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Journal of Physiology (2001), 536.2, pp. 495-503
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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The two-electrode voltage clamp (TEVC) technique has been used extensively to study the electrophysiological properties of the proton-coupled intestinal peptide transporter PEPT1 expressed in Xenopus oocytes (Boll et al. 1994, 1996; Doring et al. 1996, 1998a,b; Mackenzie et al. 1996a,b; Wenzel et al. 1996; Amasheh et al. 1997; Nussberger et al. 1997; Steel et al. 1997; Fei et al. 1998, 1999). The results of these studies show that PEPT1 transports a large variety of di- and tripeptides as well as selected peptidomimetics by proton-coupled symport leading to inwardly directed transport currents. Since the TEVC technique does not allow the composition of the intracellular compartment to be defined, very little is known about the effects of intracellular factors such as pH or the presence of internal substrates on the transport properties of PEPT1. Moreover, only a very few studies employing intestinal membrane vesicles containing PEPT1 have shown a trans-stimulation phenomenon when substrates were provided on the 'trans' side (Takahashi et al. 1998). Whether PEPT1 also transports peptides electrogenically in the reverse (inside to outside) direction has never been analysed. Moreover, kinetic constants derived for the inward and outward transport modes could help us understand the basic features of this novel proton-dependent electrogenic carrier that possesses an extraordinarily broad substrate spectrum. In the present study, we used the giant patch clamp (GPC) technique in the inside-out configuration and the classical TEVC approach to determine the basic properties and kinetic constants of substrate translocation by PEPT1 in both transport directions and under identical experimental conditions. Our results show that PEPT1 binds its substrates also at the cytosolic face and, depending on pH and membrane potential, its apparent substrate affinity can be even higher on the inside than on the outside. However, when transportable substrates are present on both sides of the membrane in sufficiently high concentrations, the direction and rate of transport are dependent solely on the membrane potential, and transport occurs symmetrically.
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METHODS |
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Xenopus laevis oocytes were collected under anaesthesia (immersion in a solution of 0.7 g l-1 3-aminobenzoic acid ethyl ester; Sigma); the frogs were killed with an anaesthetic overdose after the final collection. Oocytes were treated with 2.5 mg ml-1 collagenase for 90 min and were separated manually thereafter. They were incubated in Barth's solution containing (mM): NaCl (88), KCl (1), MgSO4 (0.8), CaCl2 (0.4), Ca(NO3)2 (0.3), NaHCO3 (2.4) and Hepes (10) (pH 7.5) at 17 °C overnight. Thereafter, stage V/VI oocytes were injected with 25 ng rabbit PEPT1 cRNA in a 50 nl volume and incubated for 3-6 days at 17 °C.
Two-electrode voltage clamp experiments were performed as described previously (Amasheh et al. 1997). In short, the oocyte was placed in an open chamber (~0.5 ml total volume) and continuously superfused (~3 ml min-1) with the Barth's solution or with solutions containing the substrates to be studied. Electrodes with resistances between 1 and 10 M
s were connected to a TEC-05 amplifier (npi electronic, Tamm, Germany). Oocytes were voltage clamped at -60 mV, and current-voltage (I-V) relations were measured using short (100 ms) pulses separated by 200 ms pauses in the potential range -160 to +80 mV. I-V measurements were made immediately before and 20-30 s after substrate application when current flow reached steady state. The current evoked by PEPT1 at a given membrane potential was calculated as the difference between the currents measured in the presence and absence of substrate. Data were stored in a computer and I-V relations were calculated with a Visual Basic (VBA) routine written in Microsoft Excel. Positive currents denote positive charges flowing out of the oocyte.
For giant patch clamp experiments, the vitelline layer was removed mechanically after a short incubation of oocytes in hypertonic medium (Barth's solution as above + 100 mM potassium aspartate) before the experiment. Patch pipettes of diameters 20-30 µm were prepared from thin-walled borosilicate glass capillaries (MTW-150, WPI, Berlin, Germany) on a Zeitz electrode puller (Zeitz Instruments, Munich, Germany) and were fire-polished on the same puller. Pipettes were filled with a solution containing (mM): NaCl (10), sodium isethionate (80), MgSO4 (1), Ca(NO3)2 (1) and Hepes (10), titrated to pH 7.5. Patch experiments were made with an EPC-9 amplifier and the PULSE program (HEKA, Lambrecht, Germany). After gigaseal formation (1-10 G), pipettes were moved to a perfusion chamber (~0.3 ml) and superfused continuously (~2 ml min-1) with the control bath solution composed of (mM): potassium aspartate (100), KCl (20), MgCl2 (4), EGTA (2) and Hepes (10) (for pH 
7.5) or Mes (10) (for pH 
6.5). During experiments, the membrane potential was clamped at -30 mV and I-V relations were measured using short (100 ms) pulses separated by 200 ms pauses in the potential range -80 to +60 mV. I-V relations were measured in the same way as described for TEVC experiments, except that the time between substrate application and measurement was 1 min. The data were evaluated with the PATCH program written and made available by courtesy of Dr Bernd Letz (HEKA, Lambrecht, Germany).
Gly-L-Gln (Sigma, Deisenhofen, Germany) and was added to the solutions in concentrations as indicated in the text. After addition of dipeptides, the pH was readjusted if necessary. The percentage of the zwitterionic form at a given pH was calculated with pK values taken from Sober (1968) for Gly-L-Gln (pK1 = 2.88 , pK2 = 8.29).
Transport parameters are defined by K0.5 (mM) and Imax (nA or pA). These parameters were calculated based on the Michaelis-Menten equation from at least three and preferably from four to five data points. Data are presented as the mean ± S.E.M. of n experiments. Statistically significant differences (P < 0.05) were determined using the Student's t test for paired or non-paired data as appropriate.
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RESULTS |
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Current-voltage relations in TEVC experiments give indications for an outwardly directed transport current
The zwitterionic dipeptide Gly-L-Gln is known as a high-affinity substrate for PEPT1 for transport in the inward direction and was also used as a test compound in the present experiments. Current-voltage relations were recorded 20 s after the start of the superfusion of oocytes with 5 mM Gly-L-Gln at two extracellular pH values (7.5 and 6.5). Since the cytosolic pH of stage VI oocytes has been reported to be between 7.4 and 7.7 (Cicirelli et al. 1983; Sasaki et al. 1992; Steel et al. 1997), the transmembrane pH gradient at an extracellular pH of 7.5 is expected to be negligible (in the range of ± 0.2 pH units), whereas at pH 6.5 the inwardly directed pH gradient of about 1 unit contributes to the driving force. As reported previously (Boll et al. 1994; Doring et al. 1996; Mackenzie et al. 1996b), at pH 6.5 the transport current remains inwardly directed in the voltage range from -160 to +80 mV. However, in the present study, we found that at pH 7.5, the direction of current became potential dependent and was outwardly directed at membrane potentials more positive than +20 mV. At lower substrate concentrations, the reversal potential was shifted to even more negative values, so that with 2 and 0.5 mM Gly-L-Gln, current direction already changed at 0 and -20 mV, respectively. These data strongly suggest that, in the absence of the driving force provided by the pH gradient, PEPT1 can transport electrogenically not only in the forward, but also in the reverse direction. Since the oocyte was clamped at -60 mV during the initial superfusion period with the dipeptide-containing solution, the substrate might have already accumulated in substantial concentrations within the oocyte, building a substrate pool for reverse transport upon the succeeding membrane depolarization during I-V recordings. In addition, endogenous peptides could also have contributed to the observed outward currents. To test the possible role of endogenous peptides, we perfused the extracellular surface with the control solution containing glibenclamide (500 µM), a known inhibitor of PEPT1. Glibenclamide did not affect basal current-voltage relations, suggesting that the cytosolic concentration of endogenous peptides is too low to cause an efflux of peptides from the oocyte that could alter the current-voltage relations measured in the presence of extracellular substrates. To further validate the assumption that an inward transport of peptides must take place before a significant outward transport can be recorded, we superfused the oocytes with 5 mM Gly-L-Gln at an elevated flow rate; and we simultaneously shortened the time between dipeptide application and the first I-V measurement to 7 s and clamped the oocyte during this time at +60 mV to reduce peptide uptake before the I-V measurement. Although, even under these conditions, the outward current measured at +80 mV could not be suppressed completely, it was significantly reduced after 7 s compared with 20 s (96 ± 31 pA versus 305 ± 26 pA, n = 4), and the zero current potential was shifted correspondingly (from 48 ± 6 mV after 7 s to 19 ± 7 mV after 20 s, n = 4). Furthermore, in some of these fast perfusion experiments, an outwardly directed current flow was observed for a few seconds immediately after switching back to substrate-free solution even at a membrane potential of -60 mV, suggesting a rapid electrogenic backflux of the substrate from a submembrane compartment (not shown).
Outward currents in giant patch clamp experiments
The TEVC experiments showed that PEPT1 was able to transport not only in not only in its normal, but also in the reverse, direction. This process was further analysed by using the giant patch clamp technique. Figure 1A shows current traces recorded at membrane potentials between -80 and +60 mV in the absence or presence of 5 mM Gly-L-Gln added to the cytoplasmic face of a membrane patch excised from a Xenopus oocyte expressing PEPT1. The corresponding I-V relations are shown in Fig. 1B. The difference between the I-V curves measured with and without substrate (Fig. 1C) is equivalent to the current evoked specifically by PEPT1-mediated peptide transport. No comparable current was obtained in water-injected oocytes (Fig. 1D) or when up to 15 mM mannitol was applied instead of Gly-L-Gln (data not shown). As demonstrated in Fig. 1C, the transport-induced current measured in the absence of a pH gradient was outwardly directed in the entire potential range investigated and approached the potential axis asymptotically at high negative potentials. This behaviour would be expected when the substrate concentration on the 'trans' side remains negligible, as in GPC experiments, in which the solute transported through a small membrane area is rapidly distributed in a large volume.
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Figure 1. Dipeptide-induced outward current measured in giant patch clamp experiments A, current traces recorded in a typical giant patch clamp experiment in response to potential pulses between -80 and +60 mV in the absence (left) or presence (right) of 5 mM Gly-L-Gln on the cytosolic surface. The pH of the solutions was 7.5 on both sides of the membrane. The short spikes at the beginning and end of the pulses denote currents charging the membrane capacitance. B, I-V relations calculated from the traces shown in A. | ||
The outward transport current elicited by 5 mM dipeptide on the cytosolic surface depended on the cytosolic pH. It reached its maximum at pH 7.5 (i.e. in the absence of a transmembrane pH gradient). Elevation of the pH to 9.0 reduced the current to 26 ± 5 %, whereas by reducing the pH to 6.5, transport currents remained unchanged at 96 ± 4 % of maximum. Transport rates obtained between pH 9.0 and pH 7.5 closely followed the changes in the percentage of the substrate present in its zwitterionic form (16.4 % to 86.0 %) at any given pH, suggesting that the neutral dipeptide species is the preferred substrate form. At pH 6.5, Gly-L-Gln is present almost exclusively in its neutral form (Table 1) and therefore the relative decline in transport currents at pH 6.5 cannot be explained by the charge of the substrate, but suggests that a higher H+ activity (lower pH) on the cytoplasmic surface causes inhibition of transport. Current measurements at pH values below 6.5 were not possible, since a low cytoplasmic pH together with depolarizing membrane potentials (above 0 mV) opened, in most patches, a large membrane conductance that showed only little or no recovery (not shown).
Kinetic parameters of substrate-evoked currents in the inward and outward transport directions
Membrane potential and pH dependencies of maximal transport currents (Imax) and apparent substrate affinities (K0.5) were calculated from the Michaelis-Menten kinetics determined with substrate concentrations ranging between 0.2 and 10 mM (inward transport) and 0.5 and 20 mM (outward transport). The results are shown in Table 1 and in Figs 2A and B for the inward and in Figs 3A and B for the outward transporting mode.
Inward transport direction in TEVC experiments. As expected for an electrogenic cotransporter, Imax showed a pronounced potential dependency, but was only modestly dependent on bath pH and ranged from 82 ± 2 % (at pH 5.5, n = 8) to 128 ± 4 % (at pH 8.5, n = 13) compared with that at pH 7.5 set to 100 % (n = 52). When Imax currents were replotted as the percentage of that measured at -160 mV, no effect of external pH on maximal transport currents was obtained (Fig. 2A). In contrast, apparent K0.5 values were dependent on both membrane potential and pH (Fig. 2B). K0.5 displayed, at each pH, a potential-dependent minimum, and this minimum was shifted with decreasing pH towards depolarizing membrane potentials (-140 mV at pH 8.5, -120 mV at pH 7.5, -40 mV at pH 6.5 and +20 mV at pH 5.5). A further depolarization below these potentials induced a sharp reduction in apparent substrate affinity.
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Figure 2. Kinetic parameters of inwardly directed Gly-L-Gln transport as a function of membrane potential and extracellular pH Data from TEVC experiments, n = 6-52. A, Imax values have been normalized to that measured at -160 mV at the pH values indicated. The dashed line represents the extrapolation of the linear section of the I-V curve onto the x-axis (for further details see Discussion). B, K0.5 values measured at different pH values and membrane potentials. Apparent K0.5 values could be calculated reasonably well only for pH ranges and membrane potentials as shown in the figure. | ||
Outward transport direction in GPC experiments. Imax measured at +60 mV was found to be independent of the cytosolic pH: Imax was 106 ± 8 % at pH 6.5 (n = 6) and 111 ± 11 % at pH 8.5 (n = 8) when compared with that at pH 7.5 (n = 11) set to 100 %. Due to the stimulation of a large membrane conductance at low cytosolic pH, no valid data could be obtained at pH 5.5. Similar to the inward transport direction, Imax was potential dependent but also displayed a distinct pH profile (Fig. 3A). The potential dependency was greatest at pH 6.5 and decreased with increasing pH. Apparent K0.5 values were essentially independent of membrane potential at pH 6.5 and 7.5, but decreased with membrane depolarization at pH 8.5 (Fig. 3B). The lowest K0.5 value was measured at pH 7.5, being 3.28 ± 0.40 mM at +60 mV. All substrate affinities calculated from the kinetics measured in GPC experiments were 5-20 times lower than the corresponding values for the inward transport under similar experimental conditions in the voltage clamp experiments (Fig. 2B versus 3B and Table 1).
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Figure 3. Kinetic parameters of outwardly directed Gly-L-Gln transport as a function of membrane potential and cytosolic pH Data from GPC experiments, n = 6-11. The extracellular (pipette) pH was 7.5. A, Imax values have been normalized to that measured at +60 mV at the pH values indicated. B, K0.5 values were nearly independent of membrane potential at pH 6.5 and 7.5, but increased modestly with membrane hyperpolarization at pH 8.5. Note that all K0.5 values are considerably higher than the corresponding values measured in TEVC experiments. | ||
Inward currents measured in giant patch clamp 'inside-out' configuration
To assess whether the outward current recorded in TEVC experiments at positive membrane potentials was indeed caused by electrogenic substrate efflux due to substrate accumulation on the cytosolic surface, we simulated this situation in giant patches in which the composition of the cytosolic solution can be controlled. However, this method requires the use of a cytosolic inhibitor to record the 'zero line', by blocking the inward current generated by the substrate continously present in the pipette (i.e. outside). Recently, glibenclamide has been reported to non-competitively inhibit PEPT1 from the extracellular surface with an inhibition constant of Ki 
25 µM (Sawada et al. 1999). We therefore tested whether glibenclamide also blocks transport when applied on the cytosolic surface and thus whether it can be used to measure inward currents in the 'inside-out' configuration. Inward currents generated by 5 mM Gly-L-Gln added to the pipette solution were progressively inhibited by increasing concentrations (50-500 µM) of glibenclamide on the cytosolic surface (Fig. 4). From these data, we calculated an apparent Ki of 61 ± 16 µM (n = 7) for this inhibitor on the inside. Addition of glibenclamide to a cytosolic solution containing 5-20 mM Gly-L-Gln inhibited the outward transport currents with a similar Ki (73 ± 12 µM, n = 4). As inhibition occurred even in the presence of high concentrations of the dipeptide, glibenclamide also acts non-competitively on the cytosolic side. In the subsequent experiments, 20 mM Gly-L-Gln was present in the pipette (external) solution and 500 µM glibenclamide was used to disclose transport currents in the absence or presence of increasing concentrations (5, 10 and 20 mM) of Gly-L-Gln in the bath. The current-voltage relations measured in the presence of glibenclamide were nearly indentical in the absence and presence of Gly-L-Gln on the cytosolic side, showing that glibenclamide inhibited both inwardly and outwardly directed current components (data not shown). Figure 5 demonstrates that increasing concentrations of Gly-L-Gln on the cytosolic surface progressively reduced the inwardly directed transport currents. The slope measured in the linear section of the I-V relationship (between -120 and -60 mV) also decreased (from 0.147 to 0.095 pA mV-1). Above a concentration of 10 mM, an outward current was obtained at positive membrane potentials. Thus, the presence of > 10 mM Gly-L-Gln on the cytosolic surface obviously mimics the situation observed in TEVC experiments, in which outward currents at positive membrane potentials occur when the substrate concentration rises in the oocyte. When 20 mM Gly-L-Gln was present on both sides of the membrane, the I-V relationship became nearly linear and crossed the potential axis close to 0 pA. The small residual current present at 0 mV was probably caused by the still incomplete substrate saturation (about 86 %) of the transporter at the cytosolic face, but this could not be tested experimentally, since Gly-L-Gln and glibenclamide together were not soluble at higher concentrations.
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Figure 4. Inhibition of the inward transport current by glibenclamide in giant patch clamp experiments Inward current was generated by 5 mM Gly-L-Gln added to the pipette solution (extracellular side) and was inhibited by increasing concentrations (as shown on the left hand side of the main graph) of glibenclamide on the cytosolic side. Glibenclamide (500 µM) caused a nearly complete inhibition of the peptide-generated inward current. The inset graph (right) shows the best fit of calculated currents (at -120 mV) to the Michaelis-Menten equation. The calculated parameters for this experiment are Ki = 39 µM and Imax = 69 pA. | ||
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Figure 5. The influence of increasing cytosolic substrate concentrations on the inward current in giant patch clamp experiments Inward current was induced by 20 mM Gly-L-Gln in the pipette solution and was measured in the presence of 0, 5, 10 and 20 mM of the same substrate added to the cytosolic side. Each line shown represents the mean of six experiments and was calculated as the difference in current-voltage relations measured in the absence and presence of 500 µM glibenclamide. Addition of increasing concentrations of Gly-L-Gln to the cytosolic side reduced inward currents at negative membrane potentials, induced an increasing outward current at positive membrane potentials, reduced slope and increased linearity for the resulting I-V relationships. | ||
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DISCUSSION |
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Here we demonstrate for the first time that the proton-coupled peptide transporter PEPT1 translocates dipeptides electrogenically not only in its 'normal', outside to inside, direction, but also in the reverse direction. Transport characteristics and kinetic constants were obtained under almost identical experimental conditions (oocytes from the same batches; on the 'trans' side, low substrate concentration and a pH of about 7.5; symmetrical membrane potential) in GPC and TEVC experiments, allowing differences in binding and transport on both membrane sides to be compared.
Bidirectional transport shows complete symmetry at saturating substrate concentrations and in the absence of a pH gradient. Under these conditions, the membrane potential solely determines the direction and rate of transport. At negative membrane potentials and dependent on pH, the substrate binding domain in its outward-facing direction shows generally a higher affinity (0.2 to 3 mM) than when facing the internal side (3 to 30 mM), suggesting major conformational differences of the substrate binding pocket in its inside- and outside-facing states. Similar asymmetric substrate affinities were also found for the GABA transporter GAT1 and the sodium-coupled glucose transporter SGLT1 in giant patch clamp experiments (Lu & Hilgemann, 1999; Sauer et al. 2000). This asymmetry in apparent substrate affinity is important in view of the physiological function of PEPT1, as it allows the substrate to be released into the cytosol when the electrochemical gradient drives inward transport. However, our data suggest that under certain conditions, for example in the absence of a membrane potential or inward positive membrane potentials, apparent substrate affinity at the internal binding site can be even higher than at the external binding site. Thus, when the substrate concentration rises at the internal site, a backflux can counteract the influx and contribute to overall currents measured. Internal substrate binding affinity is essentially independent of membrane potential at pH 6.5 and 7.5, but decreases to above 20 mM when the cell interior becomes alkaline (pH 8.5). Substrate binding affinity to PEPT1 in its outward facing state increases with hyperpolarizing potentials and lower pHout, and becomes essentially independent of membrane potential at pH 5.5. In contrast, maximal transport currents are pH independent, they increase with a distinct initial nonlinear behaviour between +40 and -80 mV, and then they increase proportionately with increases in membrane potential between -80 and -160 mV.
The differences in the characteristics of PEPT1 between the influx and efflux modes are not only evident in the apparent K0.5 values, but also in their dependence on pH and membrane potential. If we compare the effects of pH on internal and external substrate binding, it is obvious that extracellular affinity decreases dramatically when the membrane is depolarized, but the potential effect becomes less prominent with higher external proton activity. This might be explained by an increased proton binding affinity at negative membrane potential to a titratable group at some deep position within the potential field of the membrane. This increased proton binding affinity may in turn increase substrate binding affinity, assuming an ordered mechanism of ligand binding with proton first followed by substrate as proposed previously (Mackenzie et al. 1996b). The internal substrate and/or proton binding site is less affected by pH over a wide range of membrane potentials except when H+ activity is extremely low (pH 8.5).
Although it has been reported that there is a symmetry of H+ binding to the intra- and extracellular sites of PEPT1 (Nussberger et al. 1997), our data demonstrate that the effects of pH on substrate binding to the transporter protein are not identical at both sites. This discrepancy may result from the different approaches used to assess the pH effects on transport. Whereas we could define and control pH and substrate concentration on both sides of the oocyte membrane, Nussberger et al. (1997) used pH-sensitive microelectrodes in oocytes expressing PEPT1 to record pH changes induced by various manoeuvres and relate them to alterations in peptide transport kinetics. As pH microelectrodes can only assess the pH in the oocyte interior, the observed effects on apparent substrate affinity of PEPT1 may not represent the pH in the vicinity of the membrane at the internal substrate binding domain.
One of the consequences of the observed asymmetry are the potential-dependent alterations in the apparent substrate affinity ratios on the external and the internal sides of the membrane. In the absence of a membrane potential and at an identical pH of 6.5 at both surfaces, the substrate K0.5 outside is around 0.4 mM, whereas it is 5 mM on the inside. Changing the pH to 7.5 on both sides decreases the affinity outside to around 20 mM while it increases it slightly on the inside. In this situation, the binding affinity is up to five times higher on the inside than on the outside, which favours a back-transport of dipeptides - even at lower internal than external substrate concentrations. This explains the shift in reversal potential towards negative values observed in TEVC experiments measuring outward currents at lower external substrate concentrations. If an inside negative potential of only -20 mV is imposed, the affinity for substrate binding at pH 7.5 is around 4 mM; however, this increases to 0.2 mM when pHout is reduced to 6.5, while the binding affinity on the inside remains unchanged. Under physiological conditions, this asymmetry enables PEPT1 to function as an net absorptive carrier, and both the inside negative membrane potential and the lower external pH allow the protein to have a higher apparent substrate affinity for inward transport than for efflux. However, at saturating substrate concentrations, pHout no longer plays a role and the rate of transport is solely dependent on the magnitude of the inside negative cell potential.
The characteristic I-V relationships for saturating Gly-L-Gln concentrations shown in Fig. 2A demonstrate two overlapping transport components. Whereas inward currents show a linear dependence on membrane potential between -80 and -160 mV, normalized currents asymptotically fade off towards positive potentials with around 30 % of the Imax current remaining. This suggests that, in addition to the membrane potential, the substrate gradient (
S) per se must also play a role in the current responses. The potential-dependent component calculated from the extrapolation of the linear section of the I-V response onto the potential axis reveals a slope of 0.63 % Imax per millivolt and an intercept at 0 mV (see dashed line in Fig. 2A). Since this intercept remains constant when external H+ activities are varied, it can be concluded that PEPT1 does not possess any substrate-dependent proton leak current. At potentials more positive than -80 mV, the additional current component that is driven by the substrate gradient becomes increasingly prominent. This S-dependent fraction contributes less than 20 % to the total current at -60 mV, but this increases to 100 % when the membrane is depolarized to 0 mV. The reason for this characteristic potential dependency might be the fast dissipation of S when the potential-dependent and S-dependent components work in parallel (e.g. at -60 mV), whereas a much slower or even incomplete decline of S occurs when the potential-driven current is negligible (at 0 mV). At membrane voltages of 0 to +40 mV, the S-driven and the potential-driven currents even flow in opposite directions. In this situation, substrate and protons entering from outside, driven by S, will partly be exported again, resulting in a reduced net inward transport current. This backflux is favoured by the relatively high internal apparent substrate affinity of around 4 mM that is unaffected by membrane voltage between -20 and +60 mV (at pH 7.5). As the membrane potential becomes more positive inside, the balance is shifted towards a reduced influx now occurring against the electrochemical potential, but increased outward transport along the electrochemical potential. When the membrane is further depolarized above +60 mV, positive inward currents are no longer detectable. These findings demonstrate that substrate gradients cannot be neglected in TEVC experiments, neither with regard to their contribution to inwardly directed currents (the S component) nor with regard to the reversal of transport direction by depolarizing voltage jumps.
In GPC experiments, accumulation of substrate on the 'trans' side is expected to be negligible due to the low number of carriers relative to the distribution volume in the patch pipette. Consequently, the I-V relationships are less affected by S in the potential range studied (especially in the absence of a pH gradient, i.e. at pH 7.5, Fig. 3A). The potential-dependent outwardly directed transport currents in GPC experiments at pH 7.5 amount to ~0.70 % Imax per millivolt and are therefore very similar to the value calculated for the inward direction. When a high concentration of substrate is present on both sides of the membrane patch (Fig. 5), there is no S-driven component and the I-V relationship obtained for postive as well as negative potentials is almost linear.
From this part of the study, we conclude that the I-V relationships observed for PEPT1 reflect a linear membrane potential-dependent transport component and a S-dependent component that declines as the voltage is increased and substrate accumulates inside the cell. The magnitude of the S-dependent transport component depends, of course, on the time point at which the recording of the I-V relationship is performed. If time proceeds and the transporter is able to build up a high substrate concentration at the internal surface, then S might become large enough to overcome the asymmetry of the apparent substrate affinity differences and mediate a significant backflux of substrate to the outside, even against the membrane potential.
In summary, electrogenic dipeptide transport by PEPT1 occurs bidirectionally and utilizes two driving forces (membrane voltage and substrate gradient). The observed asymmetry of substrate binding affinity in the inward or outward facing state of the carrier is subject to alterations in membrane potential and pH. This establishes its physiological function as a net accumulative transporter in the normal cellular setting. However, under certain conditions, such as a low membrane voltage, the absence of a pH gradient and higher intracellular substrate concentrations, PEPT1 may even serve as an electrogenic dipeptide-proton efflux symporter. With saturating substrate concentrations on both sides of the cell membrane, both direction and rate of transport depend solely on membrane potential.
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Acknowledgements
The authors acknowledge the expert technical assistance of Mr Rainer Reichlmaier (TEVC studies) and Mr Adelmar Stamfort (GPC studies; contribution to VBA programming). This work was supported by Grant Da 190/5-2 from the Deutsche Forschungsgemeinschaft.
Corresponding author
G. Kottra: Institute of Nutrition, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany.
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, 0 mM Gly-L-Gln; 
, 5 mM Gly-L-Gln. C, the current produced by the PEPT1-mediated transport of the dipeptide is calculated as the difference between the traces shown in B. Note that current remains outwardly directed over the entire potential range studied. D, in water-injected control oocytes, Gly-L-Gln does not produce any transport current (symbols as in B).