Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain

Hong Wang, You-Jun Fei, Vadivel Ganapathy, and Frederick H. Leibach

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have cloned a peptide transporter from rat brain and found it to be identical to rat kidney PEPT2. In the present study we characterize the transport function of the rat brain PEPT2, with special emphasis on electrophysiological properties and interaction with N-acetyl-L-aspartyl-L-glutamate (NAAG). When heterologously expressed in HeLa cells and in SK-N-SH cells, PEPT2 transports several dipeptides but not free amino acids in the presence of a proton gradient. NAAG competes with other peptides for the PEPT2-mediated transport process. When PEPT2 is expressed in Xenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides of differing charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAG is a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of the transport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively charged peptides is 1, the transport nonetheless is associated with transfer of positive charge into the oocyte, as indicated by peptide-induced inward currents.

N-acetyl-L-aspartyl-L-glutamate; electrophysiology; charged peptides

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PROTON-COUPLED PEPTIDE transporters belong to a unique gene family of transport proteins whose members are found not only in the animal kingdom but also in bacteria and plants (12). These transporters mediate the transport of small peptides consisting of two, three, or four amino acids, and the transport process is active, energized by a transmembrane electrochemical proton gradient (11, 19, 26). In animals, peptide transport has been functionally described primarily in the small intestine and the kidney, where the process functions in the assimilation of dietary protein digestion products and in the reclamation of peptide-bound amino nitrogen from the glomerular filtrate, respectively (1, 16-18). Recently, two distinct peptide transporters have been cloned, one from the intestine and the other from the kidney (12, 20, 21). The intestinal peptide transporter, designated PEPT1, is a low-affinity transporter, and its primary structure has been deduced by expression and molecular cloning studies in three different animal species, namely rabbit (8, 13), rat (27, 33), and human (22). The renal peptide transporter, designated PEPT2, is a high-affinity transporter, and its primary structure has also been deduced by molecular cloning studies in rabbit (7), rat (34), and human (23). PEPT1 and PEPT2 are products of different genes, the PEPT1 gene located on human chromosome 13q24-q33 (22) and the PEPT2 gene located on human chromosome 3q13-q21 (31). More recently, a third peptide transporter, designated PHT1, has been cloned from rat brain (42). Interestingly, PHT1 shows very little homology to PEPT1 and PEPT2, but it does transport small peptides in a proton gradient-dependent manner. PHT1 is expressed in the brain and in the eye but not in the intestine or in the kidney. Among the above-mentioned three peptide transporters, only PEPT1 has been characterized functionally by detailed electrophysiological approaches (2, 8, 13, 24, 25, 30, 35).

Northern blot hybridization with PEPT2 cDNA as the probe has indicated that mRNA species hybridizing to the probe are abundantly expressed in the brain (7, 34). However, it is not clear whether these hybridizing transcripts represent PEPT2 mRNA or structurally related but hitherto unidentified transcripts. Therefore, the present study was undertaken to establish the identity of the transcripts in the brain that are recognized by the PEPT2 cDNA probe in Northern blot analysis. To this end, we screened a rat brain cDNA library using the human PEPT2 cDNA as the probe and isolated a positive full-length cDNA clone. Sequence analysis of the cDNA has revealed that it is identical to the already cloned PEPT2. We report here the electrophysiological characteristics of the rat brain PEPT2 and the interaction of this transporter with the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) and other charged peptides. Furthermore, the rat brain PEPT2 does not interact with histidine or any other amino acid, a characteristic that is in contrast to the rat brain PHT1, which interacts with histidine in addition to peptides.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. [2-14C]glycyl-[1-14C]sarcosine (Gly-Sar; specific radioactivity, 109 mCi/mmol) was custom synthesized by Cambridge Research Chemicals (Billingham, Cleveland, UK). [1-14C]glycyl-L-proline (6.4 mCi/mmol), glycyl-L-[U-14C]phenylalanine (25 mCi/mmol), and [1-14C]glycylglycine (9.1 mCi/mmol) were purchased from Amersham Radiochemical Center (Arlington Heights, IL). L-[3-3H]threonine (15.2 Ci/mmol) was also from Amersham Radiochemical Center. [3,4-3H]glutamine (59 Ci/mmol), [1-3H]glycine (35 Ci/mmol), [4,5-3H]leucine (60 Ci/mmol), and [ring-2,5-3H]histidine (57 Ci/mmol) were purchased from DuPont-NEN (Boston, MA). NAAG was obtained from Research Biochemicals (Natick, MA), and all other peptides were obtained from Sigma (St. Louis, MO).

Isolation of a peptide transporter cDNA clone from a rat brain cDNA library. A cDNA library was constructed using poly(A)+ RNA isolated from rat brain. The SuperScript plasmid system (Life Technologies, Gaithersburg, MD) was employed for this purpose. This system was chosen because the cDNA inserts can be directionally cloned into the vector pSPORT so that the inserts are under the control of the T7 promoter in the vector. This feature is essential for functional expression of the cloned cDNAs using the Xenopus laevis expression technique as well as the vaccinia virus expression technique. Double-stranded cDNA obtained by reverse transcription of the poly(A)+ RNA was modified at both termini by adapter sequences containing an Not I site at one terminus and an Sal I site at the other terminus to introduce directionality. The resultant cDNAs were size-fractionated by gel filtration chromatography (Sephacryl S-500 HR) to eliminate small cDNA fragments shorter than 0.5 kb. The cDNAs were then ligated to Not I/Sal I-digested pSPORT vector. The transformation of ligated cDNAs into Escherichia coli was done by electroporation using Electro MAX DH10B competent cells as host cells.

Screening of the cDNA library was done by colony hybridization of the plasmid cDNA library grown on nylon transfer membranes (MicroSeparation Systems, Westboro, MA). The probe was a 1.7-kb fragment released from the coding region of human PEPT2 cDNA (23) by EcoR I digestion. The probe was labeled with [alpha -32P]dCTP by random priming using a commercially available oligolabeling kit (Pharmacia). Hybridization was carried out at 65°C in a solution containing 5× SSPE (1× SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA), 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA for 24 h. Washing was done under low-stringency conditions that involved washing three times, each time for 30 min, at room temperature in a solution containing 2× SSC (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, and 0.5% SDS). Positive clones were identified, and the colonies were purified by secondary and tertiary screenings. Sequencing of the isolated cDNA was done by the dideoxy chain termination method using the Sequenase 2.0 kit (United States Biochemicals, Cleveland, OH).

Functional expression in HeLa cells and in SK-N-SH cells. Functional expression was achieved using the vaccinia virus expression technique (5). Subconfluent HeLa cells and SK-N-SH cells (American Type Culture Collection, Manassas, VA) were first infected with a recombinant vaccinia virus (VTF 7-3) encoding T7 RNA polymerase and then transfected with either the vector carrying the full-length cDNA or the vector alone (control) in the presence of Lipofectin (Life Technologies). Peptide or amino acid transport activity was assayed 10-12 h after transfection by measuring the uptake of radiolabeled peptides or amino acids. The uptake medium for peptide or amino acid uptake measurements was 25 mM MES-Tris (pH 6.0) containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, and 5 glucose. The incubation time for uptake measurements was 5 min. HeLa cells and SK-N-SH cells do not possess endogenous peptide transport activity and hence are highly suitable for heterologous expression analysis of putative peptide transporter cDNAs.

Functional expression in X. laevis oocytes and electrophysiological studies. Oocytes isolated from X. laevis (Nasco, Fort Atkinson, WI) were partially digested with collagenase A in a Ca2+-free medium and then manually defolliculated. Mature (stage V-VI), defolliculated oocytes were used for injection with cRNA preparations. The plasmid-cDNA was linearized by Not I digestion, and the cDNA insert was transcribed by using bacteriophage T7 RNA polymerase. The Ambion MEGAscript kit was used for this purpose. RNAase inhibitor and mRNA cap analog [7-methyl diguanosine triphosphate, mG(5)ppp(5)G] were included. Final concentration of the capped cRNA was adjusted to 1 µg/µl. Oocytes were injected with 50 ng cRNA or 50 nl of water. Electrophysiological measurements in cRNA- or water-injected oocytes were carried out 4-6 days after cRNA injection.

Electrophysiological characteristics of the cDNA-induced transport activity in the oocytes were studied using the two-microelectrode voltage-clamp technique. In this technique, the membrane potential was clamped at -50 mV. One microelectrode was used to monitor the actual membrane potential and the second microelectrode was used to pass currents into the oocyte in such a way that the membrane potential remained clamped at -50 mV. The current required to be passed through the second electrode is the measured parameter of the cRNA-induced transporter activity. Oocytes were superfused at room temperature with the medium containing (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 3 HEPES-MES-Tris in the pH range 5-8. All substrate solutions were made in these buffers. The substrate-induced currents were averaged over three sweeps. The oocytes were thoroughly washed before exposure to the next testing substrate, and several consecutive currents were measured using the same oocyte. The experiments were repeated in at least three independent oocytes for data analysis. The data were fitted to the equation I = ImaxSnH/(K1/2nH + SnH), where I is the substrate-evoked current (i.e., the difference in the steady-state current measured in the presence and absence of the substrate), Imax is the maximal current, S is the substrate concentration, nH is the Hill coefficient, and K1/2 is the substrate concentration at which the substrate-evoked current is one-half of Imax (Michaelis-Menten constant). The dependence of the substrate-evoked current on the peptide concentration as well as on the proton concentration was investigated. To study the relationship between membrane potential and substrate-evoked current, step changes in membrane potential (called testing membrane potential) were applied, each for a duration of 100 ms, in 20-mV increments over the range +50 mV to -150 mV. The voltage-jumping protocol was first applied in the absence of substrate and then in the presence of substrate. The substrate-specific current at each testing membrane potential was determined as the difference between the currents recorded in the presence and in the absence of substrate. Kinetic analysis was performed using a commercially available computer program (SigmaPlot, Jandel Scientific, San Rafael, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and structural characterization of rat brain PEPT2 cDNA. Screening of the rat brain cDNA library (~6 × 106 colonies) using a fragment of human PEPT2 cDNA as the probe resulted in the identification of 22 positive colonies. Twelve of these colonies were purified by secondary and tertiary screening, and the cDNA inserts of the plasmids were characterized by size and sequence analyses. All 12 cDNA inserts were found to be identical except for size differences resulting from truncations of variable length at the 5' end. The largest of these cDNAs was 3.9 kb long, and this cDNA was subjected to complete sequence analysis. It was found to be identical to the PEPT2 cDNA cloned by Saito et al. (34) from a rat kidney cDNA library. The only difference was that the rat brain PEPT2 cDNA was shorter by 146 bp in the 5' untranslated region.

Functional characterization of the rat brain PEPT2 cDNA. To demonstrate that the rat brain PEPT2 cDNA codes for a functional peptide transporter, the cDNA was expressed in HeLa cells using the vaccinia virus expression technique. Transport of four dipeptides (Gly-Sar, Gly-Gly, Gly-Phe, and Gly-Pro) and five amino acids (Gly, Thr, Leu, Gln, and His) was measured in HeLa cells that were transfected with either the pSPORT-cDNA construct or the pSPORT plasmid alone (Table 1). It was found that the cDNA induced the transport of all four peptides severalfold but that the transport of the free amino acids was not affected. Transport measurements in these experiments were made at an extracellular pH of 6.0, a condition that creates an inwardly directed proton gradient across the HeLa cell plasma membrane. It was clearly evident from these results that the PEPT2 cDNA isolated from rat brain encodes a functional proton-coupled peptide transporter.

                              
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Table 1.   Uptake of peptides and amino acids in HeLa cells transfected with either empty vector or rat brain PEPT2 cDNA

Successful isolation of the PEPT2 cDNA from the brain tissue reported here raises interesting questions as to the possible function of this peptide transporter in the brain. NAAG, a dipeptide derivative, is present in high concentrations in mammalian brain. This peptide has been suggested to function as a neurotransmitter (28, 38). Available evidence suggests that NAAG is a selective agonist of specific subtypes of glutamate receptors (37, 40, 41). One of the mechanisms of inactivation of this putative neurotransmitter is hydrolysis by a peptidase (4, 9, 32) that is associated with neuronal and glial plasma membranes (6, 10). In addition to this extracellular degradative pathway, NAAG is also known to be taken up into neuronal cells by a transport pathway whose identity remains unknown (39). Because NAAG is a dipeptide derivative and the brain tissue expresses PEPT2, we tested the possibility that NAAG may be a substrate for PEPT2. We first studied the ability of NAAG to inhibit the transport of radiolabeled Gly-Sar in HeLa cells that functionally express PEPT2. Figure 1A shows that NAAG is an inhibitor of PEPT2-mediated Gly-Sar transport. Significant inhibition was observed at a concentration of 1 mM. The IC50 value (i.e., the concentration of NAAG necessary to inhibit 50% of Gly-Sar transport) for the inhibition was ~3 mM. Kinetic analysis revealed that NAAG is a competitive inhibitor of PEPT2-mediated Gly-Sar transport (Fig. 1B). In the absence of NAAG, Gly-Sar transport in PEPT2-expressing HeLa cells occurred via a single, saturable mechanism with a K1/2 of 35 ± 6 µM and a maximal velocity of 1.07 ± 0.07 nmol · 106 cells-1 · 5 min-1. In the presence of 4 mM NAAG, the K1/2 for Gly-Sar increased to 265 ± 48 µM, with no significant change in the maximal velocity (1.26 ± 0.17 nmol · 106 cells-1 · 5 min-1).


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Fig. 1.   A: influence of increasing concentrations of N-acetyl-L-aspartyl-L-glutamate (NAAG) on Gly-Sar transport in PEPT2-expressing HeLa cells. Transport of 10 µM [14C]Gly-Sar (5-min incubation) was measured in HeLa cells transfected with pSPORT-rat brain PEPT2 cDNA construct at pH 6.0 in presence of 0-10 mM NAAG. B: kinetics of inhibition of PEPT2-mediated Gly-Sar transport by NAAG in HeLa cells. Transport of Gly-Sar was measured in PEPT2-expressing HeLa cells in either absence (open circle ) or presence (bullet ) of 4 mM NAAG. Concentration range for Gly-Sar was 25-400 µM, and incubation time was 5 min. Transport measured in HeLa cells transfected with vector alone was subtracted to account for nonspecific transport. Results are given as Eadie-Hofstee plots (V vs. V/S, where V is Gly-Sar transport in pmol · 106 cells-1 · 5 min-1 and S is Gly-Sar concentration in µM).

HeLa is a human cervical epithelial cell line, and it has been extensively used in the functional characterization of a variety of cloned transporters after heterologous expression. To rule out the possibility that the observed functional features of the brain PEPT2 expressed in HeLa cells may be unique to the cell type, we studied the function of the brain PEPT2 after heterologously expressing the transporter in SK-N-SH cells. SK-N-SH is a human neuroblastoma cell line, and we thought that it would be desirable to establish the key functional features of the brain PEPT2 in this brain-derived cell line. We studied the ion dependence, substrate specificity, and saturation kinetics of the transporter in SK-N-SH cells. As is the case with HeLa cells, SK-N-SH cells do not possess detectable endogenous peptide transport activity. The rat brain PEPT2 cDNA could be functionally expressed in these cells by the vaccinia virus expression technique, as assessed by the cDNA-induced transport of Gly-Sar. This transport was Na+ and Cl- independent and was energized by an inwardly directed proton gradient (Fig. 2A). Competition studies showed that the cDNA-induced Gly-Sar transport was inhibitable by differently charged (neutral, anionic, and cationic) dipeptides (Ala-Val, Ala-Glu, and Ala-Lys) and by NAAG (Fig. 2B). Kinetic analysis indicated that the cDNA-induced Gly-Sar transport was saturable with a K1/2 of 84 ± 8 µM (Fig. 3).


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Fig. 2.   A: influence of Na+, Cl-, and protons on Gly-Sar transport in PEPT2-expressing SK-N-SH cells. Transport of 20 µM [14C]Gly-Sar (5-min incubation) was measured in SK-N-SH cells transfected with pSPORT-rat brain PEPT2 cDNA construct using a buffer of pH 6.0 or 8.0. Ionic composition of buffer was changed in such a way that it contained NaCl, was Na+ free, or was Cl- free. In Na+-free buffer, NaCl was isosmotically replaced by N-methyl-D-glucamine chloride. In Cl--free buffer, all Cl- salts (NaCl, KCl, and CaCl2) were isosmotically replaced by corresponding gluconate salts. B: substrate specificity of rat brain PEPT2 expressed in SK-N-SH cells. Transport (5 min) of 30 µM [14C]Gly-Sar was measured at pH 6.0 in presence or absence of Ala-Val (0.5 mM), Ala-Lys (0.5 mM), Ala-Glu (0.5 mM), or NAAG (4 mM).


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Fig. 3.   Saturation kinetics of Gly-Sar transport mediated by rat brain PEPT2 expressed in SK-N-SH cells. Transport (5 min) of Gly-Sar was measured in PEPT2-expressing cells over a Gly-Sar concentration range of 25-400 µM. Transport measured in cells transfected with vector alone was subtracted to account for nonspecific transport. Inset: Eadie-Hofstee plot (V/S vs. V).

Electrophysiological characteristics of PEPT2-mediated transport of differently charged peptides. We investigated the function of PEPT2 expressed in X. laevis oocytes using electrophysiological approaches. For this investigation, we chose three dipeptides of differing charge as representative substrates for PEPT2: Ala-Val (a neutral dipeptide), Ala-Glu (an anionic dipeptide), and Ala-Lys (a cationic dipeptide). Figure 4 describes the activation of PEPT2-mediated transport of all three peptides by protons in the extracellular medium. Although the three peptide substrates are differently charged under the experimental conditions, the activation of transport by protons was hyperbolic in all three cases, indicating a proton-to-peptide coupling ratio of 1. The Hill coefficient for protons, which is indicative of the coupling ratio, was 1.0 ± 0.4 for Ala-Glu, 1.0 ± 0.1 for Ala-Lys, and 1.3 ± 0.2 for Ala-Val. There was very little influence of membrane potential on the Hill coefficient. Thus it appears that each of the three peptides is cotransported with one proton. The K1/2 for the activation of the transport process by protons was in the range 0.75-2.25 µM.


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Fig. 4.   Top: influence of external proton concentration on peptide-induced currents (I) in PEPT2-expressing X. laevis oocytes for Ala-Glu, Ala-Lys, and Ala-Val. Concentration of peptides was 75 µM, and range of proton concentrations tested was 31.6 nM to 10 µM (i.e., pH 5.0-7.5). Bottom: Hill coefficient values (nH) for proton at different testing membrane potentials (Vtest) for the 3 dipeptides.

Further kinetic studies on the proton and membrane potential dependence of the transport of the three peptides revealed significant similarities (Fig. 5). The K1/2 values for all three peptides were influenced markedly by the external proton concentration as well as by the membrane potential (Fig. 5, A-C). Although the K1/2 did not appear to be affected significantly by external pH in the physiological range of membrane potential (-50 to -70 mV), appreciable changes in the K1/2 were seen depending on the external pH under hyperpolarizing membrane potentials. Interestingly, in the case of the charged peptides Ala-Glu and Ala-Lys, there was no change in the K1/2 when external pH was switched from 6.0 to 5.5, but further reduction in the external pH to 5.0 greatly increased the K1/2. In contrast, in the case of the neutral peptide Ala-Val, increases in K1/2 were seen when the external pH was switched from 6.0 to 5.5 and also when it was switched from 5.5 to 5.0. 


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Fig. 5.   A-C: influence of external pH and Vtest on Michaelis-Menten constant (K1/2) for the 3 dipeptides Ala-Glu (A), Ala-Lys (B), and Ala-Val (C). D-F: influence of Vtest on peptide-induced currents (Imax) at different pH for Ala-Glu (D), Ala-Lys (E), and Ala-Val (F). For each pH, Imax at a Vtest of -150 mV was taken as 1 for comparison of influence of Vtest on magnitude of Imax at all 3 pH values.

Despite the differences in the net charge of the three peptide substrates, the current-membrane potential relationship was similar in all three cases (Fig. 5, D-F). The currents increased as the testing membrane potential was hyperpolarized from -50 mV and decreased as the testing membrane potential was depolarized from -50 mV. The currents in all three cases approached zero when the testing membrane potential was in the range between 0 and +50 mV.

Electrophysiological characteristics of PEPT2-mediated transport of NAAG. The results obtained with PEPT2-expressing HeLa cells show that NAAG competes with Gly-Sar for interaction with PEPT2. This suggests but does not prove that NAAG is a transportable substrate for PEPT2. To demonstrate directly that PEPT2 does indeed transport NAAG, we employed the X. laevis oocyte expression system and analyzed the transport-associated currents. Figure 6A shows the transport-associated currents when PEPT2-expressing oocytes were superfused with NAAG (5 mM) at pH 7.5 or 5.5. The presence of NAAG in the perifusion medium at pH 5.5 induced marked inward currents. The same concentration of NAAG, however, induced much less inward current when the pH of the perifusion medium was 7.5 instead of 5.5. In contrast, NAAG failed to induce detectable currents at pH 7.5 or at pH 5.5 in water-injected oocytes (Fig. 6B). These data show that PEPT2 mediates the transport of NAAG in a proton-dependent manner and that the transport process results in the transfer of positive charge into the oocytes. We also analyzed the influence of various proton concentrations in the external medium on the magnitude of 5 mM NAAG-induced currents (Fig. 6C). The magnitude of the currents was found to be hyperbolically related to external proton concentrations (pH range 5.0-7.5). The Hill coefficient for protons, which is a measure of the proton-to-NAAG stoichiometry, was 0.8 ± 0.1. Thus the number of protons that is cotransported with NAAG appeared to be 1. 


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Fig. 6.   Influence of external proton concentration on 5 mM NAAG-induced I in oocytes injected with either PEPT2 cRNA or water. A: tracings of 5 mM NAAG-induced I at pH 7.5 or 5.5 in PEPT2-expressing oocytes. ND96 buffer consists of (in mM) 96 NaCl, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, and 5.0 HEPES (pH adjusted to 7.4 with Tris). B: tracings of I in water-injected oocytes under same experimental conditions described in A. C: dependence of 5 mM NAAG-induced inward I on proton concentration in external medium. Proton concentration was varied in range between 31.6 nM (pH 7.5) and 10 µM (pH 5.0).

Figure 7A shows that superfusion of PEPT2-expressing oocytes with increasing concentrations of NAAG at pH 5.5 induced inward currents of increasing magnitude. Figure 7B describes the relationship of current and testing membrane potential for NAAG transport. The currents were found to increase with increasing concentrations of NAAG and also with increasingly hyperpolarizing membrane potential. Similar relationships between the magnitude of the currents and NAAG concentration were evident at all testing membrane potentials studied. With a given concentration of NAAG, the currents increased in magnitude when the testing membrane potential was increasingly hyperpolarized. These results show that hyperpolarization dramatically enhances NAAG transport mediated by PEPT2.


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Fig. 7.   A: influence of increasing concentrations of NAAG on NAAG-induced I in PEPT2-expressing oocytes at pH 5.5. B: I-Vtest relationship at pH 5.5 for various concentrations of NAAG in PEPT2-expressing oocytes. C: saturation kinetics of NAAG-induced I at pH 5.5 in PEPT2-expressing oocytes with different Vtest. D: influence of Vtest on K1/2 for NAAG. K1/2 values were calculated by measuring I at different Vtest at pH 5.5 in presence of various concentrations of NAAG (0.1-5 mM).

The transport of NAAG by PEPT2 was found to be saturable at all testing membrane potentials studied (Fig. 7C). The relationship between NAAG concentration and the magnitude of the currents was hyperbolic, and the data were found to fit best to a transport model involving a single saturable system. We also determined the K1/2 for NAAG. The K1/2 was found to be dependent on the testing membrane potential to a significant extent (Fig. 7D).

It must be mentioned here that there was significant variation in the magnitude of peptide-induced currents among different PEPT2-expressing oocytes. This difference was most likely due to variation in the expression levels of the transporter protein in different oocytes. However, there was no qualitative difference in the functional features of PEPT2 studied in different oocytes. All of the key features, including the proton dependence, proton-to-peptide stoichiometry, current-membrane potential relationship, substrate affinities, and dependence of K1/2 for peptides on membrane potential, were reproducible in each oocyte expressing PEPT2.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although Northern blot analysis has indicated the presence of transcripts hybridizing to PEPT2 cDNA probes in rat brain, the exact identity of these transcripts has never been established. Furthermore, peptide transport activity has not been demonstrated in the brain tissue by any experimental approach. Therefore, the present results constitute the first evidence for the expression of functionally competent PEPT2 transcripts in the brain. Yamashita et al. (42) recently isolated from a rat brain cDNA library a cDNA clone that codes for a transporter capable of mediating the uptake of small peptides as well as of the free amino acid histidine. There is, however, very little homology at the structural level between this peptide/histidine transporter (PHT1) and PEPT2. Furthermore, although both PHT1 and PEPT2 mediate proton-coupled transport of peptides, PEPT2 does not interact with histidine as PHT1 does. To date, PEPT2 has been cloned only from the renal tissue, where it is believed to function in the reabsorption of peptides from the glomerular filtrate.

Here we report on the functional characteristics of PEPT2 heterologously expressed in two different mammalian cell lines as well as in X. laevis oocytes. The two mammalian cell lines are HeLa (a human cervical epithelial cell line) and SK-N-SH (a human neuroblastoma cell line). The key functional features, such as the ionic dependence, substrate specificity, and saturation kinetics, were found to be similar for PEPT2 expressed in both cell types. This indicates that the functional characteristics of PEPT2 are not influenced by the cell type in which it is heterologously expressed. Electrophysiological characterization of PEPT2 function was carried out in X. laevis oocytes. Because the function of PEPT2 was assessed for the present study using intact cells, the observed characteristics are attributed to PEPT2 expressed in the plasma membrane of these cells. This is also evident from the studies with X. laevis oocytes, in which the PEPT2 function was monitored using peptide-induced changes in membrane potential across the oocyte plasma membrane.

The results presented here clearly demonstrate that the neuropeptide NAAG is a transportable substrate for the proton-coupled peptide transporter PEPT2, which is expressed in the brain. PEPT2 exhibits much higher affinity for its substrates than does PEPT1 (31). The K1/2 for the interaction of NAAG with PEPT2 is, however, in the low millimolar range, which suggests that the blocking of the alpha -amino group by acetylation greatly reduces the affinity. Nonetheless, the observed values may be physiologically relevant. NAAG is present in the mammalian nervous system at very high concentrations (15). The concentrations of NAAG have been shown to be in the range 0.2-4.5 mM in different regions of the nervous system. However, it is premature to speculate on the physiological role of PEPT2 in the disposition of NAAG in the brain. The precise localization of PEPT2 in the brain and whether this localization coincides with the regions of high NAAG concentrations remain still to be determined. Furthermore, there is no information available on the magnitude of the proton gradients across the plasma membranes of any of the cell types in the nervous system. Because PEPT2 is expected to function to a significant extent even in the absence of a transmembrane proton gradient, it is possible that it plays a role in the transport of NAAG. Whether such a putative function is involved in the uptake or release of NAAG will depend on relative transmembrane electrochemical gradients for NAAG and protons. A preliminary report by Nickolaus et al. (29) shows that PEPT2 expression in the brain is primarily restricted to neuronal cells in the hippocampal region and to epithelial cells of the choroid plexus. This new information is likely to fuel further research to identify the precise function of PEPT2 in the brain.

The most interesting finding with respect to the interaction of NAAG (net charge, -3) and the other three peptides (net charges, -1, +1, and 0) with PEPT2 is that the transport process induced inside-negative currents irrespective of the net charge of the peptide. The proton-to-peptide stoichiometry, however, appeared to be 1:1 in all cases. Similar findings have been reported recently by Amasheh et al. (3) for PEPT2 cloned from rabbit kidney. A simple transport model assigning the charge translocation during the transport cycle solely to the movement of the peptide substrate and proton does not therefore explain the observed findings. The transport protein itself is likely to be charged, and the interaction of differently charged peptide substrates might occur preferentially with differently charged states of the transport protein. The observations with respect to the electrophysiology of the PEPT2-mediated transport of differently charged peptides bear striking similarities to the observations made by three independent groups of investigators in the case of PEPT1-mediated transport of differently charged peptides (2, 24, 35). PEPT1 is known to contain essential histidyl residues (14, 36) that can exist either in a protonated form or in a nonprotonated form in the pH range 5.0-7.0, depending on the microenvironment of the involved histidyl residue. Similarly, PEPT2 also possesses essential histidyl residues that are obligatory for the transport function (14). Recently, Nussberger et al. (30) have proposed a model for the operational mechanism of PEPT1 with respect to the translocation of proton and peptide and for the association of the transport process with inward negative currents irrespective of the net charge of the translocated peptide substrate. This model implicates the involvement of differently charged amino acids in the substrate-binding pocket of the PEPT1 protein. A similar situation might exist in the case of PEPT2-mediated translocation of differently charged peptides.

    ACKNOWLEDGEMENTS

We thank Sarah A. Taylor and Ida O. Walker for expert secretarial assistance.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28389.

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. §1734 solely to indicate this fact.

Address reprint requests to F. H. Leibach.

Received 3 March 1998; accepted in final form 18 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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