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Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California
Submitted 21 January 2008 ; accepted in final form 25 March 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-aminolevulinic acid (-ALA) and bestatin induced inward currents, indicating that they are transported. Variations in transport rate were due to differences in affinity and in turnover rate: for example, cefadroxil was transported with higher apparent affinity but at a lower maximum velocity than Gly-Sar. Transport rates were highest at pH 5 and decreased significantly as the external pH was increased. Our results strongly suggest that the protein does not operate as a cotransporter in tissues where there is little or no pH gradient, such as choroid plexus, lung, or mammary gland. In the absence of substrates, rapid voltage jumps produced hPEPT2 capacitive currents at pH 7. These transients were significantly reduced at pH 5 but recovered on addition of substrates. The seven-state ordered kinetic model previously proposed for hPEPT1 accounts for the steady-state kinetics of neutral drug and dipeptide transport by hPEPT2. The model also explains the capacitive transients, the striking difference in pre-steady-state behavior between hPEPT2 and hPEPT1, and differences in turnover numbers for Gly-Sar and cefadroxil. No functional differences were found between the common variants hPEPT2*1 and hPEPT2*2. hPEPT2 substrates and drugs; kinetic model of H+/oligopeptide cotransport; polymorphisms
-aminolevulinic acid (6, 14). PEPT1, found in the intestine as well as the kidney, is a low-affinity/high-capacity transporter, while PEPT2, which is also expressed in choroid plexus, mammary gland and lungs, has been characterized as a high-affinity/low-capacity system (4). Previously, we described the mechanisms of human PEPT1 (hPEPT1) function (11, 16). Here, we present the first detailed kinetic study of dipeptide and drug transport by human PEPT2 (hPEPT2). The human renal H+/oligopeptide cotransporter hPEPT2 (Fig. 1) is comprised of 729 amino acid residues, with 12 predicted transmembrane domains (9). There are two main variants of hPEPT2 that differ in three amino acid positions, hPEPT2*1 and hPEPT2*2 (Fig. 1), and are distributed evenly among the population at 44–47% (http://pharmacogenetics.ucsf.edu). In the present work, we used tracer uptakes and electrophysiological analysis to investigate and compare the interactions between hPEPT2 variants, the dipeptide glycylsarcosine, and a selection of drugs. We examined the steady-state and pre-steady-state kinetics of both variants in the absence and presence of substrates. Finally, we used our model for H+/dipeptide cotransport (see Fig. 8 and Ref. 16) to interpret the results and gain insights into functional differences between hPEPT1 and -2, and the effect of substrates on the transport mechanism.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All unlabeled chemicals, reagent grade, were purchased from Sigma (St. Louis, MO) except for glycylsarcosine (Gly-Sar), cephalexin, and cefadroxil, which were from MP Biomedicals (Solon, OH). Amoxicillin, ampicillin, cefadroxil, and cephalexin were used at concentrations up to their maximal solubility at pH 5.0–6.0 (5, 10, 10, and 20 mM, respectively). -Aminolevulinc acid (-ALA) and bestatin were used at 0.5 mM, and N-acetyl-Asp-Glu (NAAG) was tested at 0.1 mM; higher concentrations of these drugs induced nonspecific current responses in control oocytes (16). [Glycyl-2-3H]Gly-Sar (specific activity 60 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Restriction endonucleases were from New England Biolabs (Ipswich, MA).
Construction of hPEPT2 Variants
hPEPT2*1 and hPEPT2*2 cDNAs (in pTLN plasmid) were provided by the University of California-San Francisco (UCSF) Pharmacogenetics Core Facility. For expression in Xenopus oocytes, the hPEPT2 cDNAs were subcloned into a pBluescript plasmid containing the polyadenylated tail and the 5'- and 3'-untranslated regions of human Na+/glucose cotransporter (hSGLT1) (8). Briefly, the pTLNs were digested with Nco I and Dra I to obtain the 2.4-kb hPEPT2 cDNAs, and the pBluescript plasmid was digested with PshA I to remove the hSGLT1 coding sequence. The desired fragments were gel purified and ligated as described previously (16). Competent XL1-Blue cells (Stratagene, La Jolla, CA) were transformed by electroporation, and colonies were selected in a medium with ampicillin and tetracycline. Plasmid DNA was prepared using purification kits by Qiagen (Valencia, CA). The fidelity of the new clones was verified by restriction analysis and sequencing.
cRNA Synthesis
hPEPT2 plasmids were linearized with BamH I and transcribed in vitro using the Ambion T3 MEGAScript kit and RNA cap analog (Applied Biosystems, Foster City, CA). The polyadenylated cRNAs were purified by means of the Ambion MicroPoly(A)Purist kit.
Expression of hPEPT2s in Oocytes
Mature female Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). All animal protocols followed guidelines approved by the University of California Chancellor's Committee on Animal Research and the National Institutes of Health. Frogs were anesthetized with 0.1% Tricaine (Sigma) buffered with 0.1% NaHCO3, a portion of the ovary was surgically removed, and the frogs were killed by an overdose of Nembutal (60 mg for 60 min). Stage V-VI oocytes were selected and maintained at 18°C in modified Barth's solution, supplemented with antibiotics (16). Oocytes were injected 1 day after isolation with 50 ng of hPEPT2*1 or hPEPT2*2 cRNA and incubated at 18°C for 4–7 days. Experiments were performed at 20–22°C. Noninjected oocytes served as controls.
Gly-Sar Uptake Assays
Oocytes were incubated for 30 min in the presence of 5 µM to 2 mM Gly-Sar (0.1 µM [3H]Gly-Sar) in a medium containing (in mM) 100 NaCl or choline chloride, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES/Tris (pH 7.5) or 10 2-(N-morpholino)ethanesulfonic acid (MES)/Tris (pH 6.0). Competition assays were performed using the neuropeptide NAAG and selected cephalosporins (cefadroxil and cephalexin), penicillins (ampicillin and amoxicillin), peptidomimetic drugs (bestatin), and nonpeptidic compounds (-ALA) (16).
Electrophysiology
A two-microelectrode voltage-clamp system was used to measure substrate-induced steady-state currents in hPEPT2-expressing oocytes (10, 16). Steady-state current-voltage relationships were measured in Na+ or choline media at pH 7.5, pH 7.0, pH 6.5 (buffered with HEPES/Tris), pH 6.0, pH 5.5, or pH 5.0 (buffered with MES/Tris) in the absence or presence of Gly-Sar and/or drugs. A pulse protocol was applied in which membrane potential of oocytes was held at –50 mV and stepped to a test value (Vm), from –150 to +50 mV in 20-mV increments (ON response); steady-state currents were measured at the end of 100 ms. Subsequently, the membrane potential was returned to the holding value, and currents were recorded for 600 ms (OFF response); shorter OFF pulse durations led to the underestimation of the OFF pre-steady-state currents. pClamp and Axoscope software (Molecular Devices, Union City, CA) was used for pulse protocol application and data acquisition, and continuous current data were recorded with a chart recorder. Unless otherwise noted, all experiments were repeated on at least three oocytes from different donor frogs.
Data Analysis
The kinetic parameters of radiotracer uptake and substrate-related inward currents were calculated by nonlinear regression (SigmaPlot 10.0; Systat Software, San Jose, CA). Data were fitted to Eq. 1
 | (1) |
1.
Pre-steady-state currents in response to step jumps in membrane potential were isolated by fitting the total current across the oocyte membrane (It) to Eq. 2 (11)
 | (2) |
m, t is time, Ipss is the initial hPEPT2 pre-steady-state current with time constant pss, and Iss is the steady-state current. Transporter-mediated transients [Ipssexp(–t/pss)] were determined by subtraction of the capacitive and steady-state components. At each voltage, the equivalent charge transfer (Q) was calculated by integrating the pre-steady-state currents with time. Pre-steady-state data fitting was performed by means of Clampfit 8.2 (Molecular Devices). Differences between hPEPT2s were evaluated by Student's t-test, using SigmaPlot 10.0.
Mathematical Modeling
The seven-state kinetic model for hPEPT2 (see Fig. 8) was solved as described for hPEPT1 (see Supplemental Materials and Ref. 16; supplemental data are available at the online version of this article). Starting from the set of rate constants used for hPEPT1, we obtained a numerical solution for the 16 rate constants and 3 voltage dependence parameters for hPEPT2 that provides a global fit to our experimental data for H+/substrate cotransport and the pre-steady-state kinetics (see Table 2). In our simulations, the number of transporters N was varied to account for the different levels of protein expression between oocytes. Model predictions are presented alongside experimental data (see Figs. 3 and 5–7; Table 1), and the N values are given in the legends.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Uptake of [3H]Gly-Sar by hPEPT2*1 (P2*1) or hPEPT2*2 (P2*2) transporters expressed in oocytes was proton dependent, as it increased up to eightfold when the external pH was lowered from 7.5 to 6.0 (Fig. 2A). Replacement of Na+ by choline did not affect transport. In noninjected oocytes, influx of [3H]Gly-Sar was 20% (at pH 7.5) or 3% (at pH 6.0) that of cRNA-injected oocytes. At pH 6.0, the apparent affinity of Gly-Sar uptake (K0.5GS) was 51 ± 15 (P2*1) and 64 ± 18 µM (P2*2), and the maximal rate of influx (JmaxGS) was 352 ± 19 (P2*1) and 312 ± 18 pmol/h·oocyte (P2*2).
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-ALA caused a greater inhibition in P2*2 (75%) than in P2*1 (60%) (P < 0.05). NAAG (0.1 mM) had no significant inhibitory effect. Gly-Sar uptake into noninjected oocytes was not modified by any of the drugs (not shown). Electrophysiology
Substrate selectivity.
Figure 3A shows a record from a representative experiment, in which a P2*1-expressing oocyte voltage-clamped at –50 mV was exposed to 0.5 mM Gly-Sar or -ALA at pH 5.0. Addition of either substrate to the superfusion buffer resulted in the generation of inward currents, 57 nA by Gly-Sar and 22 nA by -ALA. If the oocyte was superfused with 0.5 mM Gly-Sar, the addition of 0.5 mM -ALA produced a 20% increase in the current. These currents reversed on replacement with substrate-free buffer (Fig. 3A). No uncoupled H+ transport was observed in hPEPT2 oocytes: in the absence of substrate, switching the pH of the buffer from 7.5 to 5.0 resulted in an increase in baseline current (20 nA at –50 mV) and a shift in membrane potential (+10 mV) no larger than those observed in noninjected oocytes.
Gly-Sar currents were not affected by external Na+ but were significantly attenuated at low H+ concentrations. For example, currents evoked at –50 mV in P2*1 oocytes by 1 mM Gly-Sar at pH 5.0 were 131 ± 5 nA in Na+ and 122 ± 7 nA in choline, and at pH 7.5 were 12 ± 1 nA in Na+ and 14 ± 3 nA in choline. Similar results were obtained in P2*2 oocytes (not shown).
The effect of external pH on the kinetics of Gly-Sar currents is shown in Fig. 3, B and C. We measured the currents induced at –50 mV by increasing concentrations of the dipeptide (0.1–10 mM) at pH 7.0–5.0, and fit the data to Eq. 1 to estimate the apparent kinetic constants of Gly-Sar transport. In P2*1 and P2*2, K0.5GS was 0.1–0.3 mM at pH 7.0–5.5 but increased to 1.2–1.5 mM at pH 5.0 (Fig. 3B). ImaxGS increased steadily from 14 nA at pH 7.0 to 165 nA at pH 5.0 (Fig. 3C). The Hill coefficient for Gly-Sar was 1 throughout the entire pH range.
We compared the currents evoked by 10 mM Gly-Sar (ImaxGS) at pH 5.0 with those induced by the selected drugs in the same oocyte (Fig. 4A). All drugs except NAAG evoked currents, indicating that they are transported. As shown in Fig. 4A for representative P2*1 and P2*2 oocytes, the currents due to 10 mM ampicillin and 5 mM amoxicillin were 10% of ImaxGS, whereas those evoked by 20 mM cephalexin and 10 mM cefadroxil were 30 and 60%, respectively. -ALA and bestatin induced 15% of the current due to 10 mM Gly-Sar. At pH 5.5 and 6.0, these substrate selectivity profiles were maintained (not shown).
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40% of ImaxGS, in accordance with the K0.5GS for these individual oocytes, 1 (P2*1) and 0.8 mM (P2*2). I0.5 mMGS was inhibited by 20 mM cephalexin (25%), 10 mM ampicillin (35%), 5 mM amoxicillin (35%), and 0.5 mM bestatin (40%). In contrast, -ALA had additive effects. Thus I0.5 mMGS in the presence of 0.5 mM -ALA was approximately the sum of the currents induced by the substrates independently (Fig. 4, A and B). I0.5 mMGS increased on addition of 10 mM cefadroxil but was not affected by 1 mM or lower concentrations of the drug (Fig. 4B). I0.5 mMGS in the presence of 10 mM cefadroxil was the same as that evoked by the drug alone (Fig. 4A), i.e., Gly-Sar and cefadroxil were not additive. Finally, I0.5 mMGS was not affected by 0.1 mM NAAG. No differences were found between P2*1 and P2*2 (P > 0.05). Voltage and pH dependence of dipeptide and drug transport. Figures 5A and 6A show the effect of voltage and pH on the currents induced by Gly-Sar in P2*1 oocytes. Within the pH range 7.0–5.0, the current-voltage (I-V) relationships for 1–10 mM Gly-Sar approached zero at +50 mV and increased with hyperpolarization. Currents induced by 5 mM or higher concentrations of Gly-Sar increased with decreasing pH: for example, in the oocyte shown in Fig. 5A, currents induced at –110 mV by 10 mM Gly-Sar were 50 nA at pH 7.0, 170 nA at pH 6.0, and 435 nA at pH 5.0. The currents evoked at pH 5.0 by 2 mM or lower concentrations of Gly-Sar were inhibited at large negative potentials, and the voltage at which the IGS-V relationship saturated was –70 mV at 0.1 mM, –90 mV at 1 mM (Fig. 6A), and –110 mV at 2 mM (not shown). At pH 5.5, voltage-dependent inhibition was observed only for 0.25 mM or less Gly-Sar, at Vm equal to or more negative than –110 mV (not shown). At pH 6.0 or higher, the IGS-V relationships for 1–10 mM Gly-Sar did not saturate with Vm over the voltage range tested.
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2 µM at 1 mM Gly-Sar to 6 µM at 10 mM Gly-Sar (Fig. 5C). For 1–10 mM Gly-Sar, the coupling coefficient for protons was estimated to be 1 (see MATERIALS AND METHODS). Next, we compared the characteristics of steady-state Gly-Sar currents to those induced by the model drug cefadroxil (Fig. 6 and Table 1). Unless otherwise noted, experiments were carried out at pH 5.0. Figure 6, A and B, shows the concentration dependence of steady-state currents evoked by Gly-Sar (IGS; Fig. 6A) and cefadroxil (ICEF; Fig. 6B), in the same P2*1-expressing oocyte. As observed for Gly-Sar (Figs. 5A and 6A), the I-V curves for cefadroxil approached zero at +50 mV and increased with hyperpolarization. Unlike Gly-Sar, the currents evoked by 0.1–10 mM cefadroxil at pH 5.0 were not inhibited at large negative potentials (Fig. 6B).
Between –50 and –150 mV, K0.5GS increased as the membrane potential became more negative, whereas K0.5CEF was voltage independent (Fig. 6C). In P2*1 oocytes, K0.5GS increased from 2 mM at –50 mV to 7 mM at –150 mV, while K0.5CEF was 0.5 mM throughout the voltage range (Fig. 6C). ImaxGS and ImaxCEF were voltage dependent. ImaxGS increased more steeply with hyperpolarization than ImaxCEF: for example, in P2*1 oocytes, ImaxGS increased from 170 to 600 nA, and ImaxCEF increased from 60 to 100 nA (Fig. 6D). K0.5GS and K0.5CEF were higher in P2*1 than in P2*2 within the voltage range (Table 1). When normalized to the ImaxGS obtained at –150 mV (Table 1), the ImaxGS-V curves of P2*1 and P2*2 were identical; the same behavior was observed for cefadroxil (not shown). The Hill coefficient for Gly-Sar and cefadroxil was maintained at 1. Because of the low magnitude of the currents measured between +50 and –30 mV, we were unable to estimate the kinetics of Gly-Sar or cefadroxil over this range.
Pre-steady-state currents: hPEPT2 charge movements.
Pre-steady-state transient currents represent charge movements associated with voltage-dependent conformational changes in the cotransporter as it goes through the transport cycle and have been postulated to be due to the movement of charged and polar residues within the membrane electric field (10). hPEPT2 oocytes showed pre-steady-state currents (Ipss) in response to step changes in membrane potential. Figure 7A shows examples of ON Ipss measured in a representative P2*1 oocyte. These Ipss were obtained from the total currents by subtraction of the membrane bilayer capacitance transients and steady-state currents (see MATERIALS AND METHODS). hPEPT2-mediated transients were most evident at pH 7.0 in the absence of substrates (Fig. 7A, panel 1), where they were moderately reduced by the addition of high concentrations (e.g., 10 mM) of Gly-Sar or cefadroxil and increased in the presence of low (e.g., 1 mM) substrate concentrations (not shown). These hPEPT2 capacitive transients were also significantly reduced at pH 5.0 (Fig. 7A, panel 2) but recovered on addition of substrates (Fig. 7A, panels 3 and 4). Relaxation of the Ipss for the ON response consisted of a single time constant (ON). In the OFF response, relaxation of hPEPT2-mediated transients was composed of an initial fast component ( fastOFF) followed by a slower one ( slowOFF). For example, at pH 7.0, the Ipss generated at –90 mV (Fig. 7A, panel 1) relaxed with ON of 15 ms, and the equivalent OFF Ipss (not shown) relaxed with fastOFF of 7 ms and slowOFF of 165 ms.
We integrated the pre-steady-state currents with time to obtain the equivalent charge moved (Q). At each test potential, the charge transfer for the ON and OFF responses (QON and QOFF, respectively) was equal, but an apparent decrease in QOFF was observed with OFF pulse durations shorter than 600 ms. For example, in the P2*1 oocyte shown in Fig. 7, QON and QOFF at –150 mV were 11 nC, but the measured QOFF dropped to 3 nC when the OFF pulse was applied for only 100 ms. As shown in Fig. 7, B and C, for the Ipss presented in Fig. 7A, the Q values asymptotically approached zero at depolarizing voltages and increased with hyperpolarization. However, the charge-voltage (Q-V) relationships did not saturate up to –150 mV, precluding experimental estimation of Qmax and V0.5.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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H+-Coupled Dipeptide and Drug Transport
Gly-Sar transport by hPEPT2 was electrogenic, pH dependent, Na+ independent, sensitive to membrane potential, and followed saturation kinetics (Figs. 2–6). The K0.5GS determined at pH 6.0 from radiotracer uptakes (50–60 µM) were consistent with those obtained from current measurements at –50 mV (170 µM, Fig. 3B).
The rates of Gly-Sar transport increased 5-fold when the external H+ concentration was raised 100-fold from 0.1 µM (pH 7.0) to 10 µM (pH 5.0) (Fig. 5A). This indicates that hPEPT2-mediated oligopeptide transport is coupled to protons. The Hill coefficient for protons was experimentally estimated to be 1, indicating that at least one H+ is necessary for the cotransport of one peptide molecule. Simultaneous measurements of radiolabeled uptakes and inward currents at pH 6.0 in Xenopus oocytes expressing rat PEPT2 indicated a H+:substrate stoichiometry of 2:1 for neutral dipeptides (3). One possible explanation for this discrepancy between our human model and the reported 2:1 stoichiometry for rat PEPT2 may be that coupling ratio may vary depending on the species, the ligand, or the ligand concentration. As shown in Figs. 3 and 5–7, we can globally explain the kinetic properties of the hPEPT2 by a coupling ratio of 1.
This study demonstrates that the hPEPT1 drug substrates amoxicillin, ampicillin, cefadroxil, cephalexin, -ALA, and bestatin (16) are also transported by human PEPT2 (Fig. 4A). The currents generated by the drugs were only a fraction of that due to comparable concentrations of Gly-Sar, indicating that they are transported with less efficiency than dipeptides. Furthermore, the magnitude of the currents induced by the aminopenicillins (amoxicillin and ampicillin) was similar to that evoked by concentrations up to 200-fold lower of -ALA and bestatin. NAAG, a substrate for hPEPT1, was not transported by hPEPT2 (Fig. 4A).
Variations in transport can be due to differences in affinity, in turnover rate, or in both. For example, cefadroxil is transported with higher affinity but at a lower rate than Gly-Sar. At pH 5.0 and –50 mV, K0.5CEF was 0.3–0.5 mM, while K0.5GS was 1.2–1.5 mM (Fig. 6C and Table 1). The Imax for cefadroxil was 30% of that for Gly-Sar (Fig. 6D and Table 1). Because of technical limitations related to drug solubility and nonspecific effects (see MATERIALS AND METHODS), we could not obtain detailed kinetics for the remaining compounds. However, the interactions of these drugs with Gly-Sar give us insights into the substrate selectivity of hPEPT2. For example, the currents induced by 0.5 mM Gly-Sar were reduced in the presence of amoxicillin, ampicillin, and bestatin (Fig. 4B), as in hPEPT1. This may be a consequence of the drugs being transported at a much lower turnover rate than Gly-Sar. Since cefadroxil did not have inhibitory effects on Gly-Sar currents (Fig. 4B), it can be speculated that amoxicillin, ampicillin, and bestatin are transported at a turnover less than 30% that of Gly-Sar. Inhibition of currents by low turnover substrates has been observed in other transporters, such as the human Na+/glucose cotransporter hSGLT1 (Loo DDF, unpublished observations).
Transport Model for hPEPT2
The functional and structural similarities between hPEPT2 and hPEPT1 (4) suggest that they share a common mechanism. Previously, we showed that an ordered seven-state kinetic model accounts for dipeptide and drug transport by hPEPT1 (16). Can this model explain the kinetics of hPEPT2? In our model (Fig. 8), cotransport occurs by a series of conformational changes induced by ligand binding and membrane voltage. In a forward transport cycle, one external H+ binds first to the outside-oriented empty transporter [C]' (state C1) to form the complex [CH]' (state C2). The external substrate is then able to bind, and the substrate-loaded protein [SCH]' (state C3) undergoes a conformational change (C3 
C4) resulting in H+/dipeptide cotransport. Subsequently, the substrate is released on the cytoplasmic side (C4 
C5), followed by the release of H+ (C5 C6). Finally, the empty transporter reorients within the membrane (C6 C1). In a transport cycle, the voltage-dependent steps are the reorientation of the empty transporter between inward facing and outward facing conformations and the binding of protons on the external membrane surface. The pre-steady-state currents (charge movement) are associated with these partial reactions (Fig. 8). To account for the observed voltage-dependent inhibition of cotransport at low substrate concentrations (e.g., Fig. 5C of Ref. 16), we hypothesized a second H+ binding to the transporter in state C2 to form the complex [CHH]' (state C7). An additional constraint on our model is the requirement that C2 C7 does not involve charge movement.
The goodness of fit of the simulations to the experimental data is shown in Figs. 3 and 5–7 and Table 1. Thus 1) the pH dependence and H+ activation kinetics (Figs. 3, B and C, and 5), 2) the steady-state I-V and K0.5-V relations for the Gly-Sar and cefadroxil cotransport (Fig. 6 and Table 1), 3) the time course of hPEPT2 Ipss (Fig. 7A), and 4) the Q-V curves (Fig. 7, B and C) are simulated qualitatively and quantitatively at the ligand (H+ and substrate) concentrations and voltages tested.
Interpretation of hPEPT2 Transport Kinetics
Our model simulations indicate that, between pH 5.0 and 7.5, at –50 mV, and in saturating Gly-Sar concentrations, the reorientation of the empty transporter within the membrane (C6 C1) is the slowest step of the hPEPT2 transport cycle (Table 2), as in hPEPT1. The maximal transport rate, Imax, is lower in cefadroxil (Fig. 6D and Table 1) because of a lower translocation rate, k34, 25 s–1 vs. 600 s–1 for Gly-Sar. The higher apparent affinity of cefadroxil transport with respect to Gly-Sar (Fig. 6C and Table 1) is accounted for by the approximate eightfold increase in substrate binding rate, k230, from 3.6 x 104 M–1·s–1 to 2.8 x 105 M–1·s–1. In hPEPT1, the predicted lower apparent affinity of cefadroxil at pH 5.0 was explained by an eightfold decrease in k230.
PEPT2 has been described as a high-affinity/low-capacity system compared with PEPT1 (5). Accordingly, K0.5GS values for hPEPT2 were up to 10-fold lower than for hPEPT1. For example, at pH 7.0, K0.5GS at –50 mV was 0.2 mM in hPEPT2 (Fig. 3B) and 2.5 mM in hPEPT1 (11). On the other hand, it is difficult to draw conclusions about the relative capacities of hPEPT1 and -2 in the absence of information about the turnover number and density of the transporters in the cell membrane. The turnover rate of transport can be estimated as the ratio of Imax at –150 mV to Qmax (10). Imax depends on all the rate constants in the transport cycle (see Eqs. A37 and A41 of Ref. 12). Thus Imax for Gly-Sar is about threefold lower in hPEPT2 than hPEPT1 (Table 1 and Ref. 16), and this is accounted for by a threefold reduction in the substrate binding rate, k230, from to 105 M–1·s–1 in hPEPT1 to 3.6 x 104 M–1·s–1 in hPEPT2 (Table 2). On the other hand, because the Q-V relationships did not saturate within the voltage range tolerated by the oocytes, we could not estimate the values of Qmax for hPEPT2. However, when our model is applied over an extended range that allows for saturation of the Q-V relationship (e.g., +300 to –300 mV), it predicts that 1) different substrates have different turnover rates, and 2) the turnover rate for a given substrate is lower in hPEPT2 than in hPEPT1. For example, the predicted turnover rates of transport for hPEPT2 at pH 5.0 are 100 s–1 for Gly-Sar and 30 s–1 for cefadroxil, while for hPEPT1 the rates are 130 s–1 for Gly-Sar and 70 s–1 for cefadroxil.
The proton-dependent inhibition of Gly-Sar transport at hyperpolarizing voltages (Fig. 6, A and C) is explained by competition for binding between protons and substrates. A consequence of this competition is that the apparent affinity for Gly-Sar decreases steadily with hyperpolarization (Fig. 6C). Our model assumes that the inhibition at low pH and large negative potentials is due to the binding of a second proton to the transporter in state C2 to form state C7. The strength of the inhibition, represented by the pseudo-rate constant k270, appears to be influenced by the nature of the substrate. For example, cefadroxil currents were not inhibited by voltage, i.e., K0.5CEF was not affected by large negative potentials (Fig. 6, B and C), and this can be accounted for by an estimated value of k270 10-fold lower than that for Gly-Sar (Table 2). The implications are that 1) this second proton binding site has lower affinity than that for cotransported protons (partial reaction C1 C2), and 2) the apparent affinity of H+ binding to this modifier site depends on the substrate. For example, the K0.5H+ for the second site (k72/k27, Table 2) is 2 mM for Gly-Sar and 20 mM for cefadroxil.
Inhibition of transport at low substrate concentrations and large negative membrane voltages has been observed in other cation-driven cotransporters, such as the plant H+/hexose cotransporter (STP1) (2) and the rat Na+/Cl–/GABA transporter rGAT1 (18). Rationale for the influence of the substrate on cation binding may be provided by the crystal structure of LeuTAa, a homolog of GAT1, which shows a close proximity of one of the two bound Na+ to the bound substrate (20).
One of the most striking differences between hPEPT1 and hPEPT2 is the kinetics of charge movement. As in hPEPT1, hPEPT2-mediated pre-steady-state transient currents (Ipss) were observed following step changes in membrane potential in the absence of substrate (Fig. 7A, panel 1). Transient relaxation kinetics were monoexponential in the ON response and contained two time constants in the OFF response (fastOFF and slowOFF). For example, for a voltage jump from –50 to –90 mV at pH 7.0, ON 15 ms, fastOFF7 ms, and slowOFF165 ms. The model predicts 1) ON 9 ms, and 2) fastOFF6 ms and slowOFF170 ms. In hPEPT1, ON and OFF transients relaxed with a single time constant, e.g., at pH 5.0, maxON and OFF 11 ms (16).
Of additional interest, hPEPT2 Ipss were attenuated as the external pH became more acidic but recovered in the presence of Gly-Sar or cefadroxil (Fig. 7A, panels 2–4). Equivalent hPEPT2 charge translocations (Q) were small at depolarizing membrane potentials and increased with hyperpolarization (Fig. 7, B and C). On the contrary, hPEPT1 Ipss were most evident at pH 5.0 and were reduced at higher pH values and on the addition of substrates. In our model, this translated into a reduction in Qmax and a shift of V0.5 to more negative voltages, both consistent with a shift in carrier state distribution, from being predominantly in state C2 in the presence of H+ alone to favoring state C6 in the presence of saturating substrate concentrations or in the absence of H+ (see Supplemental Materials and Ref. 16). As discussed above, we could not obtain experimental estimates of Qmax or V0.5 for hPEPT2. However, our model predicts that V0.5 at pH 5.0 in the absence of substrate is +150 mV, i.e., the depolarizing voltage needed to drive most of the transporters from state C2 to state C6 must be much more positive than +150 mV. As in hPEPT1, the state redistribution in the presence of substrate, or at higher pH values, manifests as a shift in V0.5 to more negative values. This is accompanied by an apparent increase in charge, as the Q-V distribution moves into our experimental voltage range. In our model, these differences in pre-steady-state behavior between hPEPT1 and -2 are explained by differences in proton binding affinity, 4 vs. 0.2 µM (k210/k120;Table 2).
Discrepancies in charge movement between transporters originate from differences in protein structure. On alignment of the amino acid sequences of human, rat, mouse, and rabbit PEPT1s and PEPT2s, we observed only four differences in charged residues within the predicted transmembrane domains (TMDs): e.g., Ile37 (in TMD1), His125 (in TMD3), Glu398 and Lys400 (in TMD9) in hPEPT2, substituted for His7, Gln95, Gln368 and Glu370 in hPEPT1 (Fig. 1). These residues are good candidates for mutagenesis studies.
Pharmacogenetics Hypothesis
To test the hypothesis that variability in drug response among individuals is caused by sequence variations in genes involved in drug disposition (15), we compared the cellular phenotypes of hPEPT2*1 (P2*1) and hPEPT2*2 (P2*2), the two most common genetic variants of the transporter distributed at roughly 50% each (Fig. 1).
P2*1 and P2*2 showed identical functional characteristics: 1) the levels of protein expression in Xenopus oocytes, 2) the acid stimulation of [3H]Gly-Sar uptake (Fig. 2A), and 3) the pH and voltage dependence of Gly-Sar transport (Fig. 3, B and C, and Table 1). No significant differences were observed between P2*1 and P2*2 with respect to transporter-drug interactions, such as 1) transport of amoxicillin, ampicillin, cephalexin, cefadroxil, -ALA, and bestatin (Fig. 4A); 2) effect of the drugs on Gly-Sar inward currents (Fig. 4B); and 3) inhibition of radiolabeled Gly-Sar uptake by most compounds (Fig. 2B). P2*1 and P2*2 also shared identical features of charge movement (not shown). Subtle differences were found, e.g., the K0.5GS and K0.5CEF values were up to twofold higher in P2*1 than in P2*2 (Table 1). Globally, our results indicate that no differences in peptidomimetic drug renal reabsorption are to be expected between individuals expressing one or the other variant. These findings are supported by more limited studies in Chinese hamster ovary and human lung epithelial cells (1, 13).
In summary, biochemical and biophysical studies of hPEPT2 expressed in oocytes established that dipeptides and drugs are substrates for this transporter at acidic pH values and that the model previously proposed for cotransport by hPEPT1, with minor adjustments in rate constants, accounts for both the steady-state and pre-steady-state kinetics. Variations in the rate of drug transport are due to differences in both their affinity and maximum transport (turnover) rates. In addition to the proximal tubule, where the luminal pH could vary, hPEPT2 is expressed in locations in the body where the pH is close to neutral, such as choroid plexus, lung, or mammary gland. Our results on the pH and voltage dependence of hPEPT2 (see Figs. 2A and 5A) suggest that this protein does not operate as a H+/substrate cotransporter in those tissues. This does not preclude that, in the absence of a pH gradient, peptides and peptide-like drugs are transported by hPEPT2 working in uniporter mode, and this would be in agreement with the multifunctional nature of other cotransporters (19). Comparative analysis of the two most frequent genetic variants of hPEPT2 did not reveal major differences in function and thus does not provide a basis for any differences in pharmacokinetics between individuals.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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