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MOLECULAR AND GENOMIC |
1 Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1751, USA
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Abstract |
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-aminolevulinic acid (-ALA) and bestatin, and the neuropeptide N-acetyl-Asp-Glu (NAAG), were transported, as judged by their ability to evoke inward currents. When the drugs were added in the presence of the typical substrate glycylsarcosine (Gly-Sar), the inward currents were equal or less than that induced by Gly-Sar alone. This suggests that the drugs are transported at a lower turnover rate than Gly-Sar, but may also point towards complex interactions between dipeptides, drugs and the transporter. Gly-Sar and the drugs also modified the kinetics of hPEPT1 presteady-state charge movement, by causing a reduction in maximum charge (Qmax) and a shift of the midpoint voltage (V0.5) to more negative potentials. Our results indicate that the substrate selectivity of hPEPT1 is: Gly-Sar > NAAG, -ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin. Based on steady-state and presteady-state analysis of Gly-Sar and cefadroxil transport, we proposed an extension of the 6-state kinetic model for hPEPT1 function that globally accounts for the observed presteady-state and steady-state kinetics of neutral dipeptide and drug transport. Our model suggests that, under saturating conditions, the rate-limiting step of the hPEPT1 transport cycle is the reorientation of the empty carrier within the membrane. Variations in rates of drug cotransport are predicted to be due to differences in affinity and turnover rate. Oral availability of drugs may be reduced in the presence of physiological concentrations of dietary dipeptides in the gut, suggesting that oral delivery drugs should be taken on an empty stomach. The common hPEPT1 single-nucleotide polymorphisms Ser117Asn and Gly419Ala retained the essential kinetic and drug recognition characteristics of the wild type, suggesting that neither variant is likely to have a major impact on oral absorption of drugs.
(Received 16 February 2006;
accepted after revision 13 April 2006;
first published online 20 April 2006)
Corresponding author M. Sala-Rabanal: Department of Physiology, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, 53-330 CHS, Los Angeles, California 90095-1751, USA. Email: msala{at}mednet.ucla.edu
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Introduction |
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-aminolevulinic acid, or -ALA) (Doring et al. 1998) and prodrugs (valacyclovir, L-
-methyldopa) (Hu et al. 1989; Ganapathy et al. 1998). However, there is still a gap in our knowledge about drug absorption via human PEPT1, and most of the limited information available has been obtained by competition studies (Daniel, 2004). Transport by hPEPT1 is electrogenic, proton coupled, and voltage dependent (Mackenzie et al. 1996a). In addition to the steady-state inward current induced by substrates, there is an hPEPT1-mediated transient presteady-state current following step jumps in membrane potential in the absence of substrates (Mackenzie et al. 1996a). This transient current has been postulated to be due to the movement of charged and polar residues in the membrane electric field, and is associated with the two voltage-dependent partial reactions of the transport cycle: the conformational change of the empty transporter between the external and internal membrane surfaces, and the H+ binding/dissociation (Mackenzie et al. 1996a).
In the present work, we used radiolabelled tracer uptake and electrophysiological measurements to investigate the molecular interactions between human PEPT1, the dipeptide glycylsarcosine and a comprehensive selection of drugs, including ß-lactam antibiotics and antineoplastic agents. To gain insights into the mechanism of hPEPT1, we examined the kinetics of the steady-state and presteady-state currents in the presence of substrates. We revised and extended our 6-state kinetic model (Mackenzie et al. 1996a) to describe the global behaviour of the transporter in the presence of electroneutral substrates and drugs. Finally, we evaluated the functional implications of the most common genetic variants of hPEPT1, Ser117Asn and Gly419Ala (Fig. 1), which occur at a frequency of 25 and 8% (http://pharmacogenetics.ucsf.edu).
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Methods |
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All unlabelled chemicals, reagent grade, were purchased from Sigma (St Louis, MO, USA), except glycylsarcosine (Gly-Sar), cephalexin and cefadroxil, which were from MP Biomedicals (Irvine, CA, USA). Amoxicillin, ampicillin, cefadroxil and cephalexin were used at concentrations up to their maximal solubility at pH 5.0 (5, 10, 10 and 20 mM, respectively). -ALA and bestatin were tested at 0.5 mM, whereas N-acetyl-Asp-Glu (NAAG) was used at 0.1 mM; higher concentrations of these three drugs induced non-specific current responses in control oocytes (not shown). [glycyl-2-3H]Gly-Sar (specific activity 60 Ci mmol–1) was obtained from American Radiolabelled Chemicals (St Louis, MO, USA). Restriction endonucleases were from New England Biolabs (Beverly, MA, USA).
Construction of variant hPEPT1s
Ser117Asn (S117N) and Gly419Ala (G419A) were obtained by cassette exchange mutagenesis, using the wild-type (WT) hPEPT1 (subcloned in pBluescript) (Mackenzie et al. 1996a) as receptor and cDNAs for each variant (subcloned in pGEM) as donors. The variants in pGEM were provided by the UCSF Pharmacogenetics Core Facility. Double digestion of each donor construct with Mfe I and Hpa I yielded a 1.5 kb cDNA fragment containing the desired mutation. These fragments were gel-purified using the QIAEX II kit (Qiagen, Valencia, CA, USA) and subcloned into the receptor plasmid by means of the Fast-Link kit (Epicentre, Madison, WI, USA), after removal of the equivalent 1.5 kb fragment. Competent XL1-Blue cells (Stratagene, La Jolla, CA, USA) were transformed by electroporation, and colonies were selected in a medium with ampicillin and tetracycline. Plasmid DNA was prepared using purification kits by Qiagen. The fidelity of the new clones was verified both by restriction analysis and automated sequencing.
cRNA synthesis
hPEPT1 plasmids were linearized with BamHI, and transcribed in vitro using the T7 MEGAScript kit and RNA cap analogue (Ambion, Austin, TX, USA). The cRNAs were prepared as described (Mackenzie et al. 1996a).
Expression of hPEPT1s in oocytes
Mature female Xenopus laevis were purchased from Nasco (Fort Atkinson, WI, USA). 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 anaesthetized 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 (Parent et al. 1992a) supplemented with 50 mg l–1 gentamicin (Sigma), 5.75 mg l–1 ciprofloxacin (Bayer, West Haven, CT, USA) and 100 mg l–1 streptomycin sulphate/100 000 units l–1 penicillin G sodium (Gibco, Invitrogen, Carlsbad, CA, USA).
Oocytes were injected 1 day after isolation with 50 ng of hPEPT1, S117N or G419A cRNA, and incubated at 18°C for 4–7 days. Experiments were performed at 20°C. Non-injected oocytes served as controls.
Gly-Sar uptake assays
Oocytes were incubated in the presence of 5 µM to 5 mM Gly-Sar (0.1 µM
[3H]Gly-Sar) in a medium containing (mM): 100 NaCl or choline (Cho) chloride, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 Hepes/Tris (pH 7.5) or 10 2-(N-morpholino)ethanesulphonic acid (Mes)/Tris (pH 5.0). After 30 min, oocytes were rinsed and assayed for radioactivity as described (Hediger et al. 1987). Competition studies were performed using the neuropeptide NAAG and selected cephalosporins (cefadroxil and cephalexin), penicillins (ampicillin and amoxicillin), peptidomimetic drugs (bestatin) and non-peptidic compounds (-ALA).
Electrophysiology
A two-microelectrode voltage-clamp system was used to measure substrate-induced steady-state currents in hPEPT1 expressing oocytes (Loo et al. 1993; Mackenzie et al. 1996a). Steady-state current–voltage relationships were measured in Na+ pH 5.0 buffer, in the absence and in the presence of Gly-Sar and/or drugs. A pulse protocol was applied in which membrane potential (Vm) of oocytes was held at –50 mV and stepped to a test value for 100 ms before returning to the holding potential. The test potential varied from +50 to –150 mV in 20 mV increments. Steady-state currents were recorded at the end of 100 ms. pClamp and Axoscope software (Axon Instruments, Union City, CA, USA) were used for pulse protocol application and data acquisition, and continuous current data were recorded with a chart recorder. Unless otherwise noted, 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, using SigmaPlot 9.0 (Systat Software, Inc., Richmond, CA, USA). Data were fitted to eqn (1), for which J is the influx (or the evoked current I), Jmax is the derived maximum transport (or maximal current Imax), S is the substrate concentration, and KS0.5 is the substrate concentration at which transport is half-maximal.
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| (1) |
m; t is time; Ipss is the initial hPEPT1 presteady-state currents 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.
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| (2) |
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| (3) |
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Mathematical modelling
The differential equation (eqn (4)) for the evolution of states for the 7-state kinetic model for hPEPT1 (Fig. 9) was solved using Berkeley Madonna 8.0.1: (http://www.berkeleymadonna.com).
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| (4) |
(k12C1–k21C2)–(k16C1–k61C6)] (Parent et al. 1992b), where N is the number of transporters, and and describe the fraction of the membrane electric field sensed by the binding of H+ and by the empty H+-binding site during membrane translocation.
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) of transient current decay were obtained as described (Mackenzie et al. 1996b), as well as by fitting the simulated presteady-state currents. |
Results |
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Figure 2A illustrates the influx of radiolabelled Gly-Sar into Xenopus laevis oocytes expressing WT, Ser117Asn (S117N) or Gly419Ala (G419A) hPEPT1 transporters. Both variants showed similar transport rates to the WT, under all the conditions tested, indicating similar levels of protein expression in the oocyte plasma membrane. In all cases, uptake of [3H]Gly-Sar occurred in a proton-dependent manner, as it increased up to 13-fold when the external pH was lowered from 7.5 to 5.0. Replacement of Na+ by Cho did not affect the uptake (data not shown). Levels of [3H]Gly-Sar influx in non-injected oocytes were 15% (at pH 7.5) or 1.5% (at pH 5.0) of those measured in cRNA-injected oocytes. In WT, the apparent affinity constant of Gly-Sar uptake (KGS0.5) was 1.3 ± 0.3 mM, and maximal rate of influx (JGSmax) was 3970 ± 290 pmol h–1 oocyte–1.
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-ALA and bestatin, and the neuropeptide NAAG. In accordance with the KGS0.5 of 1.3 mM, influx of 5 µM radiolabelled Gly-Sar dropped about 50% in the presence of 1 mM of non-labelled dipeptide. In all groups, Gly-Sar uptake decreased 50% upon addition of 10 mM cephalexin or 2 mM cefadroxil, whereas the inhibition caused by 0.5 mM
-ALA was of 30% in the WT and of 15% in the variants. Bestatin (0.5 mM) reduced transport in G419A (25%), and only S117N was sensitive to 0.1 mM NAAG (20%). Ampicillin (10 mM) and amoxicillin (5 mM) had no significant inhibitory effect in any group. Gly-Sar influx into non-injected oocytes was not modified by any of the drugs tested (not shown). Electrophysiology
Substrate selectivity. We measured the currents induced by Gly-Sar and the selected drugs in oocytes voltage clamped at –50 mV. Figure 3A shows a continuous record from a representative experiment, in which a WT hPEPT1-expressing oocyte was exposed to 0.5 mM Gly-Sar or 20 mM cephalexin. Addition of these substrates to the perfusion buffer generated inward currents in expressing oocytes, which were reversed upon replacement with substrate-free buffer.
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30 nA at –50 mV). This H+ leak was 5% of the current induced by saturating concentration of Gly-Sar. For example, in a WT-expressing oocyte, the H+ leak at –50 mV was 60 nA, and the current evoked by 20 mM Gly-Sar was 600 nA. Gly-Sar-evoked currents (i) were unaffected by external Na+, and (ii) were significantly attenuated at pH 7.5. For example, currents evoked in a representative WT-expressing oocyte by 1 mM of Gly-Sar in Na+ pH 5.0, Cho pH 5.0, Na+ pH 7.5 and Cho pH 7.5 were 225, 220, 50 and 55 nA, respectively; a similar pH dependence and Na+ independence was found in Gly-Sar currents induced in oocytes expressing S117N and G419A (not shown).
We compared the currents generated by Gly-Sar with those evoked in the same oocyte by each of the different drugs. Experiments were carried out at pH 5.0, the optimum for Gly-Sar transport (Mackenzie et al. 1996a). Results were normalized to the current induced by 0.5 mM Gly-Sar 240 ± 14 (WT), 241 ± 22 (S117N) and 232 ± 18 nA (G419A). All drugs produced inward currents. As shown in Fig. 3B, the currents due to 0.1 mM NAAG, 0.5 mM
-ALA and 0.5 mM bestatin were 14, 25 and 30%, respectively, of that generated by 0.5 mM Gly-Sar, and the currents induced by 20 mM cephalexin and 2 mM cefadroxil were 38 and 50%, respectively. Penicillins caused a poor response: the currents evoked by 10 mM ampicillin or 5 mM amoxicillin accounted for less than 20% of the current due to 0.5 mM Gly-Sar. WT and variants displayed an identical profile of substrate selectivity, except that the currents induced by -ALA in G419A and S117N were 25 and 40% lower than in WT. At the concentrations used, none of the compounds evoked detectable currents in non-injected oocytes (not shown).
Next, we evaluated the ability of the selected compounds to interact with the currents evoked by Gly-Sar, in the same oocytes as in Fig. 3B. In the representative experiment shown in Fig. 3A, a WT oocyte was superfused with 0.5 mM Gly-Sar, first in the absence, and consecutively in the presence of 20 mM cephalexin. The drug caused an abrupt 30% decrease in the Gly-Sar current. As represented in Fig. 3C, addition of 0.1 mM NAAG, 0.5 mM
-ALA and 0.5 mM bestatin led to the inhibition of 35, 40 and 45%, respectively, of the current induced by 0.5 mM Gly-Sar. Inhibition due to 20 mM cephalexin, 5 mM amoxicillin and 10 mM ampicillin was 30, 45 and 55%, respectively. Gly-Sar currents were not inhibited by 10 mM cefadroxil. No significant differences were found among WT and variant hPEPT1s.
Voltage dependence of steady-state currents.
We investigated the characteristics of Gly-Sar-evoked currents and compared them with those induced by cefadroxil, as a model for drug transport. Figure 4 shows typical current records in a representative oocyte expressing WT hPEPT1 before (Fig. 4A) and after addition of 0.5 mM (Fig. 4B) or 10 mM (Fig. 4C) Gly-Sar. Stepping the membrane potential of hPEPT1-expressing oocytes from –50 mV to a series of test values (+50 to –150 mV) resulted in the generation of a transient, presteady-state current (ON response), distinct from the fast (
1 ms) initial spike due to the lipid bilayer capacitance, which relaxed to the steady-state with a time constant ON of 4–12 ms (see for example Fig. 6B). When the membrane potential was returned to the holding value, an equal but opposite transient current was observed (OFF response). hPEPT1 transients were reduced upon addition of Gly-Sar. As shown in Fig. 4, this was particularly evident in the OFF response.
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3 mM at –150 mV to 1 mM at –30 mV (Fig. 5C), and IGSmax decreased from 1000 (Table 1) to 300 nA. No significant differences were found among WT, S117N and G419A in the KGS0.5 values for any given voltage (Fig. 5C); at –50 mV, KGS0.5 values were (mM): 1.5 ± 0.3, 1.6 ± 0.1 and 1.2 ± 0.1, respectively (Table 1). Furthermore, when normalized to the IGSmax obtained at –150 mV (Table 1), the IGSmax–V curves of all variants were identical (not shown). Owing to the low magnitude of the currents measured between +50 to –10 mV, we were unable to estimate the kinetics of Gly-Sar over this voltage range. Due to the limited solubility of cefadroxil at pH 5.0, we could not measure currents at concentrations above 10 mM, and thus could not obtain the transport kinetics of this cephalosporin.
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WT and variants followed identical kinetics of presteady-state current relaxation. At pH 5.0, the transient currents for the ON response relaxed with time constants (ON) that ranged from 4 to 12 ms (Fig. 6B). The ON–V curve fitted a Gaussian relationship, with a maximum (ONmax) of 10–12 ms at a voltage (Vmax) of –35 mV (Table 2). The value for ONmax was consistent among oocytes, but the values of Vmax varied from batch to batch. For example, in WT and variant oocytes from a different donor frog, ONmax was 12 ms but Vmax was more positive than +20 mV. In the OFF response, was voltage independent at 10–12 ms (ON, Table 2).
To calculate the equivalent charge moved (Q), we integrated the presteady-state currents with time. At each test voltage, the charge transfer for the ON and OFF responses (QON and QOFF) was equal; for clarity, only the values of QOFF are shown (Fig. 6C). The charge–voltage (Q–V) curve was fitted to a Boltzmann relation to obtain (i) Qmax, the maximum charge transfer, and (ii) V0.5, the voltage at which half of the maximum charge has moved. At pH 5.0, Qmax was 10–12 nC and V0.5 was –25 mV (Table 2). As described for Vmax, some batch-to-batch variation was observed in V0.5; for example, in the oocytes from a different donor frog, V0.5 was –10 mV. When the pH was increased (i) Qmax decreased significantly, and (ii) V0.5 shifted in the hyperpolarizing direction. In the oocytes shown in Fig. 6, Qmax at pH 7.5 was 5 nC and V0.5 was –90 mV. Values of Q, Qmax and V0.5 were similar in WT-, S117N- and G419A-expressing oocytes (Fig. 6C and Table 2).
Effect of substrates on hPEPT1 charge transfer. Figure 7A exemplifies the effect of 1 mM Gly-Sar and 2 mM cefadroxil on the Q–V distribution in the same WT-expressing oocyte from Fig. 6. Addition of either substrate (i) led to a decrease in Qmax, and (ii) caused the shift of V0.5 to more negative potentials. Thus, in the absence and in the presence of 1 mM Gly-Sar or 2 mM cefadroxil, Qmax was 10.4 ± 0.4, 4.3 ± 0.3 and 6.2 ± 0.6 nC, and V0.5 was –27 ± 3, –41 ± 4 and –31 ± 6 mV, respectively, (standard errors of the Boltzmann fits).
The reduction in Qmax (
Qmax) and the shift in V0.5 (V0.5) induced by Gly-Sar and cefadroxil were dependent upon the external concentration of substrate. Qmax was reduced about 90% by 10 mM Gly-Sar, whereas only 50% of the maximum movable charge disappeared upon addition of 10 mM cefadroxil (Table 2). Accordingly, V0.5 was –45 mV for 10 mM Gly-Sar and –20 mV for 10 mM cefadroxil (Table 2).
We calculated the percentage of decrease in Qmax induced by each concentration of Gly-Sar and cefadroxil according to:
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Qmax0.5 were (mM): 0.6 ± 0.1 (WT, Fig. 7B), 0.7 ± 0.1 (S117N) and 0.8 ± 0.1 (G419A) (standard errors of the fits). At the concentrations of cefadroxil used, the %Qmax–[cefadroxil] relationship did not saturate (Fig. 7C).
Ampicillin, amoxicillin, cephalexin, -ALA, bestatin and NAAG also modified the kinetics of hPEPT1 charge movement; the effect of these drugs in the same oocytes from Figs 6 and 7 is shown in Fig. 8. In the presence of 0.1 mM NAAG, 0.5 mM
-ALA and 0.5 mM bestatin, Qmax was 40–50% (Fig. 8A), and V0.5 was –9, –10 and –13 mV, respectively (Fig. 8B). Upon addition of 20 mM cephalexin, 5 mM amoxicillin or 10 mM ampicillin, Qmax dropped 25, 40 and 60%, respectively (Fig. 8A) and V0.5 shifted by –10 mV (Fig. 8B). The effect of all compounds on Qmax and V0.5 was similar in oocytes expressing WT, S117N and G419A hPEPT1 (Fig. 8, Table 2).
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Discussion |
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-ALA, a medium-chain omega-amino fatty acid that has been implicated in porphyrin-based cancer diagnosis and treatment (Rubio-Aliaga & Daniel, 2002); and (iv) the neuropeptide NAAG. Based on comprehensive steady-state and presteady-state analysis, we extended our 6-state kinetic model for hPEPT1 (Mackenzie et al. 1996a) and obtained a complete set of kinetic parameters that account for the global behaviour of the transporter in the presence of neutral substrates. To test the hypothesis that variations in drug response among individuals are caused by alterations in the genes involved in drug absorption (Sadée, 1999), we evaluated the physiological implications of S117N and G419A, the two most frequent single-nucleotide polymorphisms of hPEPT1. Unless otherwise noted, experiments were carried out at pH 5.0, the pH optimum for Gly-Sar transport (Mackenzie et al. 1996a). Transporter–substrate interactions
Substrate specificity. Gly-Sar transport was electrogenic, pH dependent, Na+ independent, sensitive to membrane potential, and followed saturation kinetics (Figs 2–5), in accordance with previous results (Mackenzie et al. 1996a). The KGS0.5 obtained from radiotracer fluxes was identical to those determined from current measurements in the voltage range –30 to –70 mV (1–2 mM, Fig. 5C and Table 1; and Mackenzie et al. 1996a).
Because the radiolabelled drugs are not commercially available, we could not demonstrate directly that they are substrates for hPEPT1. However, we found that ampicillin, amoxicillin, cephalexin, cefadroxil, -ALA, bestatin and NAAG all induced inward currents (Fig. 3A–B). The direct relationship between substrate-dependent currents and transport, as measured by radiotracer uptake, has been confirmed for other carriers, such as the Na+–glucose (SGLT1), the Na+–phosphate (NaPi) and the Na+–Cl––GABA (GAT1) cotransporters (Mackenzie et al. 1998; Forster et al. 1999; Loo et al. 2000). In one case, however, it should be noted that the generation of currents has proven insufficient to demonstrate actual substrate translocation (Diez-Sampedro et al. 2003).
The currents evoked by all drugs were of lesser magnitude than those generated by equal or lower concentrations of Gly-Sar. Furthermore, the amount of current induced by the antibiotics was comparable to that evoked by concentrations up to 200-fold lower of -ALA, bestatin and NAAG (Fig. 3B). The precise interpretation of these results is a challenge, since variations in transport can be due to differences in affinity, in turnover rate, or in both Where there is a lack of detailed kinetics, it is difficult to determine the relative contribution of each factor in each case. Thus, our data allow us only to speculate that the substrate selectivity of hPEPT1 at pH 5.0 is: Gly-Sar > NAAG, -ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin.
The uptake of 5 µM
[3H]Gly-Sar was blocked by cefadroxil, cephalexin and -ALA, but not by bestatin, ampicillin, amoxicillin or NAAG (Fig. 2B). Because of solubility limitations, we were unable to obtain kinetic data for these drugs. Hillgren and coworkers (Zhang et al. 2004) reported that Gly-Sar uptake into hPEPT1-expressing HeLa cells was inhibited to a significantly higher degree by -ALA and bestatin than by cephalexin and cefadroxil. Cephalexin, cefadroxil and ampicillin are low-affinity blockers of Gly-Sar uptake into Caco-2 cells, with inhibition constants between 7 and 14 mM in the pH range 5.0–6.0 (Bretschneider et al. 1999). On the other hand, bestatin and -ALA have been characterized as high-affinity substrates (K0.5
0.5 mM) for rat and rabbit PEPT1 (Doring et al. 1998; Terada et al. 2000).
A close look at the physicochemical properties of the different substrates could help explain some of these findings. For example, ß-lactams have a tripeptide-like backbone and a more complex three-dimensional structure than Gly-Sar or -ALA (see Fig. 3 of Rubio-Aliaga & Daniel, 2002), which may complicate the interactions with the binding site or act on the translocation pathway. On the other hand, it has been suggested that substrate affinity of peptide transporters can be reduced by acetylation of the -amino group (Wang et al. 1998), which could help justify the relatively low transportability of NAAG. In addition, it has been observed that the transport rate of PEPT1 anionic substrates increases with decreasing pH, which has been attributed to a hypothetical inductive effect of protons at the substrate-binding site (Irie et al. 2005). Thus, it is possible that NAAG, which carries a large anionic charge at acidic pH (–3), requires pH values lower than 5.0 to be transported effectively.
All of the drugs used in this study appear to be transported, as judged by their ability to generate inward currents (Fig. 3B). The currents induced by the drugs in the presence of 0.5 mM Gly-Sar were in most cases less than those due to the same concentration of Gly-Sar alone (Fig. 3C). One possible explanation for this apparent inhibitory effect is that these compounds are transported at a lower turnover rate than Gly-Sar. For example, based on Fig. 2B, it can be roughly estimated that the K0.5 of the ß-lactam cephalexin at –50 mV is 10 mM. With this assumption, the inward current shown in Fig. 3B can be fitted to eqn (1) to determine that the Imax of this drug is 15% of that of Gly-Sar. Alternatively, the lack of additive effects of Gly-Sar and drugs on inward H+ transport may indicate interactions between dipetides, drugs and the transporter that result in a decrease of the total rate of H+–solute cotransport by hPEPT1.
Voltage dependence of hPEPT1. Steady-state inward currents evoked by Gly-Sar and the model drug cefadroxil at pH 5.0 were concentration dependent and increased with hyperpolarization. At <2 mM, Gly-Sar currents were inhibited by large hyperpolarizing potentials, and this inhibition was relieved at higher Gly-Sar concentrations (Fig. 5A). In the voltage range –150 to –30 mV, the estimated Gly-Sar apparent affinity constants (KGS0.5) and current maxima (IGSmax) decreased as membrane potential became more positive (Fig. 5C and Table 1; and Mackenzie et al. 1996a). For 0.5–10 mM cefadroxil, the currents were inhibited at membrane potentials more negative than –90 mV (Fig. 5B).
In the absence of substrate, hPEPT1 presteady-state currents were generated following step changes in membrane potential (Figs 4A and 6A). These transporter-mediated transients (i) were attenuated at pH 7.5 and upon addition of substrates (Fig. 4B and C), (ii) relaxed with time constants () that (a) in the ON response, followed a Gaussian distribution with membrane potential (ONmax
10–12 ms), and (b) in the OFF response, were voltage-independent (OFF
10–12 ms) (Fig. 6B and Table 2), and (iii) represented hPEPT1 charge movements that fitted a Boltzmann relation (eqn (2)) with Qmax
10–12 nC (Fig. 6C and Table 2; and Mackenzie et al. 1996a). The midpoints of the ON–V and Q–V distributions, namely Vmax and V0.5, were similar in oocytes from the same frog, but varied significantly amongst preparations. For example, V0.5 ranged from –20 mV in oocytes from one frog (Table 2) to +30 mV in oocytes from a different one (see also Mackenzie et al. 1996a).
Addition of substrates modified the kinetics of charge distribution, by (i) decreasing Qmax, and (ii) shifting V0.5 in the hyperpolarizing direction (Figs 7 and 8). The Qmax and V0.5 values were dependent upon the external concentration of substrate (Figs 7 and 8; Table 2). The Qmax and V0.5 caused by the different compounds (Fig. 8) further indicate that they bind with relative affinities: Gly-Sar > NAAG, -ALA, bestatin > cefadroxil, cephalexin > ampicillin, amoxicillin.
Transport model for hPEPT1
Description of the model.
To gain insights into the mechanism of H+–oligopeptide cotransport, we re-examined and extended our previous kinetic model (Mackenzie et al. 1996a). The goal was to determine if the 6-state kinetic model could account for the observed steady-state and presteady-state kinetics of Gly-Sar and cefadroxil transport by hPEPT1. Previously, we only simulated presteady-state currents in the absence of substrate (Mackenzie et al. 1996a). Here, we have assumed that ligand binding to hPEPT1 is ordered, with H+ binding before the substrate (Fig. 9). Our model assumes that cotransport is a series of conformational changes induced by ligand (H+ and dipeptide) binding and membrane voltage (Fig. 9). In a transport cycle, one H+ binds to the outside-orientated empty transporter [C]' (state C1) to form the complex [CH]' (state C2). The substrate-loaded protein [CHS]' (state C3) undergoes a conformational change (C3
C4) resulting in H+–dipeptide cotransport. It was necessary to include an additional state to account for the observed inhibition of cotransport by negative voltages at low substrate concentrations (Fig. 5A and B; and Mackenzie et al. 1996a): a second proton binds to the transporter in state C2 to form the complex [CHH]' (state C7), inaccessible to the substrate.
Starting from the set of rate constants used to describe the presteady-state currents in the absence of substrate (Mackenzie et al. 1996a), we sought an expanded set of rate constants to include substrate binding and translocation for a global fit to our experimental data for H+–dipeptide cotransport and the presteady-state kinetics in the presence of substrates. We also imposed an additional constraint in that the model had to be consistent with the kinetics of reverse H+–oligopeptide transport (Kottra & Daniel, 2001). We obtained a numerical solution for the 16 rate constants and three voltage-dependence parameters that account qualitatively and quantitatively for the steady-state and presteady-state experimental data (see legend to Fig. 9). For the set of constants, we examined (i) the I–V relations of the Gly-Sar and cefadroxil-coupled steady-state currents, (ii) the time course of the presteady-state currents, and (iii) the –V, and (iv) the Q–V relationships. The model predictions are compared directly with the experimental data in Figs 5–7 and Tables 1 and 2.
Fit of the model.
The predicted steady-state I–V relations for the Gly-Sar and cefadroxil cotransport fit the experimental data over the entire range of substrate concentrations and voltage (Fig. 5A and B). The voltage dependence of Gly-Sar transport (K0.5) between –30 and –150 is well described by the model (Fig. 5C and D, and Table 1), and the same behaviour is predicted for cefadroxil (Fig. 5D). For both substrates, the predicted K0.5 is at a minimum at –50 mV (see values in Table 1) and increases steadily with hyperpolarization and depolarization. In both cases, the voltage-dependent increase in K0.5 can be explained as a reduction in the fraction of transporters in state C2. Hyperpolarization drives carriers in substrate-accessible state C2 to unavailable state C7. On the other hand, depolarizing voltages decrease the rate of proton binding to the transporter (C1
C2) and increase the rate of dissociation (C2
C1).
As anticipated, the model provides an accurate description of the presteady-state kinetics of hPEPT1 at pH 5.0 in the absence of substrate (Fig. 6 and Table 2; and Mackenzie et al. 1996a). Thus, the model predicts that step changes in membrane potential generate transient currents that rise rapidly to a peak before decaying to the steady state. Presteady-state currents in the ON and OFF response are equal but opposite in sign; an example of the predicted OFF transients is presented in Fig. 6A. For both ON and OFF responses, two relaxation time constants () are predicted by the model: (i) a fast component, of less than 1 ms, which is beyond the resolution of the voltage clamp (not shown), and (ii) a slower one (ON, OFF) in the range 3–11 ms (Fig. 6B and Table 2). The predicted values of ON are voltage dependent (Fig. 6B), and the ON–V curve fits a Gaussian relationship, with ONmax
= 10.8 ms and Vmax
=
– 36 mV (Table 2). The predicted time constant for the OFF response (OFF) is voltage independent at 10.7 ms (Table 2). The Q–V curves (Fig. 6C) fit a Boltzmann equation, with Qmax
= 10.6 nC, V0.5
=
–27 mV (Table 2) and z
= 1. At pH 7.5, the predicted values of Qmax and V0.5 are 6.5 nC and –70 mV, and again this is consistent with the experimental results.
The behaviour of hPEPT1 charge movements as a function of the external concentration of electroneutral substrates such as Gly-Sar and cefadroxil is also predicted by the model. As shown in Fig. 7 and Table 2, the reduction in charge transfer by substrates is simulated both qualitatively and quantitatively. The predicted reductions in Qmax with [Gly-Sar] (Fig. 7B) and [cefadroxil] (Fig. 7C) follow hyperbolic relationships, with KQmax0.5 0.6 mM (Gly-Sar) and 2.5 mM (cefadroxil). In addition, the model correctly predicts the shift in V0.5 due to 10 mM Gly-Sar and cefadroxil (Table 2).
The results suggest that the lower transport rate of cefadroxil relative to Gly-Sar is due to (i) a lower affinity and (ii) a lower turnover rate. The values of KGS0.5 are one order of magnitude below those of KCEF0.5 (Fig. 5C and D, and Table 1). At –50 mV, the predicted KCEF0.5 is 6.2 mM, fivefold higher than KGS0.5 (Table 1). The cefadroxil current maxima (ICEFmax) are expected to be half of those for Gly-Sar (Table 1). The turnover number, calculated as the ratio of Imax at –150 mV to Qmax (Loo et al. 1993), is 130 s–1 for Gly-Sar and 70 s–1 for cefadroxil (Tables 1 and 2).
Interpretation of the model.
Our model predicts that, in a transport cycle, at saturating substrate concentration, –50 mV and pH 5.0, the rate limiting step for substrate (Gly-Sar) and drug (cefadroxil) transport by hPEPT1 is the return of the empty transporter from the internal to the external membrane surface (C6
C1) (Fig. 9). The difference in turnover rate between Gly-Sar and cefadroxil is due to a difference in Imax, the maximal transport rate. Imax is not solely determined by the rate-limiting step, but depends on all the rate constants in the transport cycle (see eqns A37 and A41 of Parent et al. 1992b). Imax is reduced in cefadroxil because of a lower binding rate k°23 (see legend to Fig. 9). Even with the reduction in k23 from Gly-Sar to cefadroxil from 105 to 1.25 x 104M–1 s–1, the cefadroxil binding rate k°23
x
[cefadroxil] is greater than k61, that is, C6
C1 remains rate limiting. The shift of V0.5 to more negative values, and the reduction of Qmax with increasing concentrations of substrate and drug are consistent with the model prediction that there is a shift in the distribution of carrier states. In the presence of H+ alone, at pH 5.0 and Vm
=
–50 mV, the occupancy probabilities are C1, 0.13; C2, 0.59; C3, 0; C4, 0; C5, 0.003; C6, 0.26; C7, 0.02. At a saturating concentration of Gly-Sar (10 mM), the occupancy probabilities are: C1, 0.04; C2, 0.06; C3, 0.09; C4, 0.02; C5, 0.10; C6, 0.69; C7, 0.002. At saturating cefadroxil (100 mM), they are: C1, 0.02; C2, 0.03; C3, 0.2; C4, 0.14; C5, 0.08; C6, 0.53; C7, 0.001. Thus, the negative shift of V0.5 and the reduction in Qmax are due to the shift of the transporter from being predominantly in state C2 in the presence of H+ alone to being in state C6 in the presence of saturating substrate concentrations, at Vm
=
–50 mV. This contrasts with kinetic models for other transporters, such as the Na+–glucose cotransporter SGLT1 and the Na+–iodide cotransporter NIS. In these, the rate-limiting step seems to be the dissociation of the driving cation into the cytoplasm (C5
C6) (Eskandari et al. 1997; Loo et al. 1998).
The set of rate constants for hPEPT1 (Fig. 9) indicate that the system is asymmetric, e.g. in the absence of a driving force provided by the pH gradient (external and internal pH 7.5), the predicted KGS0.5 values for inward and outward transport are 6 and 65 mM. This is consistent with the report by Kottra & Daniel (2001) that Gly-L-Gln transport by rabbit PEPT1 was asymmetric: in the absence of a pH gradient, the K0.5 was 0.7 mM in the inward direction and 3.3 mM in the outward direction.
The nature of the interactions (C2 C7) between the transporter and the ligands (H+, Gly-Sar and cefadroxil) in the voltage-dependent inhibition of hPEPT1-mediated transport is not clear. The data indicate that there is a competition between protons and substrates. Our model for this competition assumes that the inhibition at low pH and large negative potentials is due to the binding of a second proton to the transporter to form state C7, after the first H+-binding site is occupied. Our simulations (not shown) indicate that the substrate and/or drug can not bind to the transporter in state C7, otherwise high Gly-Sar concentrations would increase rather than relieve the inhibition by protons (see for example Fig. 5A). While the strength of the interaction, represented by the pseudo-rate constant k027, is independent of substrate concentration, it is determined by the nature of the substrate per se. We estimate k027 to be 5 x 105M–1 s–1 for Gly-Sar, and 3 x 106M–1 s–1 for cefadroxil. The higher value of k027 for cefadroxil simply reflects the relatively poorer ability of the cephalosporin to overcome the inhibitory effects of protons, due perhaps to the lower affinity of this drug.
Recently, Inui and coworkers (Irie et al. 2005) proposed a model for the transport of charged and neutral substrates by hPEPT1. In their model, two protons bind first (CoHH), and then anionic substrates can bind to the two-proton-bound transporter (Fig. 3 of Irie et al. 2005). Without further assumptions, it is not clear how their model can account for the inhibition of the electroneutral Gly-Sar transport at low pH and negative membrane voltages. Our simulations indicate that additional constraints include the requirement that the binding of the second proton does not involve a charge movement, and neutral substrates can not bind to the two-proton bound transporter. With these additional assumptions, our model is equivalent to that proposed by
) and in the presence of 1 mM Gly-Sar (•) and 2 mM cefadroxil (
). B and C, dependence of reduction of Qmax on external concentration of Gly-Sar (•, B) and cefadroxil (
