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

Background & 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 Na+/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 (Na+/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). Background & 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 Na+/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 (Na+/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). The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte luminal membrane. Clearly, the establishment and maintenance of the driving forces (transmembrane and transepithelial ion gradients) are vital for maximum absorption to proceed.1Thwaites D.T. Ford D. Glanville M. Simmons N.L. H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells.J Clin Invest. 1999; 104: 629-635Crossref PubMed Scopus (70) Google Scholar Intestinal absorptive capacity is thus dependent on the cooperative activity of discrete transport processes involved directly in absorption and on those that mediate cellular (ion) homeostasis. Dietary protein is absorbed across the luminal membrane of the human small intestine in the form of small peptides (di/tripeptides) and amino acids.2Ganapathy V. Brandsch M. Leibach F.H. Intestinal transport of amino acids and peptides.in: Johnson L.R. Physiology of the gastrointestinal tract. Raven, New York1994: 1773-1794Google Scholar All di/tripeptide absorption can be accounted for by the function of a single transporter, the H+-coupled di/tripeptide transporter hPepT1.3Liang R. Fei Y.J. Prasad P.D. Ramamoorthy S. Han H. Yang-Feng T.L. Hediger M.A. Ganapathy V. Leibach F.H. Human intestinal H+/peptide cotransporter cloning, functional expression, and chromosomal localization.J Biol Chem. 1995; 270: 6456-6463Crossref PubMed Scopus (487) Google Scholar In contrast, amino acid transport across the intestinal brush-border membrane is mediated via a number of distinct transport systems that vary in substrate selectivity and ion coupling.2Ganapathy V. Brandsch M. Leibach F.H. Intestinal transport of amino acids and peptides.in: Johnson L.R. Physiology of the gastrointestinal tract. Raven, New York1994: 1773-1794Google Scholar Recent genomic revelations have allowed most mammalian amino acid transport functions to be attributed to specific gene products: at least 52 amino acid transporter-related gene products are grouped within 12 solute carrier families.4Hediger M.A. Romero M.F. Peng J.B. Rolfs A. Takanaga H. Bruford E.A. The ABCs of solute carriers physiological, pathological and therapeutic implications of human membranetransport proteins—introduction.Pflugers Arch. 2004; 447: 1-942Crossref Scopus (766) Google Scholar Despite this wealth of information, some intestinal brush-border membrane amino acid transport systems (as characterized by functional studies) have yet to be identified at the molecular level.5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar In addition, it is not known how some amino acids (including orally delivered therapeutic agents such as d-cycloserine, d-serine, and betaine)7Evins A.E. Amico E. Posever T.A. Toker R. Goff D.C. d-Cycloserine added to risperidone in patients with primary negative symptoms of schizophrenia.Schizophr Res. 2002; 56: 19-23Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 8Tsai G. Yang P. Chung L.-C. Lange N. Coyle J.T. d-Serine added to antipyschotics for the treatment of schizophrenia.Biol Psychiatry. 1998; 44: 1081-1089Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 9Smolin L.A. Benevenga N.J. Berlow S. The use of betaine for the treatment of homocystinuria.J Pediatr. 1981; 99: 467-472Abstract Full Text PDF PubMed Scopus (90) Google Scholar are absorbed across the human small intestine. Only with knowledge of the substrate selectivity (and, therefore, the structural limitations of the active centre),10Newey H. Smyth D.H. The transfer system for neutral amino acids in the rat small intestine.J Physiol. 1964; 170: 328-343Crossref PubMed Scopus (43) Google Scholar ion dependency, tissue distribution, and membrane localization of transporters involved in both nutrient and drug absorption can rational approaches to drug design and treatment of absorptive disorders be implemented. The degree of understanding of the mechanisms involved in nutrient and drug transport across the human small intestine is limited by the lack of availability of viable human tissues and the interspecies variability observed in studies with animal small intestine.5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar, 11Munck B.G. Amino acid transport by the small intestine of the rat.Biochim Biophys Acta. 1966; 120: 97-103Crossref PubMed Scopus (40) Google Scholar, 12Munck B.G. Intestinal absorption of amino acids.in: Johnson L.R. Physiology of the gastrointestinal tract. Raven Press, New York1981: 1097-1122Google Scholar, 13Munck B.G. Transport of imino acids and non-α-amino acid across the brush-border membrane of the rabbit ileum.J Membr Biol. 1985; 83: 15-24Crossref PubMed Scopus (50) Google Scholar, 14Munck L.K. Munck B.G. Distinction between chloride-dependent transport systems for taurine and β-alanine in rabbit ileum.Am J Physiol. 1992; 262: G609-G615PubMed Google Scholar, 15Munck L.K. Munck B.G. Chloride-dependent intestinal transport of imino and β-amino acids in the guinea pig and rat.Am J Physiol. 1994; 266: R997-R1007PubMed Google Scholar Our own studies using confluent monolayers of the human intestinal epithelial cell line Caco-2 describe a low-affinity, high-capacity, proton-coupled amino acid transporter (PAT) at the brush-border membrane.16Thwaites D.T. McEwan G.T.A. Brown C.D.A. Hirst B.H. Simmons N.L. Na+-independent, H+-coupled transepithelial β-alanine absorption by human intestinal Caco-2 cell monolayers.J Biol Chem. 1993; 268: 18438-18441PubMed Google Scholar, 17Thwaites D.T. McEwan G.T.A. Cook M.J. Hirst B.H. Simmons N.L. H+-coupled (Na+-independent) proline transport in human intestinal (Caco-2) epithelial cell monolayers.FEBS Lett. 1993; 333: 78-82Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 18Thwaites D.T. Armstrong G. Hirst B.H. Simmons N.L. d-Cycloserine transport in human intestinal epithelial (Caco-2) cells mediation by a H+-coupled amino acid transporter.Br J Pharmacol. 1995; 115: 761-766Crossref PubMed Scopus (38) Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar, 20Thwaites D.T. Basterfield L. McCleave P.M.J. Carter S.M. Simmons N.L. Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.Br J Pharmacol. 2000; 129: 457-464Crossref PubMed Scopus (59) Google Scholar PAT transports the small zwitterionic amino acids glycine, proline, and alanine17Thwaites D.T. McEwan G.T.A. Cook M.J. Hirst B.H. Simmons N.L. H+-coupled (Na+-independent) proline transport in human intestinal (Caco-2) epithelial cell monolayers.FEBS Lett. 1993; 333: 78-82Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar and a number of orally administered compounds, including d-serine (used in the treatment of schizophrenia),8Tsai G. Yang P. Chung L.-C. Lange N. Coyle J.T. d-Serine added to antipyschotics for the treatment of schizophrenia.Biol Psychiatry. 1998; 44: 1081-1089Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 18Thwaites D.T. Armstrong G. Hirst B.H. Simmons N.L. d-Cycloserine transport in human intestinal epithelial (Caco-2) cells mediation by a H+-coupled amino acid transporter.Br J Pharmacol. 1995; 115: 761-766Crossref PubMed Scopus (38) Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar betaine (used in the treatment of homocystinuria),9Smolin L.A. Benevenga N.J. Berlow S. The use of betaine for the treatment of homocystinuria.J Pediatr. 1981; 99: 467-472Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar, 21Boll M. Foltz M. Anderson C.M.H. Oechsler C. Kottra G. Thwaites D.T. Daniel H. Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1.Mol Membr Biol. 2003; 20: 261-269Crossref PubMed Scopus (52) Google Scholar and d-cycloserine (used as an orally delivered antibiotic and in the treatment of schizophrenia).7Evins A.E. Amico E. Posever T.A. Toker R. Goff D.C. d-Cycloserine added to risperidone in patients with primary negative symptoms of schizophrenia.Schizophr Res. 2002; 56: 19-23Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Thwaites D.T. Armstrong G. Hirst B.H. Simmons N.L. d-Cycloserine transport in human intestinal epithelial (Caco-2) cells mediation by a H+-coupled amino acid transporter.Br J Pharmacol. 1995; 115: 761-766Crossref PubMed Scopus (38) Google Scholar, 20Thwaites D.T. Basterfield L. McCleave P.M.J. Carter S.M. Simmons N.L. Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.Br J Pharmacol. 2000; 129: 457-464Crossref PubMed Scopus (59) Google Scholar In addition, PAT transports the neurotransmitter γ-aminobutyric acid (GABA),20Thwaites D.T. Basterfield L. McCleave P.M.J. Carter S.M. Simmons N.L. Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.Br J Pharmacol. 2000; 129: 457-464Crossref PubMed Scopus (59) Google Scholar several GABA analogues (e.g., nipecotic acid, isonipecotic acid, and 3-amino-1-propanesulfonic acid)20Thwaites D.T. Basterfield L. McCleave P.M.J. Carter S.M. Simmons N.L. Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.Br J Pharmacol. 2000; 129: 457-464Crossref PubMed Scopus (59) Google Scholar that function as GABA receptor agonists or reuptake inhibitors,22Krogsgaard-Larsen P. Falch E. Larsson O.M. Schousboe A. GABA uptake inhibitors relevance to antiepileptic drug research.Epilepsy Res. 1987; 1: 77-93Abstract Full Text PDF PubMed Scopus (178) Google Scholar and the conditionally essential amino acid taurine (which in humans is derived partly from dietary sources, with a particular requirement for absorption in the neonate).23Huxtable R.J. Physiological actions of taurine.Physiol Rev. 1992; 72: 101-163Crossref PubMed Scopus (2308) Google Scholar Because of the clear potential of this high-capacity transport system to play essential roles in both nutrient and drug absorption, it is perhaps surprising that there are apparently no reports of an H+-coupled, PAT-like amino acid transporter in studies with "real" mammalian small intestine. From the 1960s onward, several groups described a transport system in rat small intestine, named variously the sarcosine carrier,10Newey H. Smyth D.H. The transfer system for neutral amino acids in the rat small intestine.J Physiol. 1964; 170: 328-343Crossref PubMed Scopus (43) Google Scholar, 24Daniels V.G. Newey H. Smyth D.H. Stereochemical specificity of neutral amino acid transfer systems in rat small intestine.Biochim Biophys Acta. 1969; 183: 637-639Crossref PubMed Scopus (18) Google Scholar, 25De la Noue J. Newey H. Smyth D.H. Transfer of alanine isomers by rat small intestine.J Physiol. 1971; 214: 105-114PubMed Google Scholar imino acid carrier,11Munck B.G. Amino acid transport by the small intestine of the rat.Biochim Biophys Acta. 1966; 120: 97-103Crossref PubMed Scopus (40) Google Scholar, 12Munck B.G. Intestinal absorption of amino acids.in: Johnson L.R. Physiology of the gastrointestinal tract. Raven Press, New York1981: 1097-1122Google Scholar and methionine-insensitive sarcosine-glycine-proline system.26Thompson E. Levin R.J. Jackson M.J. The stimulating effect of low pH on the amino acid transferring systems of the small intestine.Biochim Biophys Acta. 1970; 196: 120-122Crossref PubMed Scopus (18) Google Scholar It is clear from examination of the literature that this rat intestinal transporter (from now on called the imino acid carrier) has a similar substrate specificity to PAT characterized in human (Caco-2) enterocytes.16Thwaites D.T. McEwan G.T.A. Brown C.D.A. Hirst B.H. Simmons N.L. Na+-independent, H+-coupled transepithelial β-alanine absorption by human intestinal Caco-2 cell monolayers.J Biol Chem. 1993; 268: 18438-18441PubMed Google Scholar, 17Thwaites D.T. McEwan G.T.A. Cook M.J. Hirst B.H. Simmons N.L. H+-coupled (Na+-independent) proline transport in human intestinal (Caco-2) epithelial cell monolayers.FEBS Lett. 1993; 333: 78-82Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 18Thwaites D.T. Armstrong G. Hirst B.H. Simmons N.L. d-Cycloserine transport in human intestinal epithelial (Caco-2) cells mediation by a H+-coupled amino acid transporter.Br J Pharmacol. 1995; 115: 761-766Crossref PubMed Scopus (38) Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar, 20Thwaites D.T. Basterfield L. McCleave P.M.J. Carter S.M. Simmons N.L. Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.Br J Pharmacol. 2000; 129: 457-464Crossref PubMed Scopus (59) Google Scholar Nevertheless, reference to the imino acid carrier in recent literature is limited, because most reviews describe a transporter called the IMINO carrier,27Stevens B.R. Kaunitz J.D. Wright E.M. Intestinal transport of amino acids and sugars advances using membrane vesicles.Annu Rev Physiol. 1984; 46: 417-433Crossref PubMed Scopus (300) Google Scholar, 28Mailliard M.E. Stevens B.R. Mann G.E. Amino acid transport by small intestinal, hepatic, and pancreatic epithelia.Gastroenterology. 1995; 108: 888-910Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 29Palacin M. Estevez R. Bertran J. Zorzano A. Molecular biology of mammalian plasma membrane amino acid transporters.Physiol Rev. 1998; 78: 969-1054Crossref PubMed Scopus (719) Google Scholar characterized in studies with rabbit jejunal brush-border membrane vesicles (BBMV).6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar An assumption has perhaps been made that the rat imino acid transporter and the rabbit IMINO carrier represent species-specific variants of a single transport system (the corresponding mechanism in human intestine is unclear because system PAT has generally been ignored). However, the substrate specificity and ion dependency of the imino acid and IMINO carriers are very different.5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar, 13Munck B.G. Transport of imino acids and non-α-amino acid across the brush-border membrane of the rabbit ileum.J Membr Biol. 1985; 83: 15-24Crossref PubMed Scopus (50) Google Scholar, 15Munck L.K. Munck B.G. Chloride-dependent intestinal transport of imino and β-amino acids in the guinea pig and rat.Am J Physiol. 1994; 266: R997-R1007PubMed Google Scholar The IMINO carrier has a strong preference for imino acids6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar and excludes many substrates of the imino acid carrier, including α-aminoisobutyric acid, glycine, β-alanine, and GABA5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar (all good substrates for PAT).16Thwaites D.T. McEwan G.T.A. Brown C.D.A. Hirst B.H. Simmons N.L. Na+-independent, H+-coupled transepithelial β-alanine absorption by human intestinal Caco-2 cell monolayers.J Biol Chem. 1993; 268: 18438-18441PubMed Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar, 20Thwaites D.T. Basterfield L. McCleave P.M.J. Carter S.M. Simmons N.L. Gamma-aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.Br J Pharmacol. 2000; 129: 457-464Crossref PubMed Scopus (59) Google Scholar In addition, the IMINO carrier has a strong preference for l-proline over d-proline, whereas the imino acid carrier and PAT do not discriminate between these l- and d-enantiomers.5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar, 19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar It is interesting to note that of all the classic amino acid transport systems described functionally, the molecular identity of any imino acid/IMINO carrier(s) has yet to be revealed. The human proton-coupled amino acid transporter 1 (hPAT1), or solute carrier SLC36A1, is a complementary DNA (cDNA) isolated recently from a Caco-2 cDNA library that mediates PAT-like function when expressed in heterologous cell types.30Chen Z. Fei Y.J. Anderson C.M.H. Wake K.A. Miyauchi S. Huang W. Thwaites D.T. Ganapathy V. Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2.J Physiol. 2003; 546: 349-361Crossref PubMed Scopus (134) Google Scholar The substrate specificity of hPAT1 is identical to that of the endogenous PAT system in Caco-2 cells.19Thwaites D.T. McEwan G.T.A. Simmons N.L. The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.J Membr Biol. 1995; 145: 245-256Crossref PubMed Scopus (68) Google Scholar, 30Chen Z. Fei Y.J. Anderson C.M.H. Wake K.A. Miyauchi S. Huang W. Thwaites D.T. Ganapathy V. Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2.J Physiol. 2003; 546: 349-361Crossref PubMed Scopus (134) Google Scholar The similarity in substrate specificity among hPAT1, endogenous PAT, and the rat imino acid carrier suggests that hPAT1 may represent the molecular identity of the human form of the rat imino acid carrier. However, initial comparisons suggest that there are apparently incontrovertible differences in ion dependency. hPAT1, when expressed in isolation (in human retinal pigment epithelial (HRPE) cells or Xenopus laevis oocytes), functions in an H+-coupled, pH-dependent, Na+-independent manner.30Chen Z. Fei Y.J. Anderson C.M.H. Wake K.A. Miyauchi S. Huang W. Thwaites D.T. Ganapathy V. Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco-2.J Physiol. 2003; 546: 349-361Crossref PubMed Scopus (134) Google Scholar, 31Boll M. Foltz M. Rubio-Aliaga I. Daniel H. A cluster of proton/amino acid transporter genes in the human and mouse genomes.Genomics. 2003; 82: 47-56Crossref PubMed Scopus (46) Google ScholarIn contrast, the imino acid and IMINO carriers are described as being Na+ dependent.5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar There is, however, an additional difference between rabbit and rat: transport in intact intestinal tissues is Na+ and Cl− dependent in the rabbit (and guinea pig) but is only partially Na+ dependent (60%) and is Cl− independent in the rat.5Munck B.G. Munck L.K. Rasmussen S.N. Polache A. Specificity of the imino acid carrier in rat small intestine.Am J Physiol. 1994; 266: R1154-R1161PubMed Google Scholar, 14Munck L.K. Munck B.G. Distinction between chloride-dependent transport systems for taurine and β-alanine in rabbit ileum.Am J Physiol. 1992; 262: G609-G615PubMed Google Scholar, 15Munck L.K. Munck B.G. Chloride-dependent intestinal transport of imino and β-amino acids in the guinea pig and rat.Am J Physiol. 1994; 266: R997-R1007PubMed Google ScholarThe Na+ dependence of the rabbit IMINO carrier is unequivocal: this has been shown with jejunal BBMV.6Stevens B.R. Wright E.M. Substrate specificity of the intestinal brush-border proline/sodium (IMINO) transporter.J Membr Biol. 1985; 87: 27-34Crossref PubMed Scopus (69) Google Scholar In contrast, there are no reports of the Na+ dependence of the imino acid carrier with rat intestinal BBMV, and although a slight Na+ dependence in l-proline uptake was observed in human intestinal BBMV from both adult32Rajendran V.M. Ansari S.A. Harig J.M. Adams M.B. Khan A.H. Ramaswamy K. Transport of glycyl-l-proline by human intestinal brush border membrane vesicles.Gastroenterology. 1985; 89: 1298-1304Abstract PubMed Scopus (44) Google Scholar and fetal33Malo C. Multiple pathways for amino acid transport in brush border membrane vesicles isolated from the human fetal small intestine.Gastroenterology. 1991; 100: 1644-1652Abstract PubMed Scopus (32) Google Scholar tissues, no overshoot was reported. The partial Na+ dependency of imino acid transport in studies with intact tissue preparations is reminiscent of that observed with the di/tripeptide transporter (PepT1).34Ganapathy V. Leibach F.H. Is intestinal peptide transport energized by a proton gradient?.Am J Physiol. 1985; 249: G289-G294Google Scholar, 35Thwaites D.T. Kennedy D.J. Raldua D. Anderson C.M.H. Mendoza M.E. Bladen C.L. Simmons N.L. H+/dipeptide absorption across the human intestinal epithelium is controlled indirectly via a functional Na+/H+ exchanger.Gastroenterology. 2002; 122: 1322-1333Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 36Kennedy D.J. Ganapathy V. Leibach F. Thwaites D.T. Optimal absorptive transport of the dipeptide glycylsarcosine is dependent on functional Na+/H+ exchange activity.Pflugers Arch. 2002; 445: 139-146Crossref PubMed Scopus (93) Google Scholar, 37Anderson C.M.H. Mendoza M.E. Kennedy D.J. Raldua D. Thwaites D.T. Inhibition of intestinal dipeptide transport by the neuropeptide VIP is an anti-absorptive effect via the VPAC1 receptor in a human enterocyte-like cell line (Caco-2).Br J Pharmacol. 2003; 138: 564-573Crossref PubMed Scopus (24) Google Scholar The PepT1 clone is an H+-coupled transporter that functions independently of extracellular Na+ after heterologous expression.2Ganapathy V. Brandsch M. Leibach F.H. Intestinal transport of amino acids and peptides.in: Johnson L.R. Physiology of the gastrointestinal tract. Raven, New York1994: 1773-1794Google Scholar, 36Kennedy D.J. Ganapathy V. Leibach F. Thwaites D.T. Optimal absorptive transport of the dipeptide glycylsarcosine is dependent on functional Na+/H+ exchange activity.Pflugers Arch. 2002; 445: 139-146Crossref PubMed Scopus (93) Google Scholar The driving force (the H+ electrochemical gradient) for H+-coupled nutrient transport in the small intestine is present at the luminal surface as an area of low pH (pH 6.1–6.8) called the acid microclimate.38McEwan G.T.A. Daniel H. Fett C. Burgess M.N. Lucas M.L. The effect of Escherichia coli STa enterotoxin and other secretagogues on mucosal surface pH of rat small intestine in vivo.Proc R Soc Lond B. 1988; 234: 219-237Crossref PubMed Scopus (70) Google Scholar, 39Daniel H. Fett C. Kratz A. Demonstration and modification of intervillous pH profiles in rat small intestine in vitro.Am J Physiol. 1989; 257: G489-G495PubMed Google ScholarIn intact tissue preparations, dipeptide transport shows partial Na+ dependence, whereas no Na+ dependence is observed in studies that use intestinal BBMV.34Ganapathy V. Leibach F.H. Is intestinal peptide transport energized by a proton gradient