar at pH 5.0 in two oocytes (from the
same batch of oocytes) injected with cRNA encoding the human (
)
and rabbit (
) PEPT1 H
/oligopeptide
cotransporters; evoked currents were normalized using the current
evoked at -150 mV, which for hPEPT1 was -650 nA and for
rabbit PEPT1, -1910 nA.
Figure 2:
Proton-dependent activation of
oligopeptide transport via hPEPT1. A, representative data (at V = -110 mV) showing the
dependence of the 1 mM Gly-Sar-evoked current (I
) upon external proton concentration
([H
]
), determined at pH 7.5,
6.5, 6.0, 5.5, and 5.0 in a single hPEPT1 cRNA-injected oocyte. Data
were fitted to . The Hill coefficient (n
) calculated using the data for -110 mV
was 1.1 ± 0.1, an apparent stoichiometry of 1
H
: 1 Gly-Sar; at each voltage, n
approximated to 1. B, voltage dependence of the apparent
affinity constant (K
) for
H
, at a fixed Gly-Sar concentration (1
mM).
At a fixed Gly-Sar
concentration (1 mM), I increased in
magnitude as a hyperbolic function of extracellular proton
concentration ([H
]
), with Hill
coefficient (n
) for H
1;
representative data at -110 mV is presented (Fig. 2A). At hyperpolarizing V
(-90 to -130 mV), the apparent affinity constant of
hPEPT1 for H
(K
)
was largely voltage-insensitive and approached an asymptote of
50
nM at hyperpolarizing limits (Fig. 2B). From
-70 mV, K
increased markedly
with depolarization to
1 µM at -30 mV. Switching
the superfusing Na
medium from pH 7.5 to 5.5 resulted
in a shift in baseline current of 15 ± 1 nA (mean ± S.E., n = 6 oocytes), no greater than that observed for
vehicle-injected oocytes, 12 ± 3 nA (n = 4).
The apparent affinity constant for Gly-Sar (K) at pH 7.0 increased from 1.2 to
2.5 mM as the cell was depolarized from -150 to
-50 mV (Fig. 3A). Such a decrease in substrate
affinity with depolarization is commonly observed for other
transporters, e.g. SGLT1 (7) and SGLT2(10) ,
and since [H
]
K
(see Fig. 2A) it
is probable that the K
/V
relationship (at pH 7.0) is a reflection of the voltage
dependence of K
. However, the
voltage dependence of K
at pH 5.0 was very
different: K
at -150 mV was
4
mM and fell with depolarization, reaching a minimum of 0.3
mM at +30 mV. The relationship of K
to V
at pH 6.0 and 5.5 (data not shown) bore
intermediate resemblance to that at pH 5.0 and at 7.0 (Fig. 3A). The Hill coefficient for Gly-Sar was 1 at
each V
tested. The relationship of the maximal
Gly-Sar-evoked current (I
) to V
at pH 5.0 (Fig. 3B) followed the
shape of the I/V
relation for 20 mM Gly-Sar (Fig. 1). At -150 mV, relative to K
, I
was essentially independent of pH between pH 5.0 and 7.0 (Fig. 3B); however, with depolarization, I
was significantly attenuated at
diminishing [H
]
. The I/V
relationship was shifted left with increasing
pH (at pH 7.0, the 1 mM Gly-Sar-evoked currents approached a
zero current asymptote by
+10 mV), but the slopes of the I/V
curves were similar.
Figure 3:
Kinetics of the Gly-Sar-evoked currents
for hPEPT1. A, voltage dependence of the apparent affinity
constant (K) for Gly-Sar. The
kinetic parameters of Gly-Sar transport were determined by measuring
the currents evoked by Gly-Sar at 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20
mM, each at pH 7.0, 6.0, and 5.0, in a single cRNA-injected
oocyte, and derived according to . B, voltage
dependence of the maximum current evoked by Gly-Sar, I
.
The I/V relationships for 1 mM Gly-Sar were explored in the pH range 5.0-7.5 (Fig. 4A): at pH 7.5 the Gly-Sar-evoked currents were
markedly voltage-dependent between -150 and -50 mV, and by
-30 mV diminished close to zero. At subhyperpolarized potentials (V
-90 mV) the 1 mM Gly-Sar-evoked current increased with decreasing pH from 7.5 to
5.0, but at -150 mV the evoked current was greater at pH 6.0 than
at pH 5.0, as a consequence of the increased K
observed with hyperpolarization
at pH 5.0 (see Fig. 3A). Selected data from Fig. 3were re-plotted as a function of
[H
]
(Fig. 4, B and C). At 0.1 µM H
, K
was lower at -150 mV than
at -50 mV; however, at -150 mV, increasing
[H
]
resulted in an increase in K
whereas, at -50 mV, K
fell by 72% between 0.1 and 1.0
µM H
and remained constant as
[H
]
was increased further to 10
µM. At -150 mV, the I
fell only modestly (28%) as [H
]
was reduced 100-fold from 10 to 0.1 µM (Fig. 4C). For -50 mV, although the drop in I
was proportionately more marked
than at -150 mV, I
only fell
significantly once [H
]
was well
below the K
(
0.4
µM, for 1 mM Gly-Sar).
Figure 4:
Characteristics of the Gly-Sar-evoked
currents. A, the effect of pH on the current evoked by 1
mM (subsaturating) Gly-Sar, in a separate oocyte from that
used in Fig. 3. Selected data from Fig. 3were replotted
as a function of external proton concentration: B, K, at -50 and -150 mV
(additional data for K
at pH 5.5
from a second oocyte were included); C, I
at -50 and -150
mV.
Figure 6:
Presteady-state kinetic parameters for
hPEPT1 described as a function of extracellular protons.
Presteady-state kinetic parameters were derived from data in Fig. 5. A, effect of external proton concentration upon
the maximal value,
. B,
the voltage at which
is maximal, V
A significant fraction of the dietary amino nitrogen is
absorbed as intact oligopeptides rather than free amino
acids(11) . Ganapathy and Leibach(2) , and more
recently Meredith and Boyd (1) have reviewed
H-coupled oligopeptide transport in the epithelia of
kidney, intestine, lung, and placenta, and the blood-brain barrier, in
which dipeptides and tripeptides are accepted as substrates.
Oligopeptide transport activity is evidently served by more than one
transport protein both in rabbit intestine (12) and
kidney(13) , and a second H
/oligopeptide
transporter (hPEPT2) from human kidney was recently cloned (14) and characterized(15) . Kinetic analysis
demonstrated that Gly-Sar and Gly-Pro share a common
pH-gradient-dependent carrier system in rabbit intestinal brush-border
membrane vesicles (16) but a multiplicity of transport systems
was suggested by the additive effects of several other
oligopeptides(12) . In this latter study, the authors used a
potential-sensitive fluorescent dye to demonstrate that oligopeptide
transport was electrogenic; furthermore, imposing a valinomycin-induced
K
diffusion potential could almost double Gly-Pro
transport(17) .
Expression of the cloned rabbit intestinal
H/oligopeptide cotransporter rPEPT1 in Xenopus oocytes (5) revealed that Gly-Sar transport (K
1.9 mM) was
electrogenic and independent of Na
,
Cl
, and K
, and that rPEPT1
transported a broad range of dipeptides, tripeptides, and
-lactam
antibiotics with K
0.1-5 mM.
Surprisingly rPEPT1 displayed high apparent affinity for the anionic
dipeptide alanyl-aspartate (K
140
µM), whereas in rabbit intestinal brush-border membrane
vesicles neutral dipeptides, or those bearing a single positive charge,
were generally favored(18) . The human homologue (4) exhibited higher affinity for Gly-Sar than rPEPT1 but
similar substrate scope: Gly-Sar transport in hPEPT1-transfected HeLa
cells (K
= 0.3 mM,
pH 6.0) was inhibited by several dipeptides, tripeptides, and
-lactam antibiotics, but not by free amino acids (4) .
Gly-Sar transport via rPEPT1 proceeded with a pH optimum of 5.5, and
intracellular pH recording yielded the first convincing evidence that
oligopeptide uptake was coupled to a H flux(5) . Boll et al.(6) confirmed that
uptake of the antibiotic cefadroxil was also
H
-coupled, but with a pH optimum of 6.5. The Hill
coefficient for H
obtained for Gly-Sar uptake (5) was 1, consistent with Hill plots for H
activation of Gly-Gln uptake mediated by both the high and low
affinity systems in rabbit renal brush-border membrane
vesicles(13) .
Whereas Fei et al.(5) investigated the voltage dependence of oligopeptide transport by rPEPT1 using two-microelectrode voltage-clamp and concluded that Gly-Sar transport was almost completely independent of voltage (in the range -150 to +50 mV), Boll et al.(6) found transport of both Gly-Sar and cefadroxil to be voltage-dependent, with the Gly-Sar-evoked current saturating with hyperpolarization (see below).
The present study was designed to
elucidate the molecular mechanisms by which hPEPT1 transports small
peptides. By measuring evoked currents in Xenopus oocytes
expressing the human H/oligopeptide transporter
hPEPT1, we have demonstrated that Gly-Sar transport via hPEPT1 is
electrogenic and coupled to an inward H
current. For
both the human and rabbit PEPT1 transporters we observed that evoked
currents for saturating Gly-Sar concentrations (
20 mM)
were voltage-dependent over the entire V
range
tested (-150 to +50 mV). For rPEPT1 it is unclear why there
were differences between this study and that of Fei et
al.(5) ; certainly the poor expression of rPEPT1 obtained
by Fei et al. (<100 nA), compared with up to 2,000 nA in
this study (Fig. 1B), made it difficult to detect any
changes in the evoked current with V
. However, the
data shown in Fig. 4F of (5) can be
alternatively explained: the 10 mM (subsaturating)
Gly-Sar-evoked current was V
-dependent between
+50 mV and -110 mV, with a region of negative dependence
upon V
below -110 mV, similar to that
observed for subsaturating Gly-Sar concentrations in hPEPT1 (Fig. 1A). In contrast to that obtained by Boll et
al.(6) for 10 mM Gly-Sar, the I/V
relationship for rPEPT1 for 20 mM Gly-Sar showed no
evidence of saturating with V
by -150 mV (Fig. 1B), but this is consistent with the
concentration-dependent shift in the V
at which
the evoked currents saturate for hPEPT1 (Fig. 1A, see
below).
We found that H/Gly-Sar cotransport in
oocytes expressing hPEPT1 obeyed Michaelis-Menten-type kinetics. The
apparent affinity constant for Gly-Sar (K
) was
0.7 mM at V
-50 mV (in the pH range 5.0-6.0), of
the same order as that obtained from radiotracer fluxes for hPEPT1
expressed in non-voltage-clamped HeLa cells (0.3 mM at pH
6.0)(4) , and slightly lower than the K
reported for rPEPT1 (1.9 mM at -60 mV, pH 5.5)(5) . At each V
, Hill coefficients were
1 for H
activation of the Gly-Sar-evoked current (Fig. 2A), and also for Gly-Sar activation, suggesting
1 H
:1 Gly-Sar transport stoichiometry.
Following
step changes in V in the presence of
H
, but in the absence of Gly-Sar, we observed
presteady-state (transient) currents similar to those observed for
several other cloned transport proteins expressed in oocytes, e.g. the intestinal Na
/glucose cotransporter SGLT1 (9) (see Table 1). These charge movements for hPEPT1 were
fitted to the Boltzmann relation (, Fig. 5C). We observed a modest dependence of maximal
charge transfer (Q
) upon
[H
]
(Fig. 6D)
whereas in contrast the dependence of V
(the
membrane potential at 50% Q
) upon
[H
]
was very dramatic: V
was linearly dependent upon pH with a slope of
+80 mV per 10-fold increase in
[H
]
(Fig. 5C and Fig. 6C). This represented a larger shift in V
due to changes in activator concentration than
for human SGLT1, but it was less marked than the
[Na
]
-dependent shifts in V
observed for the low affinity SGLT2, the
Na
/glutamate cotransporter (EAAT2) and the
Na
/Cl
/GABA cotransporter (GAT1). The
transient currents associated with hPEPT1 relaxed with time constants
of 2-10 ms, broadly comparable to those for SGLT1, SGLT2, SMIT
(canine Na
/myo-inositol cotransporter), the
plant H
/hexose cotransporter (STP1) and EAAT2;
however, current transients for GAT1 decay much more slowly (Table 1).
appeared to follow a bell-shaped
relationship to membrane potential (Fig. 5B); lowering
[H
]
also reduced the extent to
which
varied with V
. Increasing
pH from 5.0 to 6.0 reduced
from
10 to
8 ms;
and the membrane potential at which
was obtained
also appeared to shift
80 mV for a 10-fold increase in
[H
]
, consistent with the slope
of V
/pH (Fig. 6).
The partial
reactions involved in the presteady-state currents are illustrated in Fig. 7B; each reaction step was treated as a function
of the first order (k, k
,
and k
) or pseudo-first order (k
) rate constants. Modelling of the partial
reaction followed the principles described for rabbit and human SGLT1 (9, 19) , modified for one ion-binding site. According
to this model presteady-state currents are due to (i)
binding/dissociation of H
to/from the cotransporter
and (ii) a conformational change of the unloaded carrier between the
external and internal membrane interfaces. The phenomenological
constant
` describes the fraction of the membrane field between
extracellular H
and the H
-binding
site at the external face,
" is its internal equivalent, and
is the fraction of the membrane field sensed by the empty binding site
on the carrier during translocation; microscopic reversibility requires
that
` +
" +
= 1(20) .
Simulations indicated that this model can quantitatively account for
the Q/V
and
/V
relationships observed for hPEPT1 at pH 5.5 (Fig. 7, C and D, see legend for the values of each constant); the
model predicted
"
0, i.e. H
binding
at the internal face is essentially independent of voltage, in common
with intracellular Na
binding for
SGLT1(9, 19) . The model also qualitatively described
(not shown) (i) the reduction in Q
as pH rises
(a reflection of the relative contribution of H
binding/dissociation to the overall charge transfer) together
with the shift in V
to more hyperpolarized V
and (ii) the reduction in
from pH 5.0 to 6.5 together with the pH-dependent shift in V
Figure 7:
Kinetic model for the operation of the
human H/oligopeptide cotransporter hPEPT1. A,
an eight-state model of hPEPT1 in which the empty carrier is negatively
charged (the apparent valence of the movable charge is -1, and
the Hill coefficient is 1). Each carrier state is identified by a number; carrier states at the external face of the membrane
are further identified by a prime and, at the internal face,
by double prime. Essentially the model is similar to that
proposed for SGLT1 (19) except that two additional states (2a
and 5a) are added since Gly-Sar may bind in the absence of protons and
an appreciable ``internal leak'' may proceed uncoupled to
protons; this substrate leak pathway (states 2a
5a) is shown as a dotted line to represent the assumption that translocation of
the fully loaded carrier (states 3
4) is the favored pathway. We
do not consider a H
leak pathway (states 2
5, see
text for justification). B, presteady-state currents observed
in the presence of external protons can be accounted for by a
three-state (states 6, 1, and 2) partial reaction since transient
charge transfers are abolished by Gly-Sar. Membrane potential affects
both the translocation of the empty carrier (states 6
1) and
proton binding with the carrier (states 1
2). The partial reaction
scheme is described by four membrane potential-dependent rate constants k
, k
, k
, and k
(9, 19) : a rate constant k
(for a reaction step x
y) is defined by its potential-independent
value (k
), V
, and ligand (H
)
concentration, as well as the coefficients
`,
", and
which describe the fraction of the electric field sensed by the
H
binding to its external site (
`) or internal
site (
") and by the empty ion binding site on the carrier during
membrane translocation (
), where
` +
" +
= 1(20) ; µ is the electrical potential FV
m/RT. C and D, model
prediction of charge transfer associated with hPEPT1, based on
simulation of the three-state model in B (see text) for which
` = 0.27,
" = 0,
= 0.73, k
= 82 s
, k
= 310 s
, k
= 1
10
M
s
, k
= 550 s
,
and [H
]
= 3.16
10
M (pH 5.5). The model predictions (solid lines) of
and Q for pH 5.5,
obtained as described previously(9, 19) , were
compared with the actual data (
) extracted from Fig. 5, B-C. C,
as a function of V
; the model also predicts a second much
faster time constant (
, not shown, see (9) )
which exceeds the resolution of the current technique. D, Q as a function of V
normalized
by Q
, the maximal charge at extreme depolarizing
potentials.
[H]
modulates charge transfer across the membrane over a broad
voltage range (Fig. 5C and Fig. 6, B and C). The apparent valence of the movable charge on the
carrier (z, which may represent the aggregate of more than one
charge transfer step) was around 1 at each pH as for other transporters
except EAAT2 (Table 1), and the Q
/V
relationship extends
over a wide voltage range at any given
[H
]
. That is, the extent of the
charge transfer (and subsequently therefore also the magnitude of the
steady-state current) is finely regulated according to V
. The value of z fell by one-third over
the [H
]
range 0.32-10
µM (Fig. 6E), so that carrier orientation
was biased over a broader V
range at high
[H
]
than at low
[H
]
, whereas for human SGLT1 z was independent of
[Na
]
(9) .
The ratio
of I at -150 mV to Q
at saturating [H
]
is an
index of the turnover rate of the transporter(9) : in two
oocytes the turnover rates were 97 and 96 s
;
however, these are underestimated since the carrier was not
voltage-saturated at -150 mV (see Fig. 1and Fig. 3B). The turnover rates for hPEPT1 and rPEPT1 are
2-10-fold greater than those determined for other transporters
(see Table 1). Using the relation Q
= C
.z.e, where C
represents the carrier density, and e the elemental
charge, at pH 5.0 (Q
= 12 nC), we
estimated hPEPT1 carrier density to be
10
per oocyte,
within an order of magnitude of the density values obtained for other
transporters.
Presteady-state data indicated that H can bind to the empty carrier in the absence of oligopeptide. The
steady-state data presented indicated that H
and
oligopeptide are translocated simultaneously in the same reaction step.
This is supported by the observation that, at least at V
more positive than -70 mV, the affinity of the carrier for
substrate (Gly-Sar) deteriorated at diminishing
[H
]
(e.g. at -50
mV, K
rose sharply at
[H
]
<1 µM, Fig. 4B)(21) . The transporter prefers to bind
H
and substrate in an orderly fashion, H
first. This conclusion is based upon the observation that the
apparent maximal transport rate (I
)
was barely attenuated when [H
]
was reduced 10-fold from 10 to 1 µM (Fig. 4C). Only once
[H
]
was reduced 100-fold (to
well below the K
) did I
fall markedly, and even then the
reduction in I
was less than would
be expected for a system in which activator and substrate binding was
random(21, 22) .
At pH 7.5, the current evoked by 1
mM Gly-Sar was 30% that at pH 5.5 (Fig. 2A) whereas [
C]Gly-Sar
uptake (at 30 µM Gly-Sar, pH 7.5) was as much as 54% that
at pH 5.5(4) . The reason for the discrepancy is not clear, but
this may indicate that there is an appreciable
H
-uncoupled flux of Gly-Sar at pH 7.5. Thus we propose
a model (Fig. 7A) in which the preferred route is for
H
to bind first (states 1
2) then Gly-Sar (states
2
3), and for H
and Gly-Sar to be translocated
simultaneously (states 3
4), but not excluding the possibility
that a H
-uncoupled Gly-Sar flux may proceed (states
1
2a
5a) at high pH. An uncoupled H
flux
(states 2
5) is unlikely since switching the superfusing
Na
medium from pH 7.5 to 5.5 (in the absence of
peptide) resulted in a shift in baseline current in oocytes expressing
hPEPT1 no greater than that observed for vehicle-injected oocytes. That K
increased markedly with
depolarization (Fig. 2B) indicated that, at least
within the voltage range -70 mV to -30 mV, H
binding is voltage-dependent. According to our model, the modest
dependence of Q
upon
[H
]
(Fig. 6D)
supports the conclusion that H
binding is
voltage-dependent, i.e. ``ion well'' effect (21) , but that reorientation of the empty, charged carrier
within the membrane field accounts for most of the charge movements
observed.
The interpretation of the data is complicated under
conditions of large hyperpolarizing potentials and high
[H]
by a nonspecific inhibitory
effect of pH upon substrate binding (illustrated best by Fig. 4A). This may be due to chemical perturbations of
the protein structure at low pH since (i) hyperpolarization and high
[H
]
will together maximize the
number of empty carriers exposed to the extracellular milieu, and (ii)
the manifestation of this pH effect is countered by increasing the
[Gly-Sar]
(see Fig. 1A).
The V at which the I/V
relationships saturated with V
moved in the
hyperpolarizing direction as [Gly-Sar]
was
increased (Fig. 1A) in contrast to SGLT1 (7) and SGLT2 (
)for which saturation with V
is reached at more depolarized V
with increasing sugar at fixed
[Na
]
. This feature of hPEPT1 is
presumably a direct result of the reduced affinity of the carrier for
Gly-Sar at low pH and hyperpolarization. Since this effect is
ameliorated by increasing substrate concentration, it may be equally
noticeable at higher pH when using a substrate of lower apparent
affinity than Gly-Sar.
hPEPT1 shares general features in common with
the Na-coupled transporters: steady-state and
presteady-state kinetics for hPEPT1 were reminiscent of those for
SGLT1(7, 9) , SGLT2 (10) ,
and
SMIT(23) . The biophysical parameters determined for hPEPT1
(and rPEPT1), and the nature of their voltage dependence, were similar
to the cloned Na
-coupled transporters (Table 1).
This observation (i) argues against the notion that protons behave
inherently differently to Na
in coupled transporters,
suggesting instead that the mechanical organization of hPEPT1 and the
mechanism of cation activation are similar to
Na
-coupled transporters; and (ii) opposes the idea
that H
-coupled transporters in mammals represent a
phylogenically ``primitive'' class of transport
proteins(24) . H
is equally effective as
Na
in driving
-methyl-D-glucopyranoside
transport via SGLT1(25) .
In summary, we have shown that
glycyl-sarcosine evoked voltage-dependent and
H-dependent currents in oocytes expressing the human
H
/oligopeptide cotransporter hPEPT1. At resting
membrane potential (
-50 mV), H
behaved as
an essential activator of oligopeptide transport and the apparent
affinity for Gly-Sar was maximal when pH was close to that measured (pH
6.0) in human proximal jejunum in situ(3) . Under
these conditions, [H
]
(1
µM) was
2.5 times greater than the measured
half-maximal [H
]
. Excluding a
nonspecific effect of low pH at hyperpolarized potentials, our data are
consistent with an ordered, simultaneous transport model in which
H
binds first. Model simulation provided single-step
rate constants which can account for the presteady-state charge
movements observed for hPEPT1 in the absence of Gly-Sar, and attributed
to reorientation of the empty carrier in the membrane and in part to
H
binding/dissociation. Investigating the kinetic
characteristics of the products of site-directed mutagenesis in hPEPT1,
and those of a second H
/oligopeptide cotransporter,
hPEPT2(14) , ought to provide insights into structure-function
relationships for this family of transport proteins.