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. 2002 Dec 13;546(Pt 2):349–361. doi: 10.1113/jphysiol.2002.026500

Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2

Zhong Chen *, You-Jun Fei *, Catriona M H Anderson †, Katherine A Wake †, Seiji Miyauchi *, Wei Huang *, David T Thwaites †, Vadivel Ganapathy *
PMCID: PMC2342508  PMID: 12527723

Abstract

The human orthologue of the H+-coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco-2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of α-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na+-independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, l-alanine, l-proline, α-aminoisobutyrate (AIB) and γ-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the d-isomers of alanine and proline. With serine and cysteine, though the l-isomers did not interact with hPAT1 to any significant extent, the corresponding d-isomers were recognized as substrates. With proline and alanine, the affinity was similar for l- and d-isomers. However, with cysteine and serine, the d-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding l-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+-coupled amino acid transport system in Caco-2 cells. Furthermore, there was a high degree of correlation (r2 = 0.93) between the relative potencies of various amino acids to inhibit the H+-coupled MeAIB transport measured with native Caco-2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 cells. These data suggest that hPAT1 is responsible for the H+-coupled amino acid transport expressed in the apical membrane of Caco-2 cells.

Throughout the animal and plant kingdoms, amino acids play vital roles in a variety of essential biological functions, including protein synthesis, neurotransmission, nitrogen metabolism and cell growth. Many of these functions depend on the entry of amino acids into the cells from the extracellular medium, a process mediated by amino acid transporters in the plasma membrane. Amino acid transport systems have been identified, characterized and named based on distinct functional characteristics such as substrate specificity, ion coupling and exchange properties (Christensen, 1990; Palacin et al. 1998; Ganapathy et al. 2001). Over recent years, many amino acid transporters have been identified at the molecular level in plants, yeast and animals (Palacin et al. 1998; Ganapathy et al. 2001; Wipf et al. 2002).

Historically, the Na+ gradient was recognized as the primary driving force for solute transport across the plasma membrane of mammalian cells (Crane et al. 1961). However, subsequent work in different laboratories identified several solute transporters in mammalian cell plasma membranes that are energized not by the Na+ gradient but by the H+ gradient. These include the peptide transporters (Ganapathy & Leibach, 1985, 1991, 1999) and the monocarboxylate transporters (Halestrap & Price, 1999). In the case of amino acids, even though most of the transport systems are either Na+-coupled or ion-independent, several studies have produced evidence for the presence of a H+-coupled amino acid transport system in the apical plasma membrane of mammalian epithelial cells (Rajendran et al. 1987; Roigaard-Petersen et al. 1987; Jessen et al. 1988, 1989, 1991; Thwaites et al. 1993a, 1994, 1995a). In human intestinal cell (Caco-2) monolayers, the apical H+-coupled amino acid transport system transports a wide range of small, unbranched, zwitterionic amino acids including glycine, alanine, imino acids (proline and hydroxyproline), methylated analogues such as sarcosine, betaine, α-aminoisobutyric acid (AIB) and α-(methylamino)isobutyric acid (MeAIB), β-amino acids (β-alanine and β-taurine), γ-aminobutyric acid (GABA), and some d-amino acids such as d-serine and d-cycloserine (Thwaites et al. 1993a,b, 1994, 1995a,b,c, 2000; Thwaites & Stevens, 1999). H+-coupled l-proline transport has been demonstrated in eel (Anguilla anguilla) intestinal cells, and is localized solely in the apical membrane (Ingrosso et al. 2000). Similarly, Na+-independent, pH-dependent transport in the mucosa-to-serosa direction for l-alanine has been demonstrated across lizard (Gallotia galloti) duodenal enterocytes (Diaz et al. 2000). In the rat small intestine, in the absence of extracellular Na+, MeAIB transfer across the small intestine is stimulated 3-fold when luminal pH is reduced from pH 7.2 to 5.6, and this Na+-independent, H+-dependent MeAIB uptake is inhibited by β-alanine. In contrast, no H+-dependent MeAIB uptake could be measured in either guinea-pig or rabbit small intestine (L. K. Munck & B G. Munck, personal communication). Interestingly, the substrate specificity of the H+-coupled amino acid transporter in Caco-2 cells and rabbit renal brush border membrane vesicles is similar to that described for the IMINO carrier in rat small intestine (Munck et al. 1994), whereas the IMINO carrier identified in either rabbit (Stevens & Wright, 1985; Munck & Munck, 1992) or guinea-pig (Munck & Munck, 1994) small intestine transports a different range of substrates.

The presence of a H+-coupled amino acid transport system (system PAT) in the small intestinal epithelium with such a broad range of transportable substrates provides a potential route for nutrient, osmolyte and drug transport across the initial barrier (i.e. the luminal brush border membrane) to solute absorption. In particular, this transport system transports a number of neuromodulatory agents such as d-serine and d-cycloserine (Thwaites et al. 1995a, c). d-Serine is the endogenous co-agonist for the activation of the N-methyl-d-aspartate receptor by glutamate (Mothet, 2001). The only other apically localized amino acid transporter that transports d-serine is ATB0,+, but this transporter is expressed predominantly in the distal regions of the intestinal tract (Hatanaka et al. 2002). The intestinal system PAT also transports GABA and its analogues, which function as GABA re-uptake inhibitors and GABA receptor agonists/ antagonists (Thwaites et al. 2000). The H+ gradient as the driving force in the small intestine for nutrient or drug absorption is physiologically relevant because such a gradient is present across the enterocyte apical membrane in the form of an ‘acid microclimate’ on the mucosal surface (Rawlings et al. 1987; Daniel et al. 1989).

Many amino acid transport systems in yeast, plants and bacteria are also driven by the electrochemical H+ gradient and over recent years a large number of H+-coupled transporters have been cloned from these sources (Wipf et al. 2002). No mammalian intestinal H+-coupled amino acid transporter has yet been identified at the molecular level. However, a recent study has reported on the isolation of a H+-coupled amino acid transporter from a rat hippocampal cDNA library (Sagne et al. 2001). This transporter was named rLYAAT1 (rat lysosomal amino acid transporter 1) due to its apparent lysosomal localization in rat brain. Subsequently, the mouse orthologue of LYAAT1 was cloned and its functional characteristics elucidated using the X. laevis expression system (Boll et al. 2002). These latter investigators named the transporter PAT1 (proton-coupled amino acid transporter 1) to describe the coupling of the transport system to the electrochemical H+ gradient. In the same report, these investigators also described the cloning of a second mammalian homologue (PAT2), which is energized by an electrochemical H+ gradient. The present study was undertaken to establish the molecular identity of the H+-coupled amino acid transporter expressed in the intestinal cell line Caco-2.

Methods

Isolation of human PAT1 cDNA from a Caco-2 cell cDNA library

The amino acid sequence of rat LYAAT1 (rPAT1; GenBank accession no. AF361239; Sagne et al. 2001) was used to search the GenBank database to see if there are any ESTs (expressed sequence tags) representing the human orthologue of PAT1. This search yielded one positive EST (accession no. AL043182), whose predicted amino acid sequence showed 80 % identity with that of rPAT1. The nucleotide sequence of this EST was used to design primers for RT-PCR to obtain a cDNA probe specific for this transporter. The forward primer was 5′-GTTATTGGTGGTCCCATTG-3′ and the reverse primer was 5′-CTGCCTGACATGCATCTTG-3′. Based on the positions of these primer sequences in the EST, the predicted size of the RT-PCR product was 649 bp. This primer pair was used to obtain a fragment of human PAT1 cDNA by RT-PCR with poly(A)-RNA from the human intestinal cell line Caco-2. The conditions for RT-PCR were as follows: 94 °C for 30 s for denaturation in the first cycle (hot start); 92 °C for 30 s for denaturation, 55 °C for 20 s for annealing, and 72 °C for 1 min for elongation in each cycle for a total of 30 cycles; 72 °C for 5 min for elongation during the last cycle. This yielded a RT-PCR product of expected size. The product was subcloned in pGEM-T vector and sequenced to establish its molecular identity. A unidirectional Caco-2 cell cDNA library was established using poly(A)-RNA isolated from Caco-2 cells. The SuperScript plasmid system (Life Technologies, Grand Island, NY, USA) was employed for this purpose. The cDNA products with sizes greater than 1 kbp were separated by size-fractionation and used for ligation at the SalI/NotI site in pSPORT1 vector. The human PAT1-specific cDNA probe derived by RT-PCR was labelled with [α-32P]dCTP by random priming using the ready-to-go oligolabelling beads (Amersham Biosciences Corp., Piscataway, NJ, USA). This probe was used to screen the Caco-2 cell cDNA library under high stringency conditions. The screening of the library was done as described previously (Kekuda et al. 1997). Positive clones were purified by secondary screening.

DNA sequencing

All of the positive clones whose insert size was greater than 2 kbp were sequenced partially at the 5′ end to establish the relationship among the clones. This approach indicated that all of the clones isolated from the library were related to one another and that the size differences of the inserts were due to truncation at the 5′ end to a varying degree. The clone with the longest insert (≈5.6 kbp) was selected for complete structural analysis. Both the sense and antisense strands of the cDNA were sequenced by primer walking. Sequencing was done by Taq DyeDeoxy terminator cycle sequencing using an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer. The sequence was analysed using the National Center for Biotechnology Information server http://www.ncbi.nlm.nih.gov/. This analysis indicated that the clone isolated from the Caco-2 cell cDNA library was structurally similar to rat PAT1 with 85 % identity in the predicted amino acid sequence. Therefore, we named this clone hPAT1 (for human proton-coupled amino acid transporter).

Northern blot analysis

The expression pattern of PAT1 mRNA in human tissues and in different regions of the gastrointestinal tract was analysed by Northern hybridization under high stringency conditions using the cloned human PAT1 cDNA as a probe and commercially available ready-to-probe blots (Clontech, Palo Alto, CA, USA). The probe (≈1.1 kb) was derived from the full-length human PAT1 cDNA by digestion with Sal I and Nsi I and it contained the first two-thirds of the amino acid coding region of the cDNA. Hybridization was carried out in ExpressHyb hybridization solution (Clontech, Palo Alto, CA, USA) for 2 h at 68 °C. The blots were washed three times (10 min each) at room temperature in a solution containing 2 × SSC (saline-sodium citrate buffer; 0.15 m NaCl and 15 mm sodium citrate, pH 7) and 0.05 % SDS (sodium dodecyl sulphate) and twice (15 min each) at 50 °C in a solution containing 0.1 × SSC and 0.1 % SDS. Hybridization signals were quantified using the STORM PhosphorImaging System (Molecular Dynamics, Sunnyvale, CA, USA). In each blot, the signal in the first lane was taken as ‘1′ for determination of relative signal intensities in the rest of the lanes.

Functional expression of hPAT1 cDNA in mammalian cells

This was done in human retinal pigment epithelial (HRPE) cells using the vaccinia virus expression system as described previously (Blakely et al. 1991; Kekuda et al. 1997; Hatanaka et al. 2002). Subconfluent HRPE cells grown on 24-well plates were first infected with a recombinant (VTF7-3) vaccinia virus encoding T7 RNA polymerase and then transfected with the plasmid carrying the full-length hPAT1 cDNA. At 12-15 h post-transfection, uptake measurements were made at 37 °C with radiolabelled amino acids. The uptake medium was 25 mm Mes/Tris (pH 5.0), containing 140 mmN-methyl-d-glucamine (NMDG) chloride, 5.4 mm KCl, 1.8 mm CaCl2, 0.8 mm MgSO4 and 5 mm glucose. Initial experiments were carried out in the uptake medium in which the buffer was 25 mm Hepes/Tris (pH 7.5) and NaCl replaced NMDG chloride isosmotically. In experiments in which the influence of pH on the transport process was investigated, transport buffers of different pH values were prepared by varying the concentrations of Tris, Hepes and Mes. The time of incubation in most experiments was 5 min. Endogenous transport was always determined in parallel using cells transfected with pSPORT1 vector alone. The transport activity in cDNA-transfected cells was adjusted for the endogenous activity to calculate the cDNA-specific transport activity. Experiments were done in triplicate and each experiment was repeated at least three times. Results are expressed as means ± s.e.m.

Uptake measurements in Caco-2 cells

Caco-2 cells (passage number 109-111) were cultured essentially as described previously (Thwaites et al. 1993a). Cell monolayers were prepared by seeding the cells at high density (4-5 × 105 cells cm−2) on 12 mm diameter Transwell polycarbonate filters (Costar). Cell monolayers were maintained in culture at 37 °C in a humidified atmosphere of 5 % CO2 in air. Experiments were performed 15-17 days after seeding and 18-24 h after feeding. The cell monolayers were washed in 4 × 500 ml volumes of modified Krebs solution (pH 7.4) containing 137 mm NMDG chloride, 5.4 mm KCl, 0.99 mm MgSO4, 0.34 mm KH2PO4, 2.8 mm CaCl2 and 10 mm glucose. The pH of the solution was adjusted at 37 °C by the addition of either Mes (10 mm, pH 5.0) or Hepes (10 mm, pH 7.4) and Tris. In all experiments, the apical solution was pH 5.0 and the basolateral solution was pH 7.4. Uptake of [3H]MeAIB (1 μCi ml−1, 20 μm) was measured for 5 min at 37 °C across the apical membrane of Caco-2 cell monolayers in the presence or absence of 10 mm unlabelled amino acids. The non-mediated diffusional component of MeAIB uptake was measured under similar conditions but in the presence of 30 mm β-alanine. Mannitol was added to maintain isosmolality between experimental solutions. At the end of the incubation period, the cell monolayers were washed in 3 × 500 ml volumes of ice-cold modified Krebs solution (pH 7.4) to remove any loosely associated radiolabel on the surface of the cell monolayer. The polycarbonate filter containing the cell monolayer was then removed from the insert for determination of radioactivity associated with the monolayer. The uptake of radiolabel measured in the presence of β-alanine, which represented the non-mediated diffusional component, was subtracted from total uptake to determine the carrier-mediated uptake. Data are expressed as percentage of control measured in the absence of inhibitors. Results are given as means ± s.e.m.

Immunolocalization of PAT1 in Caco-2 cells

An anti-hPAT1 antibody was raised in chicken to a synthetic peptide (SSTDVSPEESPSEGL) following a standard immunization procedure (Cambridge Research Biochemicals Ltd, UK). The polyclonal IgY was affinity-purified using the synthetic peptide coupled to a HiTRAP NHS-activated column (Amersham Pharmacia Biotech, UK) following the manufacturer's instructions. Caco-2 cell monolayers grown on polycarbonate filters for 14 days were fixed for 1 h in 2 % paraformaldehyde in PBS (phosphate-buffered saline) and permeabilized using 0.1 % Triton X-100 in PBS for 10 min. Cells were incubated on ice for 2 h with either the affinity-purified anti-hPAT1 antibody (1:50 dilution) alone or the anti-hPAT1 antibody plus anti-human CD98 antibody (1:50 dilution) raised in mouse (BD Biosciences, Cowley, UK). To determine the specificity of the anti-hPAT1 antibody binding, the anti-hPAT1 antibody was pre-incubated with 100 μg of the antigenic peptide. After washing in PBS, Caco-2 cell monolayers were incubated for 1 h on ice with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-chicken IgG (Sigma; 1:50 dilution) alone or with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG (ZYMED, San Francisco, CA, USA; 1:50 dilution). Caco-2 cell monolayers were mounted in Vectorshield (Vector Laboratories) and observed using a laser scanning confocal microscope (Leica, TSC, NT).

Materials

[3H]MeAIB (specific radioactivity, 85 Ci mmol−1), [14C]MeAIB (specific radioactivity, 55 mCi mmol−1), and [3H]-l-glutamate (specific radioactivity, 30 Ci mmol−1) were purchased from American Radiolabeled Chemicals (St Louis, MO, USA). [3H]Glycine (specific radioactivity, 56.4 Ci mmol−1), [3H]-l-proline (specific radioactivity, 9.9 Ci mmol−1), [3H]-l-alanine (specific radioactivity, 40 Ci mmol−1), [3H]-l-serine (specific radioactivity, 25 Ci mmol−1) and [3H]-d-serine (specific radioactivity, 6.6 Ci mmol−1) were purchased from Amersham Biociences Corp. (Piscataway, NJ, USA), Moravek Biochemicals (Brea, CA, USA) or Du Pont-New England Nuclear (Boston, MA, USA).

Statistics

Uptake measurements were made in triplicate and experiments were repeated at least three times with separate transfections. Data are presented as means ± s.e.m. for these replicates.

Results

Structural features of human PAT1 cDNA

The hPAT1 cDNA is 5585 bp long (GenBank accession no. AF516142) with a 1431 bp long open reading frame (including the stop codon) coding for a protein of 476 amino acids (Fig. 1). The open reading frame is flanked by a 15 bp long 5′ untranslated region and a 4139 bp long 3′ untranslated region. The cDNA possesses a poly(A) tail and a polyadenylation signal (AATAA) upstream of the poly(A) tail. Hydropathy analysis of the amino acid sequence of hPAT1, using the PEPPLOT program in the Genetics Computer Group, Inc. (GCG, University of Wisconsin, WI, USA) and a window of 21 amino acids, predicts the presence of nine putative transmembrane domains with the amino terminus facing the cytoplasmic side. This membrane topology model for hPAT1 is different from the one proposed for rat and mouse PAT1 by other investigators (Sagne et al. 2001; Boll et al. 2002), who predicted the presence of eleven putative transmembrane domains in PAT1. The discrepancy appears to be primarily due to the difference in the amino acid window size assigned to predict the transmembrane domain. While we assigned a 21 amino acid window size, other investigators have apparently used a window size consisting of 14-18 amino acids. The locations of the nine transmembrane domains proposed in our model correspond to the nine of the eleven transmembrane domains proposed for rat and mouse PAT1. Our model does not contain the transmembrane domains 1 and 9 predicted by Sagne et al. (2001) and Boll et al. (2002). The first hydrophobic region in PAT1 is very long and may contain either one (our model) or two (alternative model) transmembrane domains. The transmembrane domain 9 modelled for rat PAT1 is very small, consisting of only 14 amino acids (Sagne et al. 2001), and does not appear to be a transmembrane domain when a window size of 21 amino acids is applied to predict the model. However, we do not know which of these two models is correct. This would require actual experimentation with epitope-specific antibodies to define the membrane topology of the protein. The identity in amino acid sequence between hPAT1 and rPAT1 is 85 %. A recent search of the GenBank database identified a 1961 bp-long cDNA sequence (accession no. NM078483), which has been predicted to represent human PAT1. This cDNA, however, codes for a putative protein of 434 amino acids. Comparison of the amino acid sequence of this putative hPAT1 with that of hPAT1 reported in the present study indicates that the two proteins are identical except for a deletion of 42 amino acids in the former. There were additional differences in five individual amino acids, but it is not known whether these differences represent polymorphisms or arise from potential sequencing errors. There is also no information available on the influence of these sequence differences on the function of the transporter because the transport characteristics of the putative hPAT1, represented by the GenBank accession no. NM078483, have not been investigated.

Figure 1. Amino acid sequence and hydropathy analysis of hPAT1.

Figure 1

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Putative transmembrane regions are underlined and numbered.

Exon-intron organization of human pat1 gene

A search of the GenBank database with the nucleotide sequence of hPAT1 cDNA (the clone reported in the present study) as the query identified the chromosomal location and the sequence of the human pat1 gene (GenBank accession no. AC034205). The gene is located on human chromosome 5q31-33 and consists of at least 11 exons coding for the cloned hPAT1 cDNA. We could account for the entire sequence of the cDNA in the genomic sequence. Figure 2 describes the exon-intron organization of human pat1 gene based on this sequence analysis. Exon 1 does not code for the protein. The putative translation start site resides in exon 2. Exon 11 codes for a small C-terminal region of the protein and for the entire 3′-untranslated region. The sizes of the exons and introns and the sequences at the splice junctions are given in Table 1.

Figure 2. Exon-intron organization of the human pat1 gene.

Figure 2

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Exons are numbered in the gene. Numbers in the cDNA indicate the nucleotide positions of the splice junctions and the hatched areas denote the untranslated regions on the 5′ and 3′ ends of the cDNA. The exact length of the first exon is not known because of a lack of information on the transcription start site. Arrowheads and numbers in the protein indicate the positions of exon junctions and amino acid positions corresponding to these junctions, respectively. Roman numerals indicate the putative transmembrane domains (TMD).

Table 1.

Exon–intron boundaries of the human pat1 gene

Exon Intron Exon
No. Size (bp) 3′ Junction 5′ Junction Size (bp) No. 3′ Junction 5′ Junction No.
1 Unknown …CTCCAG gtcagg… ∼10980 1 …ccccag CTGCCA… 2
2 152 …CACAAC gtgagt… 4617 2 …cttcag ATGGTT… 3
3 91 …ATCGTG gtaagg… 869 3 …ccccag ATGGGT… 4
4 89 …CCGCAG gtgaga… 470 4 …ttccag GCTGAA… 5
5 96 …GGGAAG gtaact… 2028 5 …ctccag ACGTGT… 6
6 85 …AAACAG gtaggc… 422 6 …ttctag GTGATA… 7
7 219 …GTTCAG gtacat… 5746 7 …tttcag AGGATC… 8
8 99 …GGAATG gtaaga… 2817 8 …tcatag GTTCTG… 9
9 167 …CTGCTG gtacgt… 2562 9 …cggcag GTTGTA… 10
10 170 …TGACAT gtgata… 8492 10 …ctgcag GGATCT… 11
11 4396 …ATTTTT — — — — — —
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Tissue expression pattern of PAT1 mRNA

Northern blot analysis of various human tissues was performed under high stringency conditions using a commercially available multiple human tissue blot to determine the expression pattern of PAT1 mRNA in human tissues (Fig. 3A). This analysis indicated that PAT1 mRNA is expressed ubiquitously in human tissues, with maximal expression in the small intestine. Moderate expression is evident in the brain, colon, kidney, liver, lung, placenta and testis. Expression is detectable, but at very low levels, in the stomach, spleen, skeletal muscle and heart. Since the expression is most predominant in the small intestine, we used another commercially available human tissue blot to assess the expression pattern of PAT1 mRNA in different regions of the gastrointestinal tract (Fig. 3B). This analysis indicated that PAT1 mRNA is abundantly expressed all along the gastrointestinal tract between the stomach and the descending colon. The expression is comparatively low in the oesophagus, caecum and rectum. In all of these tissues, the size of the hybridization signal was 6.4 kb.

Figure 3. Northern blot analysis of PAT1 mRNA expression in human tissues (A) and in different regions of human gastrointestinal tract (B).

Figure 3

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Data represent relative intensity of the hybridization signals in different lanes with the intensity in the first lane taken as 1 in respective blots.

Functional characteristics of hPAT1

The functional features of the cloned hPAT1 were investigated in a mammalian cell heterologous expression system in which hPAT1 cDNA was expressed functionally in HRPE cells using the vaccinia virus system. We first analysed the ability of hPAT1 to transport MeAIB. For this purpose, we compared the uptake of MeAIB (15 μm) in control HRPE cells transfected with vector alone and in cells transfected with hPAT1 cDNA (Fig. 4A). The cDNA-induced uptake of MeAIB was barely noticeable in the presence of Na+ when measured at extracellular pH 7.5. However, the cDNA-induced uptake became easily detectable when measured under similar conditions but in the absence of Na+. This uptake increased severalfold when the extracellular pH was changed from 7.5 to 6.0 in the absence of Na+. The stimulation of cDNA-specific uptake of MeAIB by acid pH was evident at all time periods of incubation tested (Fig. 4B). Interestingly, the influence of acid pH was much greater at shorter periods of incubation. In fact, not only did the influence of acid pH decrease with longer periods of incubation, but the cDNA-specific uptake at acid pH also decreased significantly with incubation periods beyond 15 min. Therefore, subsequent uptake measurements were made with a 5 min incubation. Under these conditions (i.e. pH 6.0, absence of Na+, 5 min incubation), the uptake of MeAIB in hPAT1 cDNA-transfected cells was about 6-fold higher than in cells transfected with vector alone.

Figure 4. Influence of Na+- and H+-gradients on hPAT1 cDNA-mediated MeAIB uptake.

Figure 4

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A, uptake of MeAIB (15 μm) was measured for 5 min in vector-transfected HRPE cells and in hPAT1 cDNA-transfected HRPE cells either in the presence of Na+ (140 mm NaCl, pH 7.5) or in the absence of Na+ (140 mm NMDGCl to replace NaCl isosmotically, pH 7.5 and 6.0). B, uptake of MeAIB (15 μm) was measured in HRPE cells transfected with either vector alone or hPAT1 cDNA. Uptake medium contained either NaCl (i.e. in the presence of Na+) or NMDG chloride (i.e. in the absence of Na+). The cDNA-specific uptake was calculated by subtracting uptake in vector-transfected cells from uptake in hPAT1 cDNA-transfected cells. Data represent the time course of cDNA-specific uptake.

Since the change of extracellular pH from 7.5 to 6.0 showed marked influence on hPAT1-mediated uptake of MeAIB, we analysed this effect over a wider range of extracellular pH (Fig. 5). The cDNA-specific uptake was barely detectable when pH was ≥ 7.5. However, the uptake increased markedly when pH was reduced below 7.5, with maximal uptake observed at pH 5.0. When compared between vector-transfected cells and hPAT1 cDNA-transfected cells, the uptake of MeAIB, measured at pH 5.0, increased about 14-fold due to the heterologous expression of hPAT1. These data show that hPAT1-mediated MeAIB uptake is H+-dependent and that Na+ has no direct role in the uptake process. Therefore, subsequent uptake measurements were made in the absence of Na+ and at an extracellular pH of 5.0. Analysis of the dependence of the hPAT1-specific uptake activity on the concentration of H+ revealed that the relationship was hyperbolic with a Hill coefficient of 0.9 ± 0.1, suggesting that the H+:MeAIB stoichiometry for hPAT1 is 1:1.

Figure 5. Influence of extracellular pH on hPAT1 cDNA-mediated MeAIB uptake.

Figure 5

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Uptake of MeAIB (20 μm) was measured in the absence of Na+ in cells transfected with either vector alone or hPAT1 cDNA. The pH of the uptake buffer was varied by appropriately adjusting the concentrations of Mes, Hepes and Tris. Data represent only the cDNA-specific uptake.

We studied the substrate specificity of hPAT1 by assessing the potency of various amino acids (10 mm) to inhibit the uptake of [14C]MeAIB (20 μm) that was mediated specifically by hPAT1. These competition experiments were done in parallel in vector-transfected cells and in hPAT1 cDNA-transfected cells. The cDNA-specific uptake was then calculated and analysed. The data given in Fig. 6A show that, among the l-amino acids examined, only alanine and proline were very effective in competing with MeAIB for uptake via hPAT1. The inhibition of hPAT1-mediated MeAIB uptake by these two amino acids was 80 %. Serine, cysteine, valine, leucine and glutamine caused a small, but significant, inhibition (15-20 %). Other amino acids tested (threonine, phenylalanine, tryptophan, lysine and glutamate) did not produce any detectable inhibition. When d-isomers of these amino acids were examined, it was found that those of alanine and proline were equally effective as the l-isomers in inhibiting hPAT1-specific MeAIB uptake. More interestingly, while the l-isomers of serine and cysteine produced only a very small inhibition of MeAIB uptake via hPAT1, the corresponding d-isomers were able to inhibit the uptake to a marked extent (60-80 %). Glycine and hydroxy-l-proline were as effective as the d- and l-isomers of proline and alanine in competing with MeAIB. The amino acid analogues AIB and GABA were also potent inhibitors (Fig. 6B). These data show that the substrates of the cloned human PAT1 include MeAIB, AIB, GABA, glycine, l-alanine, l-proline, hydroxy-l-proline, d-proline, d-alanine, d-serine and d-cysteine.

Figure 6. Influence of d- and l-amino acids on hPAT1 cDNA-mediated MeAIB uptake.

Figure 6

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Uptake of [14C]MeAIB (20 μm) was measured at pH 5 in the absence of Na+ in cells transfected with either vector alone or hPAT1 cDNA. Unlabelled d- and l-amino acids were present at 10 mm. Data, representing only the cDNA-specific uptake, are given as percentage of control uptake measured in the absence of unlabelled amino acids.

These competition studies suggest, but do not prove, that the amino acids that compete with MeAIB are recognized by hPAT1 as substrates. To provide unequivocal evidence for the transport of these amino acids via hPAT1, we measured directly the uptake of several of these amino acids in vector-transfected cells and in hPAT1 cDNA-transfected cells by using radiolabelled amino acids (Fig. 7). These studies have demonstrated that the uptake of d-serine, glycine, l-proline and l-alanine in hPAT1-expressing cells is significantly higher than in vector-transfected control cells. In contrast, there is no detectable increase in the uptake of l-serine and l-glutamine in hPAT1-expressing cells. These data provide direct evidence for hPAT1-mediated transport of d-serine, glycine, l-proline and l-alanine. The transport of glycine via hPAT1 is saturable with a Michaelis-Menten constant (Km) of 2.3 ± 0.2 mm (Fig. 8). We then determined the affinity of hPAT1 for l- and d-isomers of cysteine, serine, proline and alanine by assessing the dose-response relationship for these amino acids to inhibit hPAT1-mediated glycine uptake (Fig. 9). The IC50 values (i.e. the concentration of the compound needed to cause 50 % inhibition) were as follows: 20.1 ± 6.8 and 3.5 ± 0.5 mm for l-cysteine and d-cysteine, respectively; 29.1 ± 7.8 and 3.8 ± 0.4 mm for l-serine and d-serine, respectively; 2.0 ± 0.6 and 2.5 ± 0.3 mm for l-proline and d-proline, respectively; 1.7 ± 0.2 and 2.9 ± 0.9 mm for l-alanine and d-alanine, respectively. These data show that while the affinity of hPAT1 is similar for the l- and d-isomers of alanine and proline, the affinity is markedly different between the l- and d-isomers of cysteine and serine. In the case of the latter amino acids, the d-isomers show 6- to 8-fold higher affinity for hPAT1 than the corresponding l-isomers. Similar studies with MeAIB, hydroxy-l-proline, AIB and GABA showed that the IC50 values for these amino acids were 1.5 ± 0.2, 4.6 ± 0.8, 2.6 ± 0.7 and 2.1 ± 0.2 mm, respectively (Fig. 10). Since the concentration of glycine used in these experiments was very low compared with its Km value (20 μmversus 2.3 mm), these IC50 values are close to the Km values for these amino acids.

Figure 7. Direct measurement of hPAT1 cDNA-mediated uptake of various amino acids.

Figure 7

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Uptake of various radiolabelled amino acids (20 μm) was measured at pH 5 in the absence of Na+ in cells transfected with either vector alone or hPAT1 cDNA.

Figure 8. Kinetics of hPAT1 cDNA-mediated glycine uptake.

Figure 8

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Uptake of glycine was measured at varying concentrations at pH 5 in the absence of Na+ in cells transfected with either vector alone or hPAT1 cDNA. Data represent only the cDNA-specific uptake. Inset, Eadie-Hofstee plot: V/S (uptake rate/glycine concentration) versus V (uptake rate).

Figure 9. Inhibition of hPAT1 cDNA-specific glycine uptake by d- and l-isomers of cysteine, serine, proline and alanine.

Figure 9

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Uptake of glycine (20 μm) was measured at pH 5 in the absence of Na+ in cells transfected with either vector alone or hPAT1 cDNA in the absence and in the presence of increasing concentrations of unlabelled d- and l-isomers of cysteine, serine, proline and alanine. Data, representing only the cDNA-specific uptake, are given as percentage of control uptake measured in the absence of inhibitors.

Figure 10. Inhibition of hPAT1 cDNA-specific [3H]glycine uptake by MeAIB, glycine, hydroxy-l-proline, AIB and GABA.

Figure 10

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Uptake of [3H]glycine (20 μm) was measured at pH 5 in the absence of Na+ in cells transfected with either vector alone or hPAT1 cDNA in the absence and in the presence of increasing concentrations of indicated amino acids. Data, representing only the cDNA-specific uptake, are given as percentage of control uptake measured in the absence of inhibitors.

To determine whether the PAT1 cloned from Caco-2 cells is responsible for H+-coupled MeAIB uptake observed in native Caco-2 cells, we measured the uptake of MeAIB in HRPE cells heterologously expressing the cloned PAT1 and in native Caco-2 cells under identical conditions (20 μm MeAIB, absence of Na+, extracellular pH 5.0, and 5 min incubation) and determined the relative abilities of 12 different amino acids (10 mm) to inhibit this MeAIB uptake. When the inhibitory potencies of these amino acids measured in hPAT1-expressing HRPE cells were plotted against those measured in native Caco-2 cells, there was a good correlation (r2 = 0.93) between the two sets of data (Fig. 11). This strongly suggests that the H+-coupled uptake of MeAIB observed in native Caco-2 cells is indeed mediated by PAT1 cloned from these cells.

Figure 11. Correlation between the potencies of various amino acids for the inhibition of MeAIB uptake in native Caco-2 cells and for the inhibition of MeAIB uptake that was mediated specifically by hPAT1 in the heterologous expression system with HRPE cells.

Figure 11

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Both in Caco-2 cells and hPAT1-expressing HRPE cells, uptake of radiolabelled MeAIB (20 μm) was measured at pH 5 in the absence and presence of various inhibitory amino acids (10 mm). In Caco-2 cells, non-mediated uptake of MeAIB, measured in the presence of excess amounts (30 mm) of β-alanine, was subtracted from total uptake to determine carrier-mediated uptake. In the heterologous expression system, hPAT1-specific MeAIB uptake was determined by subtracting the uptake in vector-transfected cells from the uptake in hPAT1 cDNA-transfected cells. Data are given as percentage of control uptake measured in the absence of inhibitors.

Finally, to provide direct evidence for the participation of PAT1 in the H+-coupled uptake of MeAIB and other amino acids across the apical membrane of Caco-2 cells, we performed immunofluorescence studies using a polyclonal antibody specific for hPAT1 in Caco-2 cell monolayers cultured on polycarbonate filters (Fig. 12). The antibody detected a protein that is expressed exclusively in the apical membrane. The immunofluorescence signal was specific because pre-incubation of the antibody with the antigenic peptide eliminated the signal. In parallel studies, we also used an antibody specific for CD98, a protein known to be localized solely in the basolateral membrane of intestinal epithelial cells. This antibody detected a protein that is expressed only in the basolateral membrane in Caco-2 cells. There was no crossover of immunofluorescence signal for PAT1 to the basolateral membrane. Similarly, there was no crossover of immunofluorescence signal for CD98 to the apical membrane. These data provide unequivocal evidence for the exclusive localization of PAT1 in the apical membrane of Caco-2 cells.

Figure 12. Immunofluorescence localization of PAT1 in Caco-2 cell monolayers.

Figure 12

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A, immunofluorescent detection of PAT1 in Caco-2 cell monolayers was performed using either the anti-hPAT1 antibody alone (i-iii) or following a pre-incubation of the antibody with the antigenic peptide (iv-vi). A series of xy sections were taken through Caco-2 cell monolayers from the apical to basal surfaces. The sections labelled as apical (i and iv) were taken at the apical surface of the monolayers. Middle sections (ii and v) were taken 5 μm below the apical surface. Basal sections (iii and vi) were taken 11.5 μm below the apical surface. Images were captured using identical conditions. B, immunofluorescent detection of PAT1 (green) and CD98 (red) in Caco-2 cell monolayers was performed using the anti-hPAT1 antibody and anti-human CD98 antibody, respectively. A cross-sectional image (xz) through a Caco-2 cell monolayer was captured using confocal laser scanning microscopy. All scale bars are 20 μm.

Discussion

Relevance of the cloned PAT1 to H+-coupled amino acid transport in Caco-2 cells

The studies reported here describe the structural features and transport characteristics of hPAT1, the first H+-coupled amino acid transporter to be cloned from a human tissue/cell. These studies were prompted by the recent successful cloning of an amino acid transporter from rat brain that is driven by a H+ gradient (Sagne et al. 2001). When this transporter was expressed in mammalian cells, the protein was found to be localized predominantly in lysosomes. This led to the naming of the transporter as rat LYAAT1 (lysosomal amino acid transporter 1) (Sagne et al. 2001). However, a fraction of the protein was targeted to the plasma membrane, which enabled the investigators to characterize the transport function of this transporter. It was concluded that LYAAT1 is a H+-coupled amino acid transporter that normally functions in lysosomes in the export of amino acids into the cytoplasm. Amino acids that arise from the lysosomal breakdown of proteins thus must find their way into the cytoplasm for subsequent entry into cellular metabolism. The participation of a H+-coupled transport system in this exit process is logical because of the existence of an outwardly directed H+ gradient across the lysosomal membrane which can provide the driving force necessary for the active export of amino acids from the lysosomes via this transporter. What caught our attention in this report was that the substrate specificity of this putative lysosomal transporter was very similar to that of the H+-coupled amino acid transporter in Caco-2 cells we described in several of our studies (Thwaites et al. 1993a,b, 1994, 1995a,b,c). Since the transport studies in Caco-2 cells were done using intact cells cultured as monolayers on polycarbonate filters, the observed transport was definitely mediated by a transport system located in the apical membrane rather than in any intracellular organelle. Furthermore, our studies with Caco-2 cells were able to demonstrate directly intracellular acidification following exposure of the cells on the apical side to certain amino acids in the presence of an acidic extracellular pH (Thwaites et al. 1993a,b, 1994, 1995a,b,c). Since the substrate specificity of rat LYAAT1 was very similar to that of the H+-coupled amino acid transporter in Caco-2 cells, we asked the question whether the transport in Caco-2 cells is actually mediated by a human orthologue of LYAAT1. This would mean that LYAAT1 may not be exclusively localized to the lysosomes and that a significant pool of the transporter protein resides in the plasma membrane mediating the cellular entry of amino acids in a H+-coupled manner. Therefore, we initiated studies to determine if Caco-2 cells express the human orthologue of LYAAT1 and, if they do, whether the transporter is responsible for H+-coupled amino acid transport across the apical membrane of these cells. While these studies were in progress, Boll et al. (2002) reported on the cloning of mouse LYAAT1, which they called PAT1 (proton-coupled amino acid transporter 1). We have chosen to refer to this transporter as PAT1 rather than LYAAT1.

The results of our studies have shown that Caco-2 cells indeed express the human orthologue of PAT1. The human PAT1 cloned from these cells is able to mediate H+-coupled transport of specific amino acids in a mammalian cell heterologous expression system. The substrates of the cloned human PAT1 include MeAIB, glycine, l-proline, l-alanine, AIB, GABA and the d-isomers of alanine, proline, cysteine and serine. The substrate specificity and affinity of human PAT1 are very similar to those previously reported in native intact Caco-2 cells. Furthermore, we could show in the present study that the affinities of various amino acids for the cloned human PAT1 correlate closely with the corresponding affinities for the H+-coupled amino acid transport system in Caco-2 cells. These data indicate strongly that the H+-coupled amino acid transport detectable in the apical plasma membrane of Caco-2 cells is indeed mediated by the human orthologue of PAT1. This conclusion is further supported by the immunofluorescence studies that showed exclusive localization of the PAT1 protein to the apical membrane of these cells. Therefore, PAT1 is not likely to be localized exclusively in the lysosomes as suggested by Sagne et al. (2001). In fact, immunofluorescence signals were not detectable in any intracellular organelle in Caco-2 cells under the experimental conditions employed in the current study. In addition, a H+-coupled amino acid transport system with comparable substrate specificity and affinity has been demonstrated in purified apical membrane vesicles from the intestine and kidney (Ingrosso et al. 2000; Jessen et al. 1988, 1989, 1991; Rajendran et al. 1987; Roigaard-Petersen et al. 1987). These findings are at odds with the suggestion of exclusive localization of PAT1 in lysosomes. There are a number of examples of proteins that are expressed in both apical and intracellular membranes in epithelial cells. It may be that a single transport protein is able to function at different membranes within a single cell type or in different membranes in different cells. The most pertinent example of a transport protein that can be localized to the apical membrane as well as in the lysosomal membrane of epithelial cells is the H+-coupled divalent metal ion transporter DMT1/NRAMP2 (Gunshin et al. 1997; Canonne-Hergaux et al. 1999; Tabuchi et al. 2000).

Is PAT1 the ‘IMINO’ carrier?

The IMINO carrier is one of the few ‘classical’ mammalian amino acid transport systems that has yet to be described at the molecular level. In humans, this carrier is likely to be linked to the hereditary malabsorption syndrome known as iminoglycinuria, which is associated with a defect in proline, hydroxyproline and glycine transport in the intestine and kidney (Wellner & Meister, 1981). However, one of the peculiarities of the IMINO system is the marked differences in functional characteristics attributed to this transporter in many studies of imino acid transport across the apical membrane of the mammalian small intestine (Stevens & Wright, 1985; Munck & Munck, 1992, 1994; Munck et al. 1994). For example, the IMINO carrier in rabbit and guinea-pig intestine is Cl−-dependent whereas the rat intestinal IMINO carrier is Cl−-independent. Furthermore, the differences in substrate specificity observed in several studies suggest that the IMINO carrier in the rat intestine is distinct from that in the rabbit and guinea-pig intestines. For a number of years, we have highlighted the close similarity in substrate specificity between the rat intestinal IMINO carrier and the H+-coupled amino acid transporter (PAT) in Caco-2 cells (Thwaites et al. 1995b,c; Thwaites & Stevens, 1999). However, there was an important difference in terms of energetics of the rat intestinal IMINO carrier and the Caco-2 cell PAT. While the rat intestinal IMINO system was Na+-dependent (Munck et al. 1994), the PAT in Caco-2 cells could function in the absence of Na+ (Thwaites et al. 1993a,c). The recent observations by B. G. Munck and L. K. Munck (personal communication) that, in the absence of extracellular Na+, there is an increase in MeAIB uptake in the rat small intestine (but not in the rabbit or guinea-pig intestine) when bulk luminal pH is reduced from pH 7.2 to 5.6, support the idea that these transporters are one and the same. In other words, rat PAT1 is probably the ‘classical’ IMINO carrier that has been described in the rat intestine. On the same basis, we suggest that the human PAT1 described here most likely represents the human IMINO carrier. A number of studies have suggested that the microclimate acid pH is resistant to changes in bulk pH. However, it should be noted that many of the studies of imino acid transport were performed in rat intestine in vitro in the presence of glucose (Munck et al. 1994). These are the conditions at which the lowest pH values (pH <6) have been measured at the mucosal surface (Lucas et al. 1980). Thus, even in the absence of a change in bulk pH, it is likely that a pH gradient was present across the apical membrane in vitro under the conditions in which imino acid transport was measured in many of the earlier studies. Removal of Na+ causes only a 60 % reduction in the uptake of MeAIB in the rat small intestine (Munck et al. 1994). Interestingly, there are a number of studies using brush border membrane vesicles prepared from rabbit small intestine (e. g. Stevens & Wright, 1985) that demonstrate the Na+-dependence of the IMINO carrier, whereas there are no reports in the literature demonstrating the Na+-dependence of the IMINO carrier using rat small intestinal brush border membrane vesicles.

So, what is the basis of the partial Na+-dependence of the rat intestinal IMINO carrier? The apparent dependence of this carrier on Na+ is reminiscent of the conflicting reports about the Na+ dependencce of the intestinal peptide transporter PEPT1 in different experimental conditions and tissue preparations. In intact tissue preparations, peptide transport shows a partial dependence on Na+, whereas in brush border membrane vesicles the same transport system shows no Na+-dependence (Ganapathy & Leibach, 1985). Similarly, the cloned PEPT1 from different species shows no Na+-dependence (Ganapathy & Leibach, 1999, 1991, 2001). We suggested that a functional coupling between the H+-coupled peptide transporter and the Na+-H+ exchanger in the intestinal brush border membrane may explain the seemingly different results in different tissue preparations with regard to the Na+-dependence of the peptide transporter (Ganapathy & Leibach, 1985, 1991). Recent studies with Caco-2 cell monolayers demonstrate that there is a close functional relationship between PEPT1 and the Na+-H+ exchanger subtype NHE3 (Thwaites et al. 1999, 2002) and that the apparent Na+ dependence of PEPT1 is due to NHE3 recycling the H+, which is cotransported with peptides via PEPT1, with the concurrent influx of Na+. A preliminary study in Caco-2 cells demonstrates that PAT1 has a similar functional coupling with NHE3 (Anderson et al. 2001). However, it has to be noted that the dependence of the IMINO carrier on Na+ is unequivocal in the rabbit small intestine (Stevens & Wright, 1985). This would mean that either rabbit PAT1 exhibits different characteristics with regard to Na+ dependence or the rabbit intestine does not express PAT1 but expresses instead a Na+-coupled IMINO carrier. Future studies with cloned rabbit PAT1 can potentially address this issue.

Physiological and pharmacological relevance of transport of d-amino acids by hPAT1

The primary physiological substrates for PAT1 in the human small intestine are likely to be glycine, l-proline and l-alanine. However, this transporter does transport certain d-amino acids, in particular d-serine, d-cycloserine, d-cysteine, d-proline and d-alanine. Even though d-amino acids are not present at significant levels in the lumen of the small intestine under normal physiological conditions, the transport of certain d-amino acids via PAT1 may have pharmacological and therapeutic relevance. d-Amino acids such as d-serine and d-cycloserine are effective in the treatment of affective disorders and cancer, respectively, when administered orally. The oral bioavailability for these drugs is quite high. It is likely that PAT1 in the small intestine is responsible for the efficient absorption of these therapeutic compounds in the intestinal tract. The expression of PAT1 in the colon suggests additional potentially significant physiological roles. Colonic bacteria produce significant quantities of d-amino acids, especially d-alanine and d-serine (Friedman, 1999). Therefore, it is possible that these bacteria-derived d-amino acids are absorbed in the colon via PAT1. This process may have biological significance because d-isomers of some amino acids, including serine and cysteine, have been shown to have nutritional, physiological and therapeutic functions (Friedman, 1999; Man & Bada, 1987).

Acknowledgments

This study was supported by the National Institutes of Health grants HL64196 and AI49849 to V.G. and an MRC Career Establishment Grant (G9801704) to D.T.T.; C.M.H.A. is supported by a BBSRC Agri-Food Committee Studentship.

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