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

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 alpha-(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, alpha-aminoisobutyrate (AIB) and gamma-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 (r(2) = 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.

Figure 1. Amino acid sequence and hydropathy analysis of hPAT1

Putative transmembrane regions are underlined and numbered.

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

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).

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

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

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

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.

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

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.

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

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.

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

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

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

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

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.

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

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.

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

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.