H+/amino acid transporter 1 (PAT1) is the imino acid carrier: An intestinal nutrient/drug transporter in human and rat.

BACKGROUND AND AIMS: Amino acid (and related drug) absorption across the human small intestinal wall is an essential intestinal function. Despite the revelation of a number of mammalian genomes, the molecular identity of the classic Na(+)-dependent imino acid transporter (identified functionally in the 1960s) remains elusive. The aims of this study were to determine whether the recently isolated complementary DNA hPAT1 (human proton-coupled amino acid transporter 1), or solute carrier SLC36A1, represents the imino acid carrier; the Na(+) -dependent imino acid transport function measured at the brush-border membrane of intact intestinal epithelia results from a close functional relationship between human proton-coupled amino acid transporter-1 and N(+) /H(+) exchanger 3 (NHE3). METHODS: PAT1 function was measured in isolation ( Xenopus laevis oocytes) and in intact epithelia (Caco-2 cell monolayers and rat small intestine) by measurement of amino acid and/or H(+) influx. Tissue and membrane expression of PAT1 were determined by reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: PAT1-specific immunofluorescence was localized exclusively to the luminal membrane of Caco-2 cells and human and rat small intestine. The substrate specificity of hPAT1 is identical to that of the imino acid carrier. In intact epithelia, PAT1-mediated amino acid influx is reduced under conditions in which NHE3 is inactive. CONCLUSIONS: The identification in intact epithelia of a cooperative functional relationship between PAT1 (H(+) /amino acid symport) and NHE3 (N(+) /H(+) exchange) explains the apparent Na + dependence of the imino acid carrier in studies with mammalian intestine. hPAT1 is the high-capacity imino acid carrier localized at the small intestinal luminal membrane that transports nutrients (imino/amino acids) and orally active neuromodulatory agents (used to treat affective disorders).

Structure, tissue expression pattern, and function of the amino acid transporter rat PAT2.

The second member of the PAT (proton-coupled amino acid transporter) family of H(+)-coupled, pH-dependent, Na(+)-independent amino acid transporters was isolated from a rat lung cDNA library. The cDNA for rat PAT2 is 2396bp in length, including 70bp of 5'UTR and a poly(A) tail. The transporter gene, consisting of 10 exons, is located on rat chromosome 10q22. The cDNA codes for a protein of 481 amino acids with 72% identity (over 449 amino acids) with rat PAT1. Tissue expression studies demonstrate that mRNA abundance is generally low with highest levels being detected in lung and spleen, with lower levels in the brain, heart, kidney, and skeletal muscle. Functional expression in either mammalian cells or Xenopus laevis oocytes demonstrates that rat PAT2 mediates pH-dependent, Na(+)-independent uptake of glycine, proline, and alpha(methyl)aminoisobutyric acid (MeAIB). In conclusion PAT2 has a limited tissue distribution, higher affinity (Michaelis-Menten constant for glycine uptake between 0.49 and 0.69mM), and distinct substrate specificity compared to PAT1.

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

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.