Stoichiometry and Kinetics of the High-affinity H+-coupled Peptide Transporter PepT2*

Proton-coupled peptide transporters mediate the absorption of a large variety of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both d-Phe-l-Ala andd-Phe-l-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. Ford-Phe-l-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H+:Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H+. This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (I pss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited I pss in response to hyperpolarization at pH 6.5–8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant I pss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarizedV m , our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.

In kidney and intestine, enzymatic degradation of proteins and peptides produces oligopeptides that are absorbed across the brush-border membrane of epithelial cells, followed by breakdown into free amino acids (1)(2)(3). The absorption is carried out by proton-driven cotransport systems, as demonstrated by studies using brush-border membrane vesicles, epithelial cells in culture, intact epithelial tubules, and recombined peptide transporters (4 -10). A large variety of diand tripeptides, as well as pharmacologically active peptidelike compounds such as ␤-lactam antibiotics and angiotensinconverting enzyme inhibitors, are transported (11)(12)(13). Peptide transporters have been cloned since 1994 (3, 14 -19) and were shown to accept many oligopeptides and peptide-derived compounds as substrates, highlighting the physiological significance of these transporters in nutrient and drug absorption. Functional studies of these membrane proteins allow a better understanding of the mechanism of substrate-transporter interaction and may help establishing therapeutic strategies involving peptide-based drugs.
An electrophysiological study of PepT2 has been carried out recently on a rabbit homologue (10). However, the H ϩ :peptide stoichiometry is still controversial (6,10,13,20). In the present study, we report novel characteristics of the rat PepT2 demonstrated by neutral, anionic, and cationic substrates under various conditions, using the two-microelectrode voltage-clamp technique. We simultaneously measured the radiolabeled peptide uptakes and the PepT2-mediated currents under voltageclamp conditions in order to determine the H ϩ :substrate stoichiometry. We also demonstrated quinapril-sensitive PepT2mediated outward currents, a proton leak, and pH-dependent presteady-state currents (I pss ).

EXPERIMENTAL PROCEDURES
Isolation of PepT2 Clone and Oocyte Preparation-A kidney cortex gt10 cDNA library was screened using a 0.52-kb PepT2 probe generated by specific PCR primers. The complete PepT2 sequence was isolated and subcloned into PTLN2 plasmid. Sequence analysis of both strands confirmed a 100% identity to the published PepT2 sequence (GenBank TM accession number D63149, Ref. 21). Xenopus laevis oocytes were prepared as described previously (22) except that oocytes were defolliculated by incubating them in the calcium-free Barth's solution containing collagenase (3 mg/ml) at 18°C for 3-3.5 h. Capped cRNA of rat PepT2 was synthesized by in vitro transcription from cDNAs in PTLN2 and injected (with 15-25 ng) into oocytes. The same volumes of H 2 O were injected as the control.
Electrophysiology-The two-microelectrode voltage-clamp experiments were performed using a commercial amplifier (Clampator One, model CA-1B, Dagan Co., Minneapolis, MN) and the pCLAMP software (Version 6, Axon Instruments, Inc., Foster City, CA). In experiments involving voltage jumps, currents or membrane potentials were recorded by digitizing at 150 s/sample and by the Bessel filtering at 10 kHz. When recording currents at a holding potential, digitization at 0.5 s/sample and filtering at 20 Hz were used. Control solutions used for extracellular perfusion contained (in mM): NaCl, 100; HEPES, 10; MES, 1 2.5; Tris base 2.5; KCl, 2; CaCl 2 , 1; MgCl 2 , 1; pH 5.0 -8.0 by N-methyl-D-glucamine or HEPES. After 3ϳ5 min of membrane potential stabilization following microelectrode impalements, the oocyte was clamped to the holding potential (V h ) of Ϫ50 mV. 100-ms voltage pulses of between Ϫ160 and ϩ60 mV, in increments of ϩ20 mV, were then applied, and steady-state currents were obtained as the average values in the interval from 80 to 95 ms after the initiation of the voltage pulses.
Voltage-clamped Tracer Measurements-Substrate-evoked currents and uptake of [4-3 H-D-Phe]-Ala, [4-3 H-D-Phe]-Glu, or [4-3 H-D-Phe]-Lys were simultaneously measured under voltage-clamp conditions, according to a method similar to the one described previously (23). The radioactive phenylalanyl-dipeptides were synthesized by Zeneca Cambridge Research Biochemicals (Northwich, Cheshire, United Kingdom) and dissolved in 10% aqueous ethanol. The specific radioactivity is 12 Ci/mmol, and the concentration is 1 Ci/l (or 83.3 M) for all three peptides. The unlabeled Phe-Glu and Phe-Lys were synthesized as described previously (24), and other peptides were purchased from Sigma. The control solution or HEPES-rich solution (in mM: NaCl, 70; HEPES, 60; MES, 2.5; KCl, 2; CaCl 2 , 1; MgCl 2 , 1; pH 5.5-6.5 by N-methyl-D-glucamine) was used for external perfusion. The uptake solution consisted of the control or HEPES-rich solution plus cold (0.5, 0.5, or 1 mM of Phe-Ala, Phe-Glu, or Phe-Lys, respectively) and hot substrate (1.33, 1.33, or 2.67 l of radiolabeled Phe-Ala, Phe-Glu, or Phe-Lys, respectively, in 200 l of solution). The hot substrates contained in the uptake solutions represented only 0.11% of the total substrates. Before starting tracer uptakes, oocyte was clamped at Ϫ80 mV and perfused with substrate-free solution. Then the perfusion was stopped, and the uptake solution (200 l) was added manually using a pipettor, which washed out the substrate-free solution. The uptake lasted 5 min in the chamber whose volume is about 200 l and terminated by perfusing (washing) the oocyte with the substrate-free solution.
Statistics and Data Analysis-Experimental results were expressed in the form of mean Ϯ S.E. (n), where n indicates the number of oocytes obtained from at least two different donors. The curve-fitting procedures were performed using the SigmaPlot program (Version 4, Jandel Scientific Software, San Rafael, CA), and each fitted parameter is expressed in the form parameter Ϯ S.E., where S.E. represents the standard error in the fitting estimates.

Substrate and Proton
Affinities-PepT2 mediates the highaffinity transport of most di-and tripeptides at physiological membrane potentials (V m ) but not at hyperpolarized V m (Fig. 1,  A and B). Apparent affinities for both glycyl-dipeptides (Gly-Glu, Gly-Leu, Gly-Lys) and D-phenylalanyl-dipeptides (Phe-Glu, Phe-Ala, Phe-Lys) were strongly voltage-dependent and decreased about 10-fold when hyperpolarizing from Ϫ50 to Ϫ160 mV (Table I and Fig. 1B). Although affinities differ largely according to the charge status of glycyl-dipeptides, the maximal currents for glycyl-dipeptides remain approximately the same in the whole voltage range (Fig. 1C). High concentrations of glycyl-dipeptides evoked currents that did not saturate by hyperpolarization, while modest or low concentrations of these substrates did saturate ( Fig. 2A). In contrast, saturation at hyperpolarization was observed for phenylalanyl-dipeptides, even at high concentrations (Fig. 2B), presumably due to relatively low substrate affinity (Table I). Depending on pH, substrate type, and substrate concentration, PepT2 may exhibit decreases in current with hyperpolarization ( Fig. 2) due to low affinities for substrates. The affinities for cationic peptides are relatively low compared with those for anionic and neutral peptides, in analogy to PepT1 (25,26).
The proton affinities were determined at various membrane potentials using glycyl-dipeptide concentrations approximately equal to five times that of their K m values. Substrate-evoked currents generally increase with an increase in [H ϩ ], which is consistent with a proton-driven transport. However, as alluded to above, currents evoked by low substrate concentrations (especially those evoked by cationic substrates) decreased with increasing [H ϩ ] and hyperpolarization (Fig. 2, D and E). The affinities for proton at V m ϭ Ϫ50 mV were determined when data were fitted to the Hill Equation. The apparent affinity constants were 2.8 Ϯ 0.5 (n ϭ 6), 0.7 Ϯ 0.1 (n ϭ 3), and 0.5 Ϯ 0.1 M (n ϭ 5) (corresponding to pH 5.5, 6.1, and 6.3) for Gly-Glu, Gly-Leu, and Gly-Lys, respectively. The Hill coefficients were 1.06 Ϯ 0.03, 1.27 Ϯ 0.09, and 1.44 Ϯ 0.06, correspondingly. This appears to suggest the coupling of a single proton ion to Gly-Glu and more than one proton ion to Gly-Leu and Gly-Lys. However, Hill coefficients obtained at other V m 1 The abbreviations used is: MES, morpholineethanesulfonic acid.
FIG. 1. Affinity constants and maximal currents for glycyldipeptides at pH 6.0 and various membrane potentials. A, dose response of averaged H ϩ -Gly-Glu cotransport currents at membrane potentials Ϫ50 mV () and Ϫ160 mV (ƒ). B, apparent affinity constants (K m ) for Gly-Lys (q), Gly-Leu (E), and Gly-Glu () were determined when currents elicited by substrates at various concentrations were measured and fitted to the Hill Equation. Inset, K m for Gly-Leu (E) shown in an expanded scale. C, maximal currents (I max ) for substrates. Data are averages derived from four to six oocytes.

TABLE I Apparent affinity constants for a variety of dipeptides
Currents due to addition of various concentrations of substrates were measured in oocytes expressing the rat PepT2 and at pH 6.0. K m values were obtained by fitting the data to the Hill equation using the Sig-maPlot 4 Program. L-Gly-L-Lys 51 Ϯ 3 560 Ϯ 10 11 were significantly different, indicating that this method is not accurate for stoichiometry evaluation. In addition, since currents at low pH experienced decreases at hyperpolarized V m (see Fig. 2F, at Ϫ100 mV), they no longer obey the Michaelis-Menten or the Hill relationships. Thus, it is inaccurate to evaluate stoichiometric ratios based on Hill coefficients. More direct approaches are necessary to determine the H ϩ :substrate stoichiometry. Determination of Stoichiometry by Tracer Method-The proton:substrate coupling ratio (stoichiometry) can be accurately determined when the proton and substrate fluxes mediated by PepT2 are measured under the same conditions. Simultaneous monitoring of transporter-specific currents and radioactive substrate uptakes from the same oocyte under voltage-clamp conditions is one of the few approaches proven to be accurate. Voltage-clamp condition is critical to monitor the PepT2-specific currents, because background currents (before substrate addition) change due to depolarization that is elicited by substrate addition.
We observed that, during the uptake period, substrate-elicited currents significantly decreased after reaching initial peak values (Fig. 3, A-C), which poses the question of whether these decreases interfere with stoichiometry determination. Decreases were less pronounced when the buffer concentration was high (60 versus 10 mM HEPES). For a given buffer concentration, decreases in current were less pronounced when PepT2-specific currents were lower. With similar initial peak currents, Phe-Glu-evoked currents exhibited more decrease than Phe-Ala-evoked currents. We also observed a spike current upon perfusing an oocyte with substrate-free solution after substrate applications. The spikes were significantly higher when the buffer concentration was lower. In the same oocytes, decreases in Gly-Leu-evoked current were also present during continuous perfusion but were accelerated when perfusion was stopped (not shown). These data indicate that decreases in current were partly due to decreased proton concentrations at the immediate proximity of the extracellular membrane. Decreases in current during perfusion may be due to intracellular substrate or H ϩ accumulation (trans-inhibition), in analogy to observations for other transporters (34,35). None of these factors are expected to compromise the accuracy of stoichiometry determination.
No significant difference in the charge:uptake ratios were observed when using either control (10 mM HEPES) or HEPESrich solution (see "Experimental Procedures"). When charge was converted into picomole, the charge to uptake ratios for Phe-Glu, Phe-Ala, and Phe-Lys were determined and averaged 1.91 Ϯ 0.04 (n ϭ 11), 2.16 Ϯ 0.02 (n ϭ 8), and 2.42 Ϯ 0.03 (n ϭ 16), respectively (Fig. 3D). Data obtained using either solution were both plotted in Fig. 3D. These results indicate that the number of protons cotransported is 3 and 2, respectively, for each anionic and neutral peptide transported. The cotransported proton number per Phe-Lys molecule is 1.4, which is significantly larger than 1, suggesting the possibility that Phe-Lys is transported either under the deprotonated (neutral) form (60%) or under the positively charged form (40%).
Quinapril-inhibited PepT2-mediated Currents-Quinapril, an angiotensin-converting enzyme inhibitor, was found to inhibit the PepT2-mediated currents in oocytes expressing PepT2 (Fig. 4, A, B, and D) but had no effects on control oocytes. The inhibition was demonstrated to be noncompetitive with an IC 50 close to 1 mM by an independent study (data from our labora- tory). 2 Both inward and outward currents were inhibited (Fig.  4, A-C), indicating that there exists a reversed transport mode. In the absence of external substrates, an inward quinaprilsensitive proton current (proton leak) was observed in oocytes expressing the rat PepT2 (Fig. 4, D and E). Similar uncoupled leak currents exist in several other transporters (see Refs. 23, 34, and 37). Together with the observation that no currents were elicited by substrate addition at high pH (see Fig. 2, C-E), our data demonstrates that H ϩ binds to PepT2 prior to the substrate. Quinapril-sensitive proton leaks exhibited similar decreases with hyperpolarization compared with currents evoked by low external substrate concentrations, indicating that this is a characteristic property of PepT2.
PepT2 Presteady-state Currents-By applying voltage jumps from Ϫ50 mV to hyperpolarized potentials, PepT2 exhibited pH-dependent presteady-state currents (I pss ) (Fig. 5). At pH 5.0 -6.0, no significant or low I pss were observed in the whole voltage range tested. The PepT2-specific charge displacement (Q, obtained by eliminating the capacitative component) at pH 7.0 did not saturate up to Ϫ160 mV (see Fig. 5E). Since Q at pH 6.0 saturated by hyperpolarization, it is obvious from the figure that the maximal charge displacement (Q max ) at pH 6.0 represents less than 15% of the value at pH 7.0. Surprisingly, at the tested pH o range (5.0 -8.0), no significant I pss were observed for the off-responses (returning from test potentials V m to V h ) (see examples in Fig. 5, A-C) and I pss at pH 6.0 recovered by addition of substrate ( Fig. 5D; steady-state currents were subtracted). These abnormal behaviors of PepT2 are not readily accounted for by previously described kinetic models (see "Discussion").
Between Ϫ160 and Ϫ80 mV, relaxation time constants () of I pss for the on-responses ranged from 10 to 40 ms. With external alkalization, the V m corresponding to the maximal shifted to more negative values (Fig. 5F), similar to other transporters such as PepT1 (9) and SGLT1 (27). At more depolarized potentials, could not be evaluated due to small I pss . was only slightly affected by intracellular acidification using a previously reported approach (9) (not shown), in contrast to of PepT1 that showed large responses (9). DISCUSSION Using biophysical approaches, we have revealed unique kinetic properties of rat PepT2 expressed in Xenopus oocytes. These include the stoichiometries for different types of substrates determined under voltage-clamp conditions, the proton leak, and the reversed transport mode as well as presteadystate currents.
Stoichiometry-The number of proton ions ("n") necessary for cotransporting one substrate molecule (stoichiometry "n") determines the concentration of intracellular substrate that cells can keep at equilibrium. A high H ϩ :substrate ratio allows maintenance of high intracellular substrate concentrations but also requires a relatively large amount of H ϩ electrochemical energy. Earlier studies on isolated intestinal tissue preparations and the Caco-2 cell line derived a H ϩ :peptide ratio of greater than 2:1 based on measurements of short circuit currents and peptide fluxes (5,28). In contrast, according to equilibrium intracellular substrate estimation (14) and Hill plot analysis (13,26,29) using Xenopus oocytes expressing low affinity PepT1, a 1:1 coupling ratio was deducted for neutral substrates. More recently, our laboratory used current and tracer measurements to study the stoichiometry of PepT1 for neutral and charged peptides (25). These experiments revealed 1:1, 2:1, and 1:1 ratios for neutral, anionic, and cationic peptides, respectively.
Studies of the high-affinity peptide transporter using rat kidney brush-border membrane vesicles (6) and oocytes expressing rabbit renal PepT2 (10, 13) revealed a Hill coefficient (n H ) close to 1 for charged and neutral peptides, suggesting a 1:1 stoichiometry. However, using brush-border membrane vesicles of rat kidney cortex, Temple et al. (20) found a n H close to 1 and 2 for neutral and anionic dipeptides, respectively, and suggested a stoichiometry of 1:1 and 2:1, correspondingly. Since n H value may depend on the substrate concentration and binding cooperativity of different protons in case of coupling to multiple protons, n H is usually not equal to the actual stoichiometric ratio. Protons are also known to have multiple effects on membrane proteins as well as on substrates (30). The evaluation of n H necessitates experiments to be performed over a wide pH range. However, proteins and/or substrates might not have the same activity in the whole range, which would significantly influence the profile of current versus proton concentration ([H ϩ ]). Although in epithelial cells PepT2 is in contact with a luminal unstirred layer, which has a pH ranging between 5.5 and 6.0 (31), and proton affinity constants of PepT2 are around pH 6.0 (see "Results"), PepT2-mediated currents decreased when pH o approached 5.0 -6.5 (Fig. 2). This means that PepT2 currents no longer obey the Michaelis-Menten or Hill relationships.
Our results from simultaneous measurements of radiolabeled peptide uptakes and PepT2-mediated currents under voltage-clamp condition show that the charge:uptake ratios are close to 2 for both anionic and neutral substrates. These data indicate that PepT2 possesses two H ϩ -and one substratebinding sites. In the case of anionic substrates, one additional proton is needed, most likely for substrate protonation before or during binding. Because of the hydrophobic environment provided by the membrane and the transporter, the charges that the loaded transporter is permitted to carry within the membrane should be well controlled and relatively constant (ϩ2 for neutral and anionic dipeptides). In the case of cationic substrates (S ϩ ) such as Gly-Lys and Phe-Lys, the charges on the loaded transporter (2H ϩ and 1S ϩ ) are ϩ3, and the cotransport might be unfavorable. The lysine residue under physiological pH range (pH 5.0 -8.5) predominantly carries one positive charge. However, PepT2 protein might help in deprotonating (neutralizing) the lysine residue even at external physiological pH and, thereby, transport the resulting neutral form of dipeptide by coupling to two protons. This process is apparently equivalent to the stoichiometry of 1H ϩ :1S ϩ and corresponds to carried charges of ϩ2. Observed charge:Phe-Lys ratio of 2.4 is consistent with the interpretation that both cationic and neutral forms of Phe-Lys are transported. The former (2H ϩ :1S ϩ ) accounts for 40% and the latter (1H ϩ :1S ϩ ) accounts for 60% of observed Phy-Lys-evoked currents. However, neither of these two coupling mechanisms satisfies both the 2:1 H ϩ :substrate stoichiometry requirement and the 2:1 charge:substrate ratio requirement, which may explain observed significantly lower affinities for cationic substrates compared with neutral or anionic substrates.
The characteristics of high stoichiometry and overall high affinity of PepT2 as compared with PepT1 are consistent with its S3 localization in the kidney (32), where PepT2 can efficiently reabsorb peptides using higher electrochemical energy of protons. In contrast, the 1:1 stoichiometry and low affinity of PepT1 allow economic and efficient substrate absorption in the intestine and early parts of renal proximal tubules.
The Reversed Transport Mode-As required by the principle of microscopic reversibility, reversed transport must occur provided that substrates are available in the intracellular side of the membrane. Usually, forward and reversed transports are not symmetrical in terms of substrate/ion affinity. Transport by SGLT1 exhibits a strong inward rectification in both cotransport and Na-leak modes, as revealed using the cut-open oocyte technique (33). Reversed transport has been demonstrated by using transporter-specific inhibitors in oocytes expressing SGLT1 (by phlorizin, Ref. Gly-Leu upon voltage pulses from a holding potential of Ϫ50 mV to final potentials ranging between Ϫ160 and ϩ60 mV, each separated by 20 mV. For clarity, only currents corresponding to V m of Ϫ160, Ϫ120, Ϫ80, Ϫ50, Ϫ20, ϩ20, and ϩ60 mV are shown. B, time course of currents in the presence of both 50 M Gly-Leu and 1.2 mM quinapril, obtained from the same oocyte as in A. C, quinapril-sensitive currents in presence 50 M Gly-Leu as obtained by subtracting the recording in B from that in A. The inset is a depiction at depolarized potentials, highlighting outward currents at depolarized potentials. D, quinapril inhibition of PepT2-mediated currents at a holding potential of Ϫ50 mV. Solid and hatched bars represent applications of 25 M Gly-Leu and 1.2 mM quinapril, respectively. E, PepT2-specific quinapril-sensitive currents in the absence of external substrate (q, also inset) were compared with currents due to addition of 50 M Gly-Leu (E) in the same oocytes. Represented data were mean values from five oocytes. versed cotransport of unknown PepT2 substrates or from proton-leak current.
Presteady-state Properties of PepT2-Transporters and channels possess charged residues within the membrane which, in response to voltage changes (jumps) applied across the membrane, move (or relax) to new equilibrium positions, thus generating electrical signals (i.e. currents). These currents vanish when the new equilibrium (steady state) is reached and are therefore called presteady-state currents (I pss ). Binding/dissociation of coupling ions or substrates are often voltage-dependent and contribute to the observed I pss . Characterization of these currents provides unique information on properties of membrane transporters.
We propose a kinetic model to help understanding how PepT2 I pss are associated with conformational changes from one steady state to another (Fig. 6). For simplicity, the model was drawn with ordered binding and mirror symmetry, in analogy to models proposed for PepT1 (9,29). At low external driving-ion concentrations and in the absence of external substrate, the transporter should be predominantly inward facing (states IЈ to IVЈ, Fig. 6). I pss elicited under these (or similar) conditions have been interpreted as being associated with conformational changes of the unloaded transporter from the inward-faced to the outward-faced configurations (27,36). Conversely, when [H ϩ ] o is high, previous kinetic models predict that the transporter is predominantly outward facing (states II and III) and that voltage jumps to depolarized V m generate positive I pss . However, these predictions were not applicable to PepT2 (see below), even though they were extensively verified in a number of transporters such as rat PepT1 (9), human PepT1 (29), and SGLT1 (27,36). The reported models for these transporters also predict that 1) the maximal charge displacement (Q max ) is independent of driving-ion concentration pro-vided that the driving ion is still present and 2) the charge movement associated with the on-response from V h to a test potential V m (Q on ) is equal to that corresponding to the off- Each configuration represents a state. Outside, from states I to IV, two proton ions and one substrate (S) orderly bind to the protein. Fully loaded configuration (state IV) undergoes a conformational change during which two H ϩ and one S are translocated across the membrane. Subsequently, the dissociation of these ions and substrate occurs, as represented by reaction steps from state IVЈ to IЈ (inside). When the free carrier changes from the inward-faced state (IЈ) to the outward-faced state (I), the whole cycle of forward transport is accomplished, which generates a net inward current. In the presence of intracellular H ϩ and substrates, the backward (reversed) transport can occur as well, producing outward currents. After binding of either one or two H ϩ , the translocation across the membrane may occur in the absence of peptide substrates, generating observed H ϩ leaks.
FIG. 5. Rat PepT2-specific presteady-state currents (I pss ). The holding potential was Ϫ50 mV. For clarity, only currents at final potentials of Ϫ160, Ϫ140, Ϫ120, and ϩ40 mV were shown. Time between two consecutive jumps was 650 ms. Steady-state currents were removed from all recordings by base-line subtraction using the Pclamp6 Program. A-C, I pss were recorded in the absence of external substrates at pH 7.0, 6.5, and 6.0, respectively. D, I pss were evoked by 250 M Gly-Glu at pH 6.0. E, charge displacements upon voltage jumps from Ϫ50 mV to various test potentials were measured by integrating the currents versus time and eliminating the capacitative component that was determined when data were fitted to the sum of two exponential functions. F, relaxation time constants () at various V m and pH values. response from V m to V h (Q off ), consistent with strict charge conservation.
As alluded to above, these predictions are not applicable to PepT2. First, Q max was greatly reduced at low pH o (Fig. 5E). Second, in contrast to Q on , Q off (from hyperpolarized V m to V h of Ϫ50 mV) was negligible at any pH o tested. Third, in contrast to previous observations that the presence of external substrate diminishes or eliminates I pss (see SGLT1, Ref. 36; the Na ϩiodide transporter, Ref. 34; PepT1, Ref. 9), PepT2-specific I pss were increased by substrate addition at low pH o (Fig. 5D). Finally, when the time interval separating two consecutive voltage jumps (V h ϭ Ϫ50 mV) was reduced from 650 to 200 or 50 ms, I pss were largely reduced after the first jump (to Ϫ160 mV), whereas there was no significant reduction in I pss when using time intervals of 650 ms or longer (not shown).
These observations indicate that hyperpolarization results in transporter inactivation, which can be reversed by depolarization (Ϫ50 mV) during a 650-ms period. Hyperpolarization appears to have inactivation effects on steady-state properties as well. At low pH o , hyperpolarization resulted in decreases in substrate-evoked current (Fig. 2), substrate affinity (Table I and Fig. 1B), and proton leak (Fig. 4E). External substrate appears to prevent the transporter from being inactivated by hyperpolarization, as we can see from increases in I pss by substrate addition (Fig. 5D) and from increases in maximal steady-state currents evoked by saturating substrate concentrations (Fig. 1C). Mechanisms underlying such a hyperpolarization-stimulated inactivation are still unclear.
Although PepT2 possesses considerable sequence homology to PepT1 (ϳ50% identity), our findings revealed profound differences in several aspects such as stoichiometry, substrate affinity, effects of hyperpolarization, and presteady-state properties. While PepT1 was shown to exhibit remarkable symmetry with respect to the effects of intra-and extracellular pH on I pss , more studies are needed to elucidate corresponding effects of pH on PepT2. It remains to be seen whether binding of an additional proton in PepT2 is the origin of some of these differences.