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 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.

Uptake of Aristolochic Acid I into Caco-2 Cells by Monocarboxylic Acid Transporters

“…Several Na + -independent, H + -dependent cotransporters, such as oligopeptide transporter (PEPT)-1, organic anion transporting polypeptide (OATP)-B and amino acid transporter (PAT)-1, localize in the brush border membranes of the human small intestine and Caco-2 cells. [20][21][22][23] However, coincubation with glycylsarcosine (a typical substrate of PEPT), estro-3-sulfate (an inhibitor of OATPs), sulfobromophtalein (an inhibitor of OATPs), L-proline (a substrate of PAT1), or γ-aminobutyric acid (a substrate of PAT1) did not decrease the uptake of AAI ( Table 1). The transport of AAI from the blood into the tubular epithelium is mediated by organic anion transporter (OAT) 1 and 3, which are expressed by basolateral membranes, 4) andcoincubation with p-aminohippuric acid (a typical substrate of OATs) did not decrease the uptake of AAI.…”

PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids

“…CG1139 and PAT1 have similar affinity for alanine [K m of 1.2±0.2 mM (R 2 =0.99; Fig. 6E) versus an IC 50 of 1.7±0.23 mM for PAT1 (Chen et al, 2003a)], glycine (estimated IC 50 =3 mM, 2.3 mM for PAT1; Fig. 6C) and proline (estimated IC 50 =8 mM, 2.0 mM for PAT1).…”

The Proton/Amino Acid Cotransporter PAT2 Is Expressed in Neurons with a Different Subcellular Localization than Its Paralog PAT1

“…PAT3 and PAT4 are orphan transporters, whereas PAT1 (also designated as LYAAT1) and PAT2 are characterized as electrogenic amino acid/proton co-transporters with a high selectivity for amino acids with apolar and small side chains (3,16). PAT1 was shown to transport glycine, L-alanine, and L-proline as well as GABA and D-serine (11,12,(17)(18)(19). It is present in almost all tissues analyzed and shows high expression levels in brain, small intestine, kidney, and colon.…”