Minimal Molecular Determinants of Substrates for Recognition by the Intestinal Peptide Transporter
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23211
ISSN1083-351X
AutoresFrank Döring, Jutta Will, Salah Amasheh, Wolfgang Clauß, H. AHLBRECHT, Hannelore Daniel,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoProton-dependent electrogenic transporters for di- and tripeptides have been identified in bacteria, fungi, plants, and mammalian cells. They all show sequence-independent transport of all possible di- and tripeptides as well as of a variety of peptidomimetics. We used the mammalian intestinal peptide transporter PEPT1 as a model to define the molecular basis for its multisubstrate specificity. By employing computational analysis of possible substrate conformations in combination with transport assays using transgenic yeast cells and Xenopus laevis oocytes expressing PEPT1, the minimal structural requirements for substrate binding and transport were determined. Based on a series of medium chain fatty acids bearing an amino group as a head group (ω-amino fatty acids, ω-AFA), we show that electrogenic transport by PEPT1 requires as a minimum the two ionized head groups separated by at least four methylene groups. Consequently, a > 500 pm < 630 pm distance between the two charged centers (carboxylic carbon and amino nitrogen) is sufficient for substrate recognition and transport. Removal of either the amino group or the carboxyl group in ω-AFA maintained the affinity of the compound for interaction with the transporter but abolished the capability for electrogenic transport. Additional groups in the ω-AFA backbone that provide more hydrogen bonding sites appear to increase substrate affinity but are not essential. The information provided here does (a) explain the capability of the peptide carrier for sequence-independent transport of thousands of different substrates and (b) set the molecular basis for a rational drug design to increase the absorption of peptide-based drugs mediated by PEPT1. Proton-dependent electrogenic transporters for di- and tripeptides have been identified in bacteria, fungi, plants, and mammalian cells. They all show sequence-independent transport of all possible di- and tripeptides as well as of a variety of peptidomimetics. We used the mammalian intestinal peptide transporter PEPT1 as a model to define the molecular basis for its multisubstrate specificity. By employing computational analysis of possible substrate conformations in combination with transport assays using transgenic yeast cells and Xenopus laevis oocytes expressing PEPT1, the minimal structural requirements for substrate binding and transport were determined. Based on a series of medium chain fatty acids bearing an amino group as a head group (ω-amino fatty acids, ω-AFA), we show that electrogenic transport by PEPT1 requires as a minimum the two ionized head groups separated by at least four methylene groups. Consequently, a > 500 pm < 630 pm distance between the two charged centers (carboxylic carbon and amino nitrogen) is sufficient for substrate recognition and transport. Removal of either the amino group or the carboxyl group in ω-AFA maintained the affinity of the compound for interaction with the transporter but abolished the capability for electrogenic transport. Additional groups in the ω-AFA backbone that provide more hydrogen bonding sites appear to increase substrate affinity but are not essential. The information provided here does (a) explain the capability of the peptide carrier for sequence-independent transport of thousands of different substrates and (b) set the molecular basis for a rational drug design to increase the absorption of peptide-based drugs mediated by PEPT1. rabbit intestinal peptide transporter human intestinal sodium glucose transporter peptide transporters ω-amino fatty acids 4-amino-butanoic acid 5-amino-pentanoic acid 6-amino-hexanoic acid 7-amino-heptanoic acid 8-amino-octanoic acid 11-amino-undecanoic acid 2-amino-octanoic acid γ-amino-butanoic acid N-α-benzyloxycarbonyl-lysine, N-α-Z-arginine, N-α-benzyloxycarbonyl-arginine potassium phosphate buffer 4-morpholineethanesulfonic acid. Dipeptides, tripeptides, as well as a number of peptide-like drugs are rapidly taken up into intestinal epithelial cells by a specific apical peptide transporter encoded by the PEPT1 gene. The cDNAs of intestinal transporters of different species have been cloned from cDNA libraries (1Fei Y.-J. Kanai Y. Nussberger S. Ganapathy V. Leibach F.H. Romero M.F. Singh S.K. Boron W.F. Hediger M.A. Nature. 1994; 368: 563-566Crossref PubMed Scopus (776) Google Scholar, 2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar, 3Liang R. Fei Y.-J. Prasad D. Ramamoorthy S. Han H. Yang-Feng T.L. Hediger M.A. Ganapathy V. Leibach F.H. J. Biol. Chem. 1995; 270: 6456-6463Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 4Saito H. Okuda M. Terada T. Sasaki S. Inui K.I. J. Pharmacol. Exp. Ther. 1995; 275: 1631-1637PubMed Google Scholar), and the proteins have been characterized with respect to their operational mode by expression in Xenopus oocytes, HeLa cells, and more recently in the methylotrophic yeast Pichia pastoris (5Döring F. Theis S. Daniel H. Biochem. Biophys. Res. Commun. 1997; 232: 656-662Crossref PubMed Scopus (35) Google Scholar). At the amino acid level the PEPT11 proteins show significant homologies to peptide transporters isolated from bacteria, fungi, and plants that all belong to the PTR family of proton-dependent peptide transporters (6Steiner H.-Y. Naider F. Becker J.M. Mol. Microbiol. 1995; 16: 825-834Crossref PubMed Scopus (205) Google Scholar). The gene products are plasma membrane carrier proteins that catalyze electrogenic uphill peptide transport by coupling of substrate translocation to the movement of H3O+ with the transmembrane electrochemical proton gradient providing the driving force (7Daniel H. J. Membr. Biol. 1996; 154: 197-203Crossref PubMed Scopus (81) Google Scholar). The physiological role of PEPT1 lies in the absorption of peptide bound amino acids from the intestinal tract after their release by enzymatic breakdown of dietary or endogenous proteins. In addition, the high availability of orally active peptide-based drugs such as almost all aminocephalosporin antibiotics, ACE-inhibitors like captopril, or peptidase inhibitors like bestatin results from their active transport mediated by PEPT1 (2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar, 7Daniel H. J. Membr. Biol. 1996; 154: 197-203Crossref PubMed Scopus (81) Google Scholar). Preliminary studies (1Fei Y.-J. Kanai Y. Nussberger S. Ganapathy V. Leibach F.H. Romero M.F. Singh S.K. Boron W.F. Hediger M.A. Nature. 1994; 368: 563-566Crossref PubMed Scopus (776) Google Scholar, 2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar, 8Amasheh S. Wenzel U. Boll M. Dorn D. Weber W.-M. Clauss W. Daniel H. J. Membr. Biol. 1997; 155: 247-256Crossref PubMed Scopus (72) Google Scholar, 9Wenzel U. Gebert I. Weintraut H. Webe W.-M. Clauss W. Daniel H. J. Pharmacol. Exp. Ther. 1996; 277: 831-839PubMed Google Scholar, 10Wenzel U. Thwaites D.T. Daniel H. Br. J. Pharmacol. 1995; 116: 3021-3027Crossref PubMed Scopus (76) Google Scholar) on the substrate specificity of PEPT1 indicated that this transporter, like its nonmammalian counterparts, transports almost all possible dipeptides, tripeptides, and numerous peptidomimetics. Free amino acids and tetrapeptides appear not to be accepted as substrates. Although transport of peptides and peptidomimetics occurs in a stereospecific manner (10Wenzel U. Thwaites D.T. Daniel H. Br. J. Pharmacol. 1995; 116: 3021-3027Crossref PubMed Scopus (76) Google Scholar), the transporter discriminates possible substrates only by differences in affinity for binding and/or maximal transport capacity. Based on the naturally occurring amino acids provided either as l- ord-amino enantiomers and by the huge number of different peptide-like xenobiotics, almost 1 × 106 potential substrates can be identified. Considering the wide distribution of this novel class of solute transporters throughout nature and their nutritional as well as pharmacological importance, identification of the minimal structural determinants of substrates affecting their affinity and capability for transport would greatly advance our understanding of these carrier proteins. As a starting point, we employed the transgenic yeast system to screen a large variety of different compounds to identify a common structural motif that determines the affinity for the binding site of PEPT1. From these studies, it became obvious that the existence of a zwitterionic amino acid functionality separated by a distinct molecular distance was the most important feature. We therefore rationalized that PEPT1 should be able to interact with ω-amino fatty acids (ω-AFA) and consequently studied binding and transport of a series of ω-AFA. Because the amino fatty acids represent simple structures, a modeling of their possible conformations by semiempirical and ab initio methods and taking unspecific solvation by water into account could be performed. The capability of PEPT1 for transport of ω-AFA was demonstrated in two different expression systems by using a radiolabeled ω-AFA as well as by measuring substrate-induced inward currents in voltage clamped Xenopus oocytes expressing the carrier. Employing the ω-AFA in combination with the modeling of these novel substrates that possess only two functional groups and a hydrocarbon backbone, we have set the basis for defining the minimal molecular requirements for substrate transport by PEPT1. ω-AFA and other chemicals were obtained from Sigma (Deisenhofen, Germany). Custom-synthesized3H-(d)-phenylalanine-(l)-alanine (3H-d-Phe-Ala) with a specific activity of 9 Ci·mmol−1 was purchased from Zeneca (Billingham, UK).14C-6-amino-hexanoic acid (14C-6-AHA), with a specific activity of 55 mCi·mmol−1, was obtained from BioTrend (Köln, Germany). 3-o-[methyl-3H]-d-glucose (3H-methyl-d-glucose) with a specific activity of 86.7 Ci·mmol−1 was obtained from DuPont (Dreieich, Germany). The P. pastoris strain was cultured as described in the manual Version E of the Pichia expression kit (Invitrogen, San Diego). Construction of the Pichia strain expressing the rabbit PEPT1 and the isogenic control strain (GS-pPIC3L) has been described previously (5Döring F. Theis S. Daniel H. Biochem. Biophys. Res. Commun. 1997; 232: 656-662Crossref PubMed Scopus (35) Google Scholar). In the transgenic yeast strain GS-PEPT1, the PEPT1-cDNA was placed under the transcriptional control of the AOX1 promotor, which is inducible by methanol. To induce PEPT1 expression, cells were initially grown in a glycerol-containing medium (1.34% yeast nitrogen base, 4 × 10−5% biotin and 1% glycerol) and then switched to a medium containing 1% methanol. Uptake of 3H-Phe-Ala or 14C-6-AHA was measured 18–36 h after induction. Cells were pelleted at 3000 × g for 10 min, washed once with 50 mmpotassium phosphate buffer (PPB, pH 7.0), and resuspended to 1 OD (one OD equals approximately 5 × 107 cells)·10 μl−1 in 50 mm PPB. Uptake measurements were performed at 22–24 °C by using a rapid filtration technique with Schleicher & Schuell filters (ME 25 type, 0.45 μm pore size) as described previously (5Döring F. Theis S. Daniel H. Biochem. Biophys. Res. Commun. 1997; 232: 656-662Crossref PubMed Scopus (35) Google Scholar). Uptake was initiated by rapidly mixing 10 μl of cell suspension with 40 μl of 50 mm PPB containing 0.1 μCi of 14C-6-AHA or3H-d-Phe-Ala. Incubation of cells was terminated by addition of 3 ml of ice-cold 50 mm PPB, followed by filtration. The filter was washed twice with 3 ml of ice-cold buffer, and radioactivity associated with the filter was determined. Kinetics of 14C-6-AHA influx at pH 7.0 was determined at 30 min of incubation in the presence of increasing concentration of substrate (0.001–10 mm), and corresponding uptake rates in control cells were subtracted. Competition studies with 3H-d-Phe-Ala or14C-6-AHA serving as substrates and selected test compounds serving as competitors (0.001–50 mm) were performed at pH 7.0. Surgically removed oocytes were separated by collagenase treatment and handled as described previously (2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar). Individual oocytes were injected with 13.6 nl of water (controls) or 13.6 nl of RNA-solution containing 13.6 ng of the complementary RNAs of PEPT1 or SGLT1 followed by incubation in Barth′s solution for 5 days at 14 °C. Flux studies with oocytes expressing PEPT1 were performed in a buffer composed of 100 mm NaCl, 3 mm KCl, 1 mm CaCl2, 1 mm MgCl2and 15 mm MES/Tris at pH 6.0. Influx of3H-methyl-glucose in oocytes expressing SGLT1 were performed in a buffer composed of 100 mm NaCl, 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 15 mm Hepes/Tris at pH 7.5. The 2-electrode voltage clamp technique was applied to characterize responses in inward current (I) to substrate addition in oocytes injected with 13.6 ng of transporter cRNA of PEPT1. Steady state currents were measured in the absence and the presence of increasing concentrations of the test compounds at pH 6.0, whereas the membrane potential in oocytes was clamped to −60 mV. Current-voltage relationships were recorded as described previously (2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar). The semiempirical calculations were performed using the AM1-SM2 solvation model (11Cramer C.J. Truhlar D.G. J. Am. Chem. Soc. 1991; 113: 8305-8311Crossref Scopus (475) Google Scholar, 12Cramer C.J. Truhlar D.G. Science. 1992; 256: 213-215Crossref PubMed Scopus (476) Google Scholar) as implemented into the SPARTAN program package (Wavefunction, Irvine, CA). Theab initio optimizations were performed on the RHF/6–31+G* level, starting from gas phase geometries as obtained with the AM1 method. The energy of solvation by water was modeled by using the polarizable continuum model (13Miertus S. Scrocco E. Tomasi J. Chem. Phys. 1981; 55: 117-119Crossref Scopus (8182) Google Scholar) implemented as the SCIPCM method into the GAUSSIAN 94 program package (14Frisch, M. J., Trucks, G. W., Schlegel, H. B., Gill, P. M. W., Johnson, B. G., Robb, M. A., Cheeseman, J. R., Keith, T., Petersson, G. A., Montgomery, J. A., Raghavachari, K., Al-Laham, M. A., Zakrzewski, V. G., Ortiz, J. V., Foresman, J. B., Cioslowski, J., Stefanov, B. B., Nanayakkara, A., Challacombe, M., Peng, C. Y., Ayala, P. Y., Chen, W., Wong, M. W., Andres, J. L., Replogle, E. S., Gomperts, R., Martin, R. L., Fox, D. J., Binkley, J. S., Defrees, D. J., Baker, J., Stewart, J. P., Head-Gordon, M., Gonzalez, C., Pople, J. A. (1995) GAUSSIAN 94, Revision B (1), Gaussian, Inc., Pittsburgh, PAGoogle Scholar). All computations were performed with an IBM RS/6000 cluster of the computing center of the Justus-Liebig-University of Giessen. All calculations (linear as well as nonlinear regression analysis) were performed by using PRISM (GraphPAD, Los Angeles, CA). For each variable, at least two independent experiments (8–10 oocytes, 2 yeast preparations) were carried out. Data are given as the mean ± S.E. Significance of differences between the uptake rates and constants calculated were determined by a nonpaired t test. Based on initial observations during screening of potential substrates, we hypothesized that amino fatty acids bearing an amino group as a head group (ω-amino fatty acids, ω-AFA) could serve as substrates of PEPT1. By using a series of ω-AFA possessing 3–10 CH2 units, we demonstrate here that these compounds indeed interact with the substrate binding site of the transporter. InP. pastoris, cells expressing PEPT1 uptake of3H-d-Phe-Ala was dose dependently inhibited by the ω-AFA when the compounds contained four or more CH2units in the backbone (Fig. 1 a). In the case of 4-ABA (or GABA), there was no noticeable affinity. The EC50 values derived from competition curves with3H-d-Phe-Ala serving as a substrate ranged for the different ω-AFA between 0.30 ± 0.04 mm and 1.14 ± 0.06 mm (Table I). The apparent affinities for the ω-AFA found are very similar to those obtained for di- and tripeptides when assayed under identical experimental conditions (5Döring F. Theis S. Daniel H. Biochem. Biophys. Res. Commun. 1997; 232: 656-662Crossref PubMed Scopus (35) Google Scholar).Table IAffinity of selected compounds for inhibition of H-d-Phe-Ala influx into P. pastoris cells expressing PEPT1CompoundEC50 valuemmω-Amino fatty acids 4-ABA (NH3-(CH2)3-COO)>50 5-APA (NH3-(CH2)4-COO)1.14 ± 0.06 6-AHA (NH3-(CH2)5-COO)0.73 ± 0.07 7-AHA (NH3-(CH2)6-COO)1.09 ± 0.03 8-AOA (NH3-(CH2)7-COO)0.30 ± 0.04 11-AUA (NH3-(CH2)10-COO)0.58 ± 0.03Amines and fatty acids Hexylamine (NH3-(CH2)5-CH3)6.19 ± 0.08 Octylamine (NH3-(CH2)7-CH3)0.37 ± 0.02 Hexanoic acid (CH3-(CH2)5-COO)2.75 ± 0.07 Octanoic acid (CH3-(CH2)7-COO)1.36 ± 0.07Other compounds 5-Amino-4-oxo-pentanoic acid0.27 ± 0.03 2-Amino-octanoic acid>50 Lysine (pH 7.0)4.89 ± 0.22 Lysine (pH 8.0)2.07 ± 0.05 Arginine (pH 7.0)14.98 ± 0.10 Arginine (pH 8.0)8.14 ± 0.08Uptake of 3H-d-Phe-Ala (2 μCi · ml−1) into the Pichia cells expressing PEPT1 (1 OD) was measured for 30 min of incubation at pH 7.0 or at the pH indicated in the presence of increasing concentrations (0.001–50 mm) of competitors. Uptake rates of 3H-d-Phe-Ala into control cells (GS-pPIC3L) were subtracted. The control value for uptake of 3H-d-Phe-Ala was 34.09 ± 2.89 pmol · 1 OD−1 · 30 min−1. EC50 values represent the mean ± S.E. μ of two independent experiments performed in triplicate. EC50 values were derived by the least-squares method based on a competition curve with one component and r 2 for regression was in all cases ≥ 0.98. Open table in a new tab Uptake of 3H-d-Phe-Ala (2 μCi · ml−1) into the Pichia cells expressing PEPT1 (1 OD) was measured for 30 min of incubation at pH 7.0 or at the pH indicated in the presence of increasing concentrations (0.001–50 mm) of competitors. Uptake rates of 3H-d-Phe-Ala into control cells (GS-pPIC3L) were subtracted. The control value for uptake of 3H-d-Phe-Ala was 34.09 ± 2.89 pmol · 1 OD−1 · 30 min−1. EC50 values represent the mean ± S.E. μ of two independent experiments performed in triplicate. EC50 values were derived by the least-squares method based on a competition curve with one component and r 2 for regression was in all cases ≥ 0.98. Because competition with a dipeptide for binding at the substrate binding site does not establish that a compound is indeed transported, we employed 14C-6-AHA as a representative structure to assess its transport characteristics in Pichia cells mediated by PEPT1. As shown in Fig. 1 b,14C-6-AHA was taken up into the transgenic yeast cells but not into the control cells. Uptake of 14C-6-AHA as a function of substrate concentration displayed saturation kinetics mediated by a single transport site (Fig. 1 b, inset). The apparent K 0.5 value for 6-AHA influx was 0.92 ± 0.07 mm, and the EC50 value for d-Phe-Ala serving as the competitor was 0.84 ± 0.12 mm. To demonstrate that the transport of ω-AFA by PEPT1 is independent from the expression system used, we additionally employed oocytes expressing PEPT1. As shown in Fig. 1 c, the EC50 values for inhibition of influx of 3H-d-Phe-Ala into oocytes by 6-AHA (0.98 ± 0.05 mm) and 8-AOA (1.27 ± 0.08 mm) were similar to those obtained in the yeast expression system. 14C-6-AHA influx into oocytes expressing PEPT1 as a function of substrate concentration also displayed saturation kinetics (Fig. 1 d) with an apparent K 0.5 value of 1.21 ± 0.32 mm. Based on these observations obtained in two different heterologous expression systems, we conclude that ω-AFAs with more than 3 CH2 units in the backbone are recognized by the substrate binding site of PEPT1 and interact with affinities that are comparable with those of dipeptides. To investigate whether the different ω-AFA are transported electrogenically by PEPT1, substrate-induced inward currents were measured using the 2-electrode voltage clamp setup. When a PEPT1-expressing oocyte in the voltage clamp mode was perfused with 2.5 mm ω-AFA, positive inward currents were obtained for all substrates except for 4-ABA (Fig. 2 a). Whereas currents induced by some of the ω-AFA were not as high as that induced by 2.5 mm Gly-Gln in the same oocyte, 8-AOA elicited a current of almost identical value. Moreover, when 6-AHA- and 8-AOA-mediated currents were determined as a function of membrane potential, we obtained I-V curves that are characteristic for PEPT1 and almost identical to those recorded for Gly-Gln (Fig. 2 b). This strongly suggests that the influx of ω-AFA is, like that of dipeptides, electrogenic in nature as a consequence of cotranslocation of H3O+ along the transmembrane potential providing the driving force. The unexpected finding that simple aliphatic chains possessing an amino and carboxyl group serve as substrates addresses the question of whether both head groups are required for transport. We therefore investigated if compounds with aliphatic chains of ≥ 5 CH2 units carrying only one terminal group, either an amino group (amines) or a carboxyl group (fatty acids), were able to interact with PEPT1. As demonstrated by competition studies, both the fatty acids and the amines inhibited 3H-d-Phe-Ala influx into PEPT1-expressing P. pastoris cells in a dose-dependent manner. The EC50 values obtained (Table I) ranged between 0.37 ± 0.02 mm (octylamine) and 6.19 ± 0.08 mm (hexylamine). In the case of the fatty acids, EC50 values of 1.36 ± 0.07 and 2.75 ± 0.07 mm were obtained. The C8 compounds therefore displayed a 2–15-fold higher affinity than the corresponding C6 compounds. To assess whether the amines and fatty acids also elicited inward currents in oocytes expressing PEPT1, cells were superperfused with 2.5 mm octylamine or octanoic acid in the voltage clamp mode. In the same oocyte in which perfusion with 2.5 mm Gly-Gln induced significant inward currents (170 nA at −60 mV), the amines and the fatty acids failed to induce measurable inward currents (Fig. 2 a). This suggested that the compounds can interact with PEPT1 but cannot be transported electrogenically. To demonstrate that the amines and fatty acids inhibited PEPT1 specifically and inhibition is not caused via nonspecific interaction with the cell membrane, we used the sodium-dependent glucose transporter SGLT1 as a control. Functional expression of SGLT1 in oocytes was established by a 50-fold increase in uptake of 3H-methyl-d-glucose in cRNA-injected oocytes compared with water-injected control cells. As shown in Table II, influx of3H-methyl-d-glucose was reduced by 88.0 ± 3.1% in the presence of unlabeled glucose and almost abolished by the specific inhibitor phloridzin. However, neither high concentrations (5 mm) of octylamine nor octanoic acid inhibited SGLT1-mediated 3H-methyl-d-glucose influx into oocytes. Taken together, the medium chain amines and fatty acids obviously interact specifically with PEPT1 but do not show electrogenic transport.Table IIUptake of H-methyl-d-glucose into Xenopus oocytes expressing SGLT1 in the presence of selected compoundsCompoundUptake% of controlNone100.0 ± 7.75 mm d-Glucose12.0 ± 3.10.5 mmPhloridzin5.5 ± 1.28-AOA115.2 ± 10.3Octylamine95.0 ± 4.3Octanoic acid109.1 ± 21.5Uptake of 3H-methyl-d-glucose (10 μCi · ml−1) into oocytes expressing SGLT1 was measured for 30 min of incubation in the absence or the presence of 0.5 mmphloridzin or 5 mm other compounds. Data are presented as the mean ± S.E. μ for the residual uptake rates compared with control uptake (3.31 ± 0.23 pmol · oocyte−1· 30 min−1) in the absence of inhibitors (percent of control). Uptake rates of 3H-methyl-d-glucose into water-injected oocytes (0.06 ± 0.02 pmol · oocyte−1 · 30 min−1) were subtracted. Data are given as the mean ± S.E. μ for 8–10 oocytes per condition and are representative for two independent experiments in triplicate.Significantly different when compared with the control (non-paired t-test). Open table in a new tab Uptake of 3H-methyl-d-glucose (10 μCi · ml−1) into oocytes expressing SGLT1 was measured for 30 min of incubation in the absence or the presence of 0.5 mmphloridzin or 5 mm other compounds. Data are presented as the mean ± S.E. μ for the residual uptake rates compared with control uptake (3.31 ± 0.23 pmol · oocyte−1· 30 min−1) in the absence of inhibitors (percent of control). Uptake rates of 3H-methyl-d-glucose into water-injected oocytes (0.06 ± 0.02 pmol · oocyte−1 · 30 min−1) were subtracted. Data are given as the mean ± S.E. μ for 8–10 oocytes per condition and are representative for two independent experiments in triplicate. Significantly different when compared with the control (non-paired t-test). That amino acids are not transported by the epithelial peptide transporters has been established in numerous studies employing radiolabeled amino acids as well as by using electrophysiological methods (1Fei Y.-J. Kanai Y. Nussberger S. Ganapathy V. Leibach F.H. Romero M.F. Singh S.K. Boron W.F. Hediger M.A. Nature. 1994; 368: 563-566Crossref PubMed Scopus (776) Google Scholar, 2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar, 8Amasheh S. Wenzel U. Boll M. Dorn D. Weber W.-M. Clauss W. Daniel H. J. Membr. Biol. 1997; 155: 247-256Crossref PubMed Scopus (72) Google Scholar). Based on our observations with the ω-AFA, one would assume that at least the dibasic amino acids lysine and arginine interact with PEPT1. Both possess the right backbone length (≥ 4 CH2 groups) and the head groups to mimic the ω-AFA,i.e. an amino group in the side chain and the carboxyl group bound to the α-carbon. However, because both amino acids carry an additional α-amino group close to the carboxyl group, one could rationalize that this specific configuration prevents lysine and arginine from interaction with PEPT1. To test this hypothesis, we used lysine and arginine derivatives with protected α-amino groups (N-α-Z-l-lysine, N-α-Z-l-arginine) as inhibitors of 3H-d-Phe-Ala influx into the transgenic Pichia cells. At a concentration of 5 mm, both amino acid derivatives significantly (p < 0.001) reduced3H-d-Phe-Ala uptake by 69.7 ± 5.8% (N-α-Z-l-lysine) and 48.6 ± 5.5% (N-α-Z-l-arginine). The free amino acids reduced dipeptide influx at 5 mm by 48.1 ± 3.5% (lysine) and by only 11.8 ± 4.4% (arginine). In addition to the use of derivatized amino acids, we compared lysine and arginine interaction with PEPT1 at pH 7.0 and 8.0. Because of different pK a values of the amino groups, the α-amino group becomes significantly more deprotonated at pH 8.0 than the amino group in the side chain. As shown in Table I, increasing pH from 7.0 to 8.0 decreased the EC50 value for lysine inhibition of dipeptide influx from 4.89 ± 0.22 to 2.07 ± 0.05 mm and the EC50 value for arginine from 14.98 ± 0.10 to 8.14 ± 0.08 mm. In comparison, the affinity of zwitterionic substrates remains generally unaffected by these changes in pH as shown previously (2Boll M. Markovich D. Weber W.-M. Korte H. Daniel H. Murer H. Pfluegers Arch. Eur. J. Physiol. 1994; 429: 146-149Crossref PubMed Scopus (198) Google Scholar, 8Amasheh S. Wenzel U. Boll M. Dorn D. Weber W.-M. Clauss W. Daniel H. J. Membr. Biol. 1997; 155: 247-256Crossref PubMed Scopus (72) Google Scholar). This suggested that the positive charge of the α-amino group may indeed impair the affinity of the amino acids for interaction with PEPT1. To mimic the configuration of the amino and carboxyl group at the α-carbon as given in amino acids, we used 2-amino-octanoic acid (2-AOA) and determined its affinity for PEPT1 in comparison to 8-AOA (Table I). When the amino group was moved from the 8 to the 2 position, the compound failed to cause any inhibition of dipeptide influx (EC50 > 50 mm), whereas 8-AOA displayed a high affinity for inhibition with an EC50 value of 0.30 ± 0.04 mm. The results of this series of experiments strongly supports the proposal that charged amino and carboxyl groups in close proximity prevent the ligands from interacting with the transporter binding site, whereas when separated by the proper spacer (≥ 4 CH2-groups), substrates can bind with a high affinity and get transported. As shown here, PEPT1 sharply discriminates in binding and transport between 4-ABA and 5-APA. We therefore decided to employ computational model analysis to predict the compounds possible conformations based on energy minimization calculations. The results are compiled in Table III. For our model calculations, glycine served as a paradigm. As expected and documented for this amino acid both experimentally (16Suenram R.D. Lovas F.J. J. Mol. Spectrosc. 1978; 72: 372-382Crossref Scopus (271) Google Scholar) and theoretically (17Gordon M.S. Jensen J.H. Acc. Chem. Res. 1996; 29: 536-543Crossref Scopus (201) Google Scholar), in the gas phase, the neutral form is the most stable one. At the highest level of theory, the zwitterionic form of glycine is calculated to be less stable by 16–20 kcal/mol and proton transfer from the amino group to the carboxyl group to yield the neutral form occurs without a barrier. Even when glycine is complexed with two water molecules, the zwitterion is calculated to be less stable but now proton transfer is hindered (18Jensen H.J. Gordon M.S. J. Am. Chem. Soc. 1995; 117: 8159-8170Crossref Scopus (439) Google Scholar). Only in the presence of a stronger basic group such as in arginine may these stabilities be reversed (19Price W.D. Jokusch R.A. Williams E.-R. J. Am
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