Proton-coupled Peptide Transporter Research Articles

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

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

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

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.

Molecular Identification of a Role for Tyrosine 167 in the Function of the Human Intestinal Proton- Coupled Dipeptide Transporter (hPepT1)

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Substrate upregulation of the human small intestinal peptide transporter, hPepT1.

1. Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl-L-glutamine (Gly-Gln) of the proton-coupled peptide transporter, hPepT1, in the Caco-2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl-[14C]sarcosine was increased 1.64 (+/- 0.34)-fold after incubation of Caco-2 cells for 3 days in a peptide-rich medium (4 mM Gly-Gln replacing 4 mM Gln). 2. A full-length Caco-2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile-662 to Val. Transcript sizes, on Northern blots of Caco-2 poly(A)+ RNA probed with a 630 bp 5' hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide-induced increase in substrate transport was accompanied by a parallel increase of 1.92 (+/- 0.30)-fold (n = 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half-life from 8.9 +/- 1.1 to 12.5 +/- 1.6 h (n = 3), but the increase in half-life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide-mediated increase in hPepT1 transcription. 3. Anti-hPepT1-specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco-2 cells. Gly-Gln supplementation of media resulted in a 1.72 (+/- 0.26)-fold (n = 5) increase in staining intensity of Caco-2 cells. 4. We conclude that Caco-2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly-Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.

Expression of the mammalian renal peptide transporter PEPT2 in the yeast Pichia pastoris and applications of the yeast system for functional analysis.

It has recently been identified the PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2 represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di- and tripeptides and of selected peptidomimetics. Here the functional expression of PEPT2 in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter. Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85-87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide 3H-D-Phe-Ala with an app. K0.5 of 0.143 +/- 0.016 mM. Inhibition of 3H-D-Phe-Ala uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained in renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPT2 expressing cell clone in which transport function can be monitored by a simple yeast growth test was established. In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast. The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPT2 or for selection of mutants generated by random mutagenesis.

Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in xenopus laevis oocytes

1. To gain information on the mode of operation of the renal proton-coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2. Using differently charged glycyl-dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH-dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2-50 microM) between pH 5.5 and 7.4 and membrane potentials of +/- 0 to -50 mV. 3. Maximal substrate-evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pHo). Potential-dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4. The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from -50 to -150 mV substrate affinities decrease 10- to 50-fold whereas apparent Imax increases almost 10-fold. 5. Even at saturating H+/H3O+ and dipeptide concentrations the I-V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6. These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.

Cloning and Functional Expression of a Brain Peptide/Histidine Transporter

Here we report the cloning and functional characterization of a rat novel peptide/histidine transporter (PHT1), which was expressed in the brain and the retina. The cDNA encodes the predicted protein of 572 amino acid residues with 12 putative membrane-spanning domains. The amino acid sequence has moderate homology with a nonspecific peptide transporter found in the plant. When expressed in Xenopus laevis oocytes, PHT1 cRNA induced high affinity proton-dependent histidine transport activity. This transport process was inhibited by dipeptides and tripeptides but not by free amino acids such as glutamate, glycine, leucine, methionine, and aspartate. Dipeptide carnosine transport activity was also confirmed by direct uptake measurement. By in situ hybridization analysis, PHT1 mRNA was widely distributed throughout whole brain. Especially, intense hybridization signals were found in the hippocampus, choroid plexus, cerebellum, and pontine nucleus. Signals were located in both the neuronal and small nonneuronal cells in these areas. PHT1 protein could contribute to uptake of oligopeptides, which function as neuromodulators, and clearance of degraded neuropeptides and be a new member in the growing superfamily of proton-coupled peptide and nitrate transporters, although its structure, localization, and pharmacological characteristics are unique among these members.

Peptide transporters.

Proton-coupled peptide transporters found in animal cells are unique because of their energetics and their ability to accept a broad spectrum of chemically diverse peptides as substrates. The abundant expression of these transporters in the small intestine and the kidney indicates their importance in the maintenance of protein nutrition. There is also evidence for additional functions of the peptide transporters which are of physiological, pharmacological and clinical relevance.

Basolateral dipeptide transport by the intestine of the teleost Oreochromis mossambicus.

Transport characteristics of [14C]glycylsarcosine ([14C]Gly-Sar) were measured in herbivorous tilapi (Oreochromis mossambicus) intestinal basolateral membrane vesicles (BLMV) purified with Percoll gradient centrifugation. Specific activity of the vesicle Na(+)-K(+)-adenosinetriphos- phatase was increased 12-fold, whereas specific activity of the brush-border enzyme alkaline phosphatase was enriched only by 0.8-fold. [14C]Gly-Sar uptake was stimulated by increasing concentrations of extravesicular protons rather than by a transmembrane proton gradient. A transmembrane K+ diffusion potential (inside negative) did not stimulate [14C]Gly-Sar uptake above that observed with short-circuited vesicles. An inwardly directed Na+ gradient had no effect on peptide uptake. Kinetic analysis of basolateral transport rate revealed that the transport occurred by a saturable process conforming to Michaelis-Menten kinetics [Kt [concentration of [14C]Gly-Sar that yielded one-half of maximal influx (Jmax)] = 13.27 +/- 3.80 mM, Jmax = 15,155 +/- 3,096 pmol.mg protein-1.6 s-1]. The basolateral transporter was insensitive to diethylpyrocarbonate (DEP), a specific inhibitor of proton-coupled peptide transport systems. [14C]Gly-Sar influx into tilapia BLMV showed cis-inhibition by several other dipeptides, suggesting that the [14C]Gly-Sar transporter was shared by other peptides too. These observations strongly suggest that the basolateral intestinal dipeptide transporter in herbivorous fishes is distinctly different from either the high- or low-affinity brush-border transporter. It is proton dependent, electroneutral, sodium independent and accepts a wide variety of dipeptides.

Electrogenic, proton-coupled, intestinal dipeptide transport in herbivorous and carnivorous teleosts

In both herbivorous tilapia (Oreochromis mossambicus) and carnivorous rockfish (Sebastes caurinus) intestinal and pyloric cecal brush-border membrane vesicles (BBMV), [14C]glycylsarcosine ([14C]Gly-Sar) uptake was stimulated by a transmembrane proton gradient. A transmembrane K(+)-diffusion potential (inside negative) stimulated [14C]Gly-Sar uptake above that observed with short-circuited vesicles, whereas an inwardly directed Na+ gradient in both fishes had no effect on peptide uptake. In tilapia, [14C]Gly-Sar influx occurred by the combination of 1) a high-affinity, saturable, proton gradient-dependent carrier system [Kt [concentration that equals one-half of maximum influx (Jmax)] = 0.56 +/- 0.08 mM; Jmax = 1,945.0 +/- 174.6 pmol.mg protein-1.10 s-1]; 2) a low-affinity, nonsaturable (within 1-10 mM), proton gradient-dependent carrier system (nonsaturable carrier-mediated transport component = 4,514.0 +/- 28.1 pmol.mg protein-1.10 s-1.mM-1); and 3) a diffusional component accounting for < 10% of total influx within the concentration range tested. Influx (10 s) of 1-10 mM [14C]Gly-Sar in tilapia intestine was significantly (P < 0.01) inhibited by 10 mM diethylpyrocarbonate, a specific inhibitor of proton-coupled peptide transport systems. [14C]Gly-Sar influx into tilapia BBMV showed cis-inhibition and trans-stimulation by Gly-Pro, suggesting that [14C]Gly-Sar and Gly-Pro shared the same mucosal peptide transporter in fish. These observations strongly suggest that intestinal transport of peptides in herbivorous and carnivorous fishes is proton gradient dependent, electrogenic, sodium independent, and qualitatively resembles the peptide transport paradigm proposed for mammals.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Dipeptide transport characteristics of the apical membrane of rat lung type II pneumocytes

The transport of a hydrolysis-resistant dipeptide, D-phenylalanyl-L-alanine (D-Phe-L-Ala), has been studied by high-performance liquid chromatography in rat lung epithelial cells and apical membrane vesicles. Time-dependent uptake of D-Phe-L-Ala into isolated type II pneumocytes was shown. Uptake was saturable, and Michaelis-Menten kinetics were fitted to the data and gave an apparent Michaelis constant (Km) of 3.4 mM and a maximum velocity (Vmax) of 7.0 nmol.mg protein-1.min-1. However, known peptide transport inhibitors unexpectedly increased intracellular D-Phe-L-Ala concentration when initial rates of peptide uptake were studied. Apical (brush-border) membrane vesicles prepared from rat lung also showed time- and concentration-dependent influx of D-Phe-L-Ala (apparent Km 2.0 mM, Vmax 0.53 nmol.mg protein-1.min-1). Influx of this neutral dipeptide into the vesicles was shown to be both electrogenic and stimulated by an inwardly directed proton gradient. Influx was inhibitable by mercuric chloride and by the amino acid residue modifying compounds N-acetylimidazole and diethylpyrocarbonate. These findings strongly suggest the presence of a proton-coupled peptide transport protein in the apical surface of the type II cell. This transporter may play a role in lung homeostasis.

Functional expression of intestinal dipeptide/β-lactam antibiotic transporter in Xenopus laevis oocytes

An intestinal active transport system specific to small peptides and peptide-like drugs such as β-lactam antiobiotics was functionally expressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal cells. The transport activity was evaluated by measuring the uptake of a tripeptide-like cephalosporin antibiotic, ceftibuten, which has high affinity for the intestinal peptide/H + co-transporter and is resittant to peptidases. Ceftibuten transport in mRNA-injected oocytes was pH dependent (a proton gradient is the driving force), stereo selective (uptake of the cis-isomer of ceftibuten was about 4-fold higher than that of the trans-isomer), saturable and temperature dependent. Furthermore, various dipeptides showed cis-inhibitory and trans-stimulatory effects on the uptake of ceftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipeptides are transported by a common carrier protein. These results are in accordance with the functional properties of native proton-coupled peptide transporter previously clarified by studies with isolated intestinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the membrane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive β-lactam antibiotic, [ 3h]benxylpenicillin, followed by SDS-PAGE analysis of the radiolabeled protein. Furthermore, an experiment with mRNA size-fractionated by sucrose density gradient centrifugation indicated that the peptide transporter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtained using a heterologous gene expression technique, confirm that intestinal absorption of β-lactam antibiotics occurs through a carrier-mediated mechanism and show that biologically stable β-lactam antibiotics can be useful probes for molecular analysis of intestinal peptide transporter.