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

Autor: You-Jun Fei, Vadivel Ganapathy, Seiji Miyauchi, David T. Thwaites, Catriona M.H. Anderson, Wei Huang, Zhong Chen, Katherine A. Wake
Rok vydání: 2003
Předmět:
Zdroj: The Journal of Physiology. 546:349-361
ISSN: 1469-7793
0022-3751
DOI: 10.1113/jphysiol.2002.026500
Popis: 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.
Databáze: OpenAIRE
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