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PubMed1998

Sodium-dependent homo- and hetero-exchange of neutral amino acids mediated by the amino acid transporter ATB degree.

We have investigated the functional characteristics of the human amino acid transporter ATB degree using the Xenopus laevis oocyte expression system. When expressed in oocytes, ATB degree mediates the uptake of neutral amino acids in an Na(+)-dependent manner. In addition, this transporter is able to mediate the efflux of intracellular neutral amino acids in exchange with extracellular neutral amino acids. This homo- and hetero-exchange of amino acids is absolutely Na(+)-dependent and conforms strictly to the substrate specificity of ATB degree. Kinetic analysis indicates that the affinity of ATB degree for a given amino acid substrate is similar whether ATB degree catalyzes the influx of the amino acid or the amino acid-induced efflux of intracellular amino acids. These results demonstrate for the first time the ability of ATB degree to function as a homo- and hetero-exchanger for its substrates.

Amino Acid Transport Systems↗
PubMed2002

Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon.

D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine.

Amino Acid Transport Systems, Basic↗
PubMed1996

GNP1, the high-affinity glutamine permease of S. cerevisiae.

Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have isolated the gene encoding the high-affinity glutamine permease and named it GNP1. The amino-acid sequence of GNP1, and its hydropathy profile of 12 transmembrane domains, closely resemble those of known amino-acid permeases. The Km of GNP1 for glutamine uptake was determined to be 0.59 mM. Cells lacking GNP1 exhibit reduced levels of glutamine transport, and are resistant to a toxic analog of glutamine, L-glutamic acid gamma-monohydroxamate. Unlike other amino-acid permeases, whose expression is nitrogen-source limited, GNP1 is expressed on both rich and poor nitrogen sources.

Amino Acid Sequence↗
PubMed1999

Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases.

Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition.

ATP-Binding Cassette Transporters↗
PubMed1989

Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases.

The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

ATP-Binding Cassette Transporters↗
PubMed2006

Selective decrease of SN1(SNAT3) mRNA expression in human and rat glioma cells adapted to grow in acidic medium.

The system N glutamine (Gln) transporter SN1(SNAT3) is overexpressed in human malignant glioma cells in situ as compared to the adjacent brain tissue or metastases from different organs [Sidoryk, M., Matyja, E., Dybel, A., Zielińska, M., Bogucki, J., Jaskólski, D.J., Liberski, P.P., Kowalczyk, P., Albrecht, J., 2004]. Increased expression of a glutamine transporter SNAT3 is a marker of malignant gliomas. NeuroReport 15, 575-578], but its role in tumor growth as compared to the other Gln transporters is unknown. One of the profound, growth-promoting effects of glial tumor in situ is acidification of the extracellular space. In the kidney SN1(SNAT3) mRNA participates in the adaptation to acidosis. In this study therefore, expression of mRNAs coding for SN1(SNAT3) and other Gln transporters was measured in human (T98G) and rat (C6) glioma cells incubated for 4h in an acidic medium (AI) (pH 6.5). MTT assay revealed no cell loss in AI cells, and intracellular pH (pHi) as measured by a fluorescent probe (BCECF-AM) was slightly alkaline in C6 and T98G cells, indicating that the cells have adapted to AI. AI significantly decreased the SN1(SNAT3) mRNA expression in C6 (a 60% decrease) and T98G cells (a 50% decrease). The decrease retreated in C6 cells 4h after transferring them back to the neutral medium. The expression of ASCT2 mRNA (system ASC), ATA1 mRNA (system A) and SN2(SNAT5) mRNA (system N) were not affected by AI in either of the cell lines. [(3)H]Gln uptake in C6 or T98G cells grown in neutral medium was mainly mediated by system ASCT2: system N contributed to only approximately 7% of the uptake. AI did not affect the total Gln uptake, and only slightly decreased the system N-mediated component of the uptake. Hence, SN1(SNAT3) does not seem to be involved in the adaptation of cultured glioma cells to acidic millieu.

Amino Acid Transport System A↗
PubMed1998

Induction of cystine transport via system x-c and maintenance of intracellular glutathione levels in pancreatic acinar and islet cell lines.

The relationship between l-cystine transport and intracellular glutathione (GSH) levels was investigated in cultured pancreatic AR42J acinar and betaTC3 islet cells exposed to diethylmaleate, an electrophilic agent known to activate cellular antioxidant responses. Cystine transport was mediated predominantly by the Na+-independent anionic amino acid transport system x-c, with influx inhibited potently by glutamate and homocysteate but unaffected by cationic or neutral amino acids. Saturable cystine transport was 10-fold higher in AR42J (531 pmol (mg protein)-1 min-1) than in betaTC3 (49 pmol (mg protein)-1 min-1) cells, and GSH levels were higher in AR42J cells. Treatment with 2-mercaptoethanol increased GSH levels in betaTC3 cells from 7.5 to 36 nmol (mg protein)-1, whilst the GSH content in AR42J cells (64 nmol (mg protein)-1) was not altered significantly. Incubation of AR42J or betaTC3 cells with homocysteate (2.5 mM, 0-48 h), a competitive inhibitor of cystine transport via system x-c, reduced intracellular GSH levels and resulted in a time-dependent (6-24 h) induction of system x-c transport activity. Treatment of AR42J cells with diethylmaleate (100 microM, 0-48 h) resulted in a time- (5-10 h) and protein synthesis-dependent induction of cystine transport, with intracellular GSH levels initially decreasing and then increasing 2-fold above control levels after 24 h. Diethylmaleate also depressed GSH levels in betaTC3 cells, but cystine transport was not elevated significantly. In both AR42J and betaTC3 cells, inhibition of gamma-glutamyl cysteine synthetase by buthionine sulphoximine (100 microM, 24 h) reduced GSH levels but had no effect on cystine transport. The present findings establish that induction of system x-c leads to changes in GSH levels in pancreatic AR42J acinar and betaTC3 islet cells, with changes in the intracellular redox state stimulating transporter expression. Induction of activity of system x-c, together with adaptive increases in GSH synthesis in response to oxidative stress, may contribute to cellular antioxidant defences in pancreatic disease.

Amino Acid Transport Systems, Basic↗
PubMed2001

Carrier-mediated processes in blood--brain barrier penetration and neural uptake of paraquat.

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringer's solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

1-Methyl-4-phenylpyridinium↗
PubMed1997

Tyrosine phosphorylation-and epidermal growth factor-dependent regulation of the sodium-coupled amino acid transporter B0 in the human placental choriocarcinoma cell line JAR.

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B(0) (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657-18661, 1996). The presence of the amino acid transport system B(0) (ATB(0)) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB(0) in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na(+)-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB(0). Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB(0) activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB(0) activity and on ATB(0) mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB(0) activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB(0) activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B(0) and that the expression of the system in these cells is stimulated by EGF.

Alanine↗
PubMed2000

Substrate specificity of the brush border K+-leucine symport of Bombyx mori larval midgut.

L-leucine uptake into membrane vesicles from Bombyx mori larval midgut was tested for inhibition by 55 compounds, which included sugars, N-methylated, alpha-, beta-, gamma-, delta-, epsilon-amino acids, primary amines, alpha-amino alcohols, monocarboxylic organic acids and alpha-ketoacids. Based on cis-inhibition experiments performed at the high pH (10.8) characteristic of the midgut luminal content in vivo, we find that the carrier binding site interacts with molecules which possess a well-defined set of structural features. Amino acids are preferentially accepted as anions and the ideal inhibitor must have an hydrophobic region and a polar head constituted by a chiral carbon atom bearing two hydrophilic groups, a deprotonated amino-group and a dissociated carboxylic group. Binding is reduced if one of the two hydrophilic groups is removed. Lowering the pH to less alkaline value (8.8) only affects the affinity of delta- and epsilon-amino acids, which are excluded from binding because of their positively charged side-chain. Modifications of the potassium electrochemical gradient increased the affinity constant values of the molecules, but have little effect on the rank of specificity. Physiological implications of the data reported are discussed.

Amino Acid Transport Systems↗
PubMed2006

Heterogeneous mutations in the SLC3A1 and SLC7A9 genes in Chinese patients with cystinuria.

Cystinuria is a recessively inherited aminoaciduria that leads to recurrent urolithiasis. It is caused by the defective transport of cystine and dibasic amino acids in the proximal renal tubules and intestinal epithelium. Two genes responsible for this, SLC3A1 and SLC7A9, are known. Patients with two SLC3A1 mutations are classified as type A cystinuria, whereas patients with two SLC7A9 mutations are classified as type B cystinuria. Few clinical and molecular data have been reported for Asian cystinuria patients. In this study, we determined the molecular basis of cystinuria in eight unrelated Chinese subjects. Coding exons and flanking introns of the SLC3A1 and SLC7A9 genes were directly sequenced after amplification by polymerase chain reaction. Five different SLC3A1 mutations were found. Two missense mutations, D210G and S547L, were novel. The other three SLC3A1 mutations (IVS6+2T>C, R181Q and R365W) have been described previously. In addition, four novel SLC7A9 mutations, C137R, c.730delG, IVS10+2_3delTG and IVS12+3insT, together with two previously reported mutations (A70V and G195R) were found. All patients except one carried compound heterozygous mutations. IVS12+3insT was detected in patients from two families. This is the first molecular genetic study on Chinese cystinuria patients. Three patients with type A cystinuria, two with type B cystinuria, and three carriers of type B cystinuria were identified. Our results suggest that the molecular basis of cystinuria is heterogeneous in our local population.

Adult↗
PubMed1994

Regulation of System B0 amino-acid-transport activity in the renal epithelial cell line NBL-1 and concomitant changes in SAAT1 hybridizing transcripts.

alpha-(Methylamino)isobutyric acid (MeAIB) insensitive Na(+)-dependent alanine transport activity in the bovine kidney cell line NBL-1 was increased upon amino acid starvation (> or = 20% over control levels). When L-phenylalanine (3 mM) was included in the starvation medium the increase was further enhanced (> or = 85% over control levels). In cells grown in control medium the Vmax, for MeAIB-insensitive Na+/alanine co-transport was found to be 6.0 +/- 0.7 nmol/3 min per mg (Km 41 +/- 12 microM) and for L-phenylalanine-treated amino-acid-starved cells the Vmax. was 21 +/- 5 nmol/3 min per mg (Km 92 +/- 40 microM). The increase in Vmax. was prevented by cycloheximide. Substrate specificity analysis identified the L-phenylalanine-induced transport system as System B0. [35S]Methionine labelling of cells during the amino acid starvation/phenylalanine treatments resulted in the differential labelling of a protein of 78 kDa. Northern-blot analysis using a SAAT1-specific probe revealed the presence of a new transcript (3.2 kb) in RNA extracted from cells incubated in amino acid starvation medium with L-phenylalanine included. The present findings suggest a novel means of control for System B0 by the use of physiological stress. It is also proposed that SAAT1 and System-B0 transcripts have considerable sequence similarity.

Alanine↗
PubMedDate not supplied

Amino acid prodrugs of acyclovir as possible antiviral agents against ocular HSV-1 infections: interactions with the neutral and cationic amino acid transporter on the corneal epithelium.

PURPOSE: The aim of this study was to explore the feasibility of improvement of ocular bioavailability of the antiviral agent acyclovir by designing amino acid prodrugs targeted to the amino acid transporters on the rabbit cornea. MATERIALS AND METHODS: Transcorneal flux of two water-soluble amino acid ester prodrugs of acyclovir (ACV), gamma-glutamate-ACV (EACV) and L-tyrosine-ACV (YACV), was studied across freshly excised rabbit cornea. Chemical and enzymatic hydrolysis studies of the two prodrugs were also conducted. RESULTS: EACV inhibited the uptake of [(3)H]L-Arg in rabbit primary corneal epithelial cells (rPCECs). The compound also exhibited longer half-life (t(1/2)) in cornea in comparison to YACV. Transcorneal flux of EACV was observed to be concentration-, energy-, and sodium-dependent and independent of pH within the range studied. EACV transport was inhibited by neutral and cationic amino acids, L-ornithine (specific for cationic amino acids), and BCH (2-aminobicyclo-[2,2,1]-heptane-2-carboxylic-acid) (specific inhibitor for L-type system and B(0,+) system). On the other hand, YACV was not recognized by this amino acid transporter as it failed to inhibit the uptake of [(3)H]Arg, and also its transport across cornea was not inhibited by arginine. YACV and EACV exhibited excellent antiviral activity against HSV-1 and 2 and Varicella-Zoster Virus (VZV) in comparison to ACV. CONCLUSIONS: Analyses of the inhibition pattern of EACV transport suggests the involvement of a single transport system; namely, B(0,+). Design of amino acid prodrugs seems to be an attractive strategy to enhance the solubility of the otherwise poorly aqueous soluble compounds and also to afford a targeted and possibly enhanced delivery of the active drug.

Acyclovir↗
PubMed2005

Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome.

In 11 patients with a recessive congenital disorder, which we refer to as "the hypotonia-cystinuria syndrome," microdeletion of part of the SLC3A1 and PREPL genes on chromosome 2p21 was found. Patients present with generalized hypotonia at birth, nephrolithiasis, growth hormone deficiency, minor facial dysmorphism, and failure to thrive, followed by hyperphagia and rapid weight gain in late childhood. Since loss-of-function mutations in SLC3A1 are known to cause isolated cystinuria type I, and since the expression of the flanking genes, C2orf34 and PPM1B, was normal, the extended phenotype can be attributed to the deletion of PREPL. PREPL is localized in the cytosol and shows homology with prolyl endopeptidase and oligopeptidase B. Substitution of the predicted catalytic residues (Ser470, Asp556, and His601) by alanines resulted in loss of reactivity with a serine hydrolase-specific probe. In sharp contrast to prolyl oligopeptidase and oligopeptidase B, which require both aminoterminal and carboxyterminal sequences for activity, PREPL activity appears to depend only on the carboxyterminal domain. Taken together, these results suggest that PREPL is a novel oligopeptidase, with unique structural and functional characteristics, involved in hypotonia-cystinuria syndrome.

Amino Acid Sequence↗
PubMed2005

Identification of novel cystinuria mutations and polymorphisms in SLC3A1 and SLC7A9 genes: absence of SLC7A10 gene mutations in cystinuric patients.

Cystinuria represents 3% of nephrolithiasis in humans with an overall prevalence of 1 in 7,000 neonates. Two genes have been reported to account for the genetic basis of cystinuria, the SLC3A1 and the SLC7A9. Recently, the possible involvement of the SLC7A10 gene in the genetic basis of the disorder was also reported. In the present study, we found a total of 15 mutations in 20 Greek cystinuric patients. Eight mutations are novel, 4 in the SLC3A1: F266S, T351I, R456C, and N516D, and 4 in the SLC7A9: 479-1G>C, Y232C, D233E, and 1399+1G>T. Furthermore, 2 polymorphisms were identified in the SLC3A1 gene and 16 polymorphic variants were also found in the SLC7A9 gene of which the 235+18C>A, 604+10G>A, and 604+24T>C are novel. Finally, no mutation was found in the SLC7A10 gene in all patients. Only, the novel 634+8C>G and the previously reported 913-11C+T polymorphisms were identified in the SLC7A10 gene. In conclusion, a spectrum of SLC3A1 and SLC7A9 mutations are responsible for the genetic basis of cystinuria in Greek patients.

Amino Acid Transport System y+↗