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PubMed2002

The regionalization of PepT1, NBAT and EAAC1 transporters in the small intestine of rats are unchanged from birth to adulthood.

The ontogenetic development of PepT1, NBAT and EAAC1 along the vertical and horizontal axes of the rat small intestine was evaluated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The proximodistal profiles of mRNA levels showed that PepT1 was evenly distributed, whereas NBAT had greater expression in the proximal part, and EAAC1 in the distal part. These regionalizations were the same from postnatal days 4 to 50. PepT1 and NBAT proteins were detected in the microvilli of enterocytes along the length of the villi. NBAT was also found in the cytoplasm. Surprisingly, EAAC1 was located exclusively in the microvilli of enterocytes in the crypt and the bases of the villi. These protein expression patterns were similar in all parts of the small intestine (proximal, median and distal), at all ages. We conclude that the expression of PepT1, NBAT or EAAC1 are differently regulated according to both the horizontal and vertical axes.

Aging↗
PubMed2002

Purification, reconstitution, and characterization of Na(+)/serine symporter, SstT, of Escherichia coli.

A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.

Amino Acid Transport Systems↗
PubMed2005

Identification of novel SLC3A1 gene mutations in Spanish cystinuria families and association with clinical phenotypes.

Cystinuria is an inherited metabolic disease characterized by an abnormal urinary excretion of cystine and dibasic amino acids, leading to kidney stone formation. Incidence of cystinuria in the Mediterranean Spanish population is one of the highest in the world. In view of the low prevalence of previously reported mutations in the SLC3A1 gene, analyses to identify novel variants were carried out on 20 cystinuria families. Additionally, we investigated the possible association between these molecular variants and clinical phenotypes. Genomic DNA from 48 cystinuria patients, 44 healthy relatives and 81 unrelated controls from the East Mediterranean coast of Spain was screened by conformation sensitive gel electrophoresis. Abnormal patterns were confirmed by nucleotide sequence determination and by further restriction fragment-length polymorphism. We only found 11 genetic variants within the SLC3A1 gene: five known polymorphisms (114C > A, 231T > A, 1136 + 3delT, 1332 + 7T > C and 1338G > A), four point mutations (M467T, R452W, I105R and Y461X), one single base pair deletion (1767delA) and one 2-bp insertion (1670insAT). Two of these genetic variants (I105R and 1670insAT) were described for the first time. All mutations but one were detected in families classified as Type I cystinuria due to the transmission pattern of the disease. Association analyses revealed that 231T > A (M467T), 1136 + 3delT and 1332 + 7T > C genetic variants were statistically related with urinary amino acid excretion in cystinuria patients. Although some molecular variants within the SLC3A1 gene were associated with clinical traits in cystinuria patients, the low detection rate of mutations in this gene strongly suggests that variation of the SLC3A1 is not the major genetic factor contributing to cystinuria in this Mediterranean population.

Adolescent↗
PubMed2006

Analysis of a 1-year-old cystinuric patient with recurrent renal stones.

Cystinuria is a hereditary disorder of cystine and dibasic amino acids (lysine, arginine, ornithine) transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. Cystine stones frequently occur in the second or third decade of life with an occasional occurrence in infancy and in old age. Herein is presented the case of a 1-year-old girl with cystinuria and recurrent urolithiasis; the genetic basis of the disease was investigated by mutational analysis of the SLC3A1 gene. The data show that the present patient has an increased cystine (923.08 microg/mL) level and was heterozygote for M467T mutation.

Amino Acid Transport Systems, Basic↗
PubMed2005

Transient neonatal cystinuria.

BACKGROUND: Cystinuria is an inherited disorder of luminal reabsorptive transport for cystine and dibasic amino acids in the renal proximal tubule. Two cystinuria genes have been identified. Mutations of SLC7A9, which encodes the luminal transport channel itself, tend to be dominant and mutations of SLC3A1 (rBAT), which encodes a transporter subunit, are always recessive. Patients who inherit two recessive mutations or two dominant mutations have equally severe forms of cystinuria. Heterozygotes excrete cystine in the normal (type I), moderate (type III), or high stone-forming (type II) range. METHODS: Infants with cystinuria were identified via the Quebec Newborn Urinary Screening Program. In a subgroup of these infants, cystinuria was severe in the first months of life, but partially resolved by 2 to 4 years postnatally. We assigned each patient a final cystinuria phenotype at 3 to 4 years. In addition, we characterized SLC3A1 gene expression in fetal and postnatal human kidney. RESULTS: Most infants with transient neonatal cystinuria are eventually classified as type III heterozygotes. All infants with mutant cystinuria genes have exaggerated neonatal cystine excretion except those who inherit two SLC3A1 mutations (type I/I cystinuria); these children have persistent severe cystinuria, implying that wildtype SLC3A1 is required for the maturational effect. Expression of SLC3A1 mRNA was found to be tenfold higher in postnatal vs. fetal kidney; SLC3A1 expression is doubled by the proximal tubule transcription factor, PAX8. rBAT is expressed in the proximal convoluted and straight tubules in both fetal and adult kidney. CONCLUSION: Maturation of SLC3A1 gene expression between midgestation and 4.5 years postnatal age may account for transient neonatal cystinuria.

Amino Acid Transport Systems, Basic↗
PubMed2005

Molecular genetic analysis of SLC3A1 and SLC7A9 genes in Czech and Slovak cystinuric patients.

Cystinuria is a frequently inherited metabolic disorder in the Czech population (frequency 1/5,600) caused by a defect in the renal transport of cystine and dibasic amino acids (arginine, lysine and ornithine). The disease is characterized by increased urinary excretion of the amino acids and often leads to recurrent nephrolithiasis. Cystinuria is classified into two subtypes (type I and type non-I). Type I is caused predominantly by mutations in the SLC3A1 gene (2p16.3), encoding heavy subunit (rBAT) of the heterodimeric transporter. Cystinuria non-I type is caused by mutations in the SLC7A9 gene (19q13.1). In this study, we present results of molecular genetic analysis of the SLC3A1 and the SLC7A9 genes in 24 unrelated cystinuria families. Individual exons of the SLC3A1 and SLC7A9 genes were analyzed by direct sequencing. We found ten different mutations in the SLC3A1 gene including six novel ones: three missense mutations (G140R), D179Y and R365P), one splice site mutation (1137-2A>G), one deletion (1515_1516delAA), and one nonsense mutation (Q119X). The most frequent mutation, M467T; was detected in 36% of all type I classified alleles. In the SLC7A9 gene we found six mutations including three new ones: one missense mutation (G319R), one insertion (611_612insA) and one deletion (205_206delTG). One patient was compound heterozygote for one SLC3A1 and one SLC7A9 mutation. Our results confirm that cystinuria is a heterogeneous disorder at the molecular level, and contribute to the understanding of the distribution and frequency of mutations causing cystinuria in the Caucasian population.

Adolescent↗
PubMed2003

Nitric oxide synthesis requires activity of the cationic and neutral amino acid transport system y+L in human umbilical vein endothelium.

L-arginine transport is mediated by the cationic/neutral amino acid transport system y+L and cationic amino acid transporters y+/CATs in human umbilical vein endothelial cells (HUVECs). System y+/CATs activity may be rate-limiting for nitric oxide (NO) synthesis, but no reports have demonstrated system y+L involvement in NO synthesis in endothelium. We investigated the role of system y+L in NO synthesis in HUVECs. Transport of 1.5 microM L-arginine was inhibited (P < 0.05) by L-lysine (K(i), 1.4 micro M), L-leucine (K(i), 1.8 micro M) and L-phenylalanine (K(i), 4.1 microM), but was unaltered (P > 0.05) by L-alanine or L-cysteine. The system y+/CATs inhibitor, N-ethylmaleimide (NEM), did not alter 1.5 microM L-arginine transport, but inhibited (92 +/- 3 %) 100 microM L-arginine transport. L-arginine transport in the presence of NEM was saturable (V(max), 0.37 +/- 0.02 pmol (microg protein)(-1) min(-1); K(m), 1.5 +/- 0.3 microM) and competitively inhibited by L-leucine in the presence of Na+ (V(max), 0.49 +/- 0.06 pmol (microg protein)(-1) min(-1); K(m), 6.5 +/- 0.9 microM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for the high-affinity transport system y+L. N(G)-Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 microM L-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y+L activity (V(max), 0.15 +/- 0.008 pmol (microg protein)(-1) min(-1); K(m), 1.4 +/- 0.3 microM) and NO synthesis. However, 25 mM D-glucose increased NO synthesis and L-arginine transport via system y+. Thus, L-arginine transport through system y+L plays a role in NO synthesis, which could be a determining factor in pathological conditions where the endothelial L-arginine-NO pathway is altered, such as in diabetes mellitus.

Amino Acid Transport System y+L↗
PubMed1981

Cellular distribution of neutral and basic amino acid transport systems in rabbit ileal mucosa.

1. An autoradiographic technique is described whereby the cellular location of tritiated amino acids can be determined following uptake by rabbit ileal mucosa. 2. Stirring solutions in contact with the intestinal mucosa during measurement of rapid influx changes the quantity, but not the distribution, of alanine taken up by the tissue. 3. Conditions predicted to favour either a high affinity system (Ly1) or a low affinity system (Ly2) were used to measure lysine distribution following uptake. Maximal uptake for both transport systems occurred in fully differentiated enterocytes at the tips of villi. Initial maturation of the Ly1 system, which was slow, was followed by a rapid phase of development. The Ly2 system lacked this rapid phase of late development. 4. The cellular distribution of alanine entering on a low affinity Na-independent neutral amino acid carrier closely resembles that determine for Ly1 system for lysine entry. 5. Arginine is a potent inhibitor of lysine uptake through the Ly2 system. Little or no diffusion of lysine appears to take place into rabbit ileal enterocytes. 6. The different distribution of the high and low affinity systems for lysine transport provides further support for their independent existence. It also suggests that more than one message exists fo the switching on of amino acid transport function in differentiating enterocytes.

Alanine↗
PubMed1998

Cloning and expression of the gene for the Na+-coupled serine transporter from Escherichia coli and characteristics of the transporter.

We cloned a gene (sstT) for the Na+/serine symporter from the chromosome of Escherichia coli by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.

Amino Acid Transport Systems↗
PubMed1998

The Saccharomyces cerevisiae YCC5 (YCL025c) gene encodes an amino acid permease, Agp1, which transports asparagine and glutamine.

The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.

Amino Acid Transport Systems↗
PubMed2005

New insights into cystinuria: 40 new mutations, genotype-phenotype correlation, and digenic inheritance causing partial phenotype.

OBJECTIVE: To clarify the genotype-phenotype correlation and elucidate the role of digenic inheritance in cystinuria. METHODS: 164 probands from the International Cystinuria Consortium were screened for mutations in SLC3A1 (type A) and SLC7A9 (type B) and classified on the basis of urine excretion of cystine and dibasic amino acids by obligate heterozygotes into 37 type I (silent heterozygotes), 46 type non-I (hyperexcretor heterozygotes), 14 mixed, and 67 untyped probands. RESULTS: Mutations were identified in 97% of the probands, representing 282 alleles (86.8%). Forty new mutations were identified: 24 in SLC3A1 and 16 in SLC7A9. Type A heterozygotes showed phenotype I, but mutation DupE5-E9 showed phenotype non-I in some heterozygotes. Type B heterozygotes showed phenotype non-I, with the exception of 10 type B mutations which showed phenotype I in some heterozygotes. Thus most type I probands carried type A mutations and all type non-I probands carried type B mutations. Types B and A mutations contributed to mixed type, BB being the most representative genotype. Two mixed cystinuria families transmitted mutations in both genes: double compound heterozygotes (type AB) had greater aminoaciduria than single heterozygotes in their family. CONCLUSIONS: Digenic inheritance is an exception (two of 164 families), with a limited contribution to the aminoaciduria values (partial phenotype) in cystinuria. Further mutational analysis could focus on one of the two genes (SLC3A1 preferentially for type I and SLC7A9 for type non-I probands), while for mixed probands analysis of both genes might be required, with priority given to SLC7A9.

Amino Acid Transport Systems, Basic↗
PubMed2001

Cloning and functional characterization of a new subtype of the amino acid transport system N.

We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50-56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na(+)-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li(+) tolerant, and pH sensitive. The transport mechanism involves the influx of Na(+) and amino acids coupled to the efflux of H(+), resulting in intracellular alkalization. Proline, alpha-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.

Amino Acid Sequence↗
PubMed2004

The molecular basis of cystinuria.

Cystinuria is an inherited form of nephrolithiasis due to failure of reabsorptive transport in the proximal tubule. Patients with classical recessive cystinuria have inherited two mutations of the SLC3A1 gene, encoding a subunit of the transport mechanism. Patients with the dominant form of cystinuria have inherited two mutations of the SLC7A9 gene, encoding the transport channel itself. A smaller subset of patients have mixed-type cystinuria, combining recessive and dominant mutant alleles. Children at risk for nephrolithiasis can be identified by the level of urinary cystine only after tubular transport has matured (age 2 years). Conservative therapy with high urine volume and urinary alkalinization is sufficient for some, but recurrent stone formation may cause renal damage and warrants prophylaxis with agents that form mixed disulfides with cystine.

Amino Acid Transport Systems, Basic↗
PubMed1999

Large neutral amino acids block phenylalanine transport into brain tissue in patients with phenylketonuria.

Large neutral amino acids (LNAAs), including phenylalanine (Phe), compete for transport across the blood-brain barrier (BBB) via the L-type amino acid carrier. Accordingly, elevated plasma Phe impairs brain uptake of other LNAAs in patients with phenylketonuria (PKU). Direct effects of elevated brain Phe and depleted LNAAs are probably major causes for disturbed brain development and function in PKU. Competition for the carrier might conversely be put to use to lower Phe influx when the plasma concentrations of all other LNAAs are increased. This hypothesis was tested by measuring brain Phe in patients with PKU by quantitative 1H magnetic resonance spectroscopy during an oral Phe challenge with and without additional supplementation with all other LNAAs. Baseline plasma Phe was approximately 1,000 micromol/l and brain Phe was approximately 250 micromol/l in both series. Without LNAA supplementation, brain Phe increased to approximately 400 micromol/l after the oral Phe load. Electroencephalogram (EEG) spectral analysis revealed acutely disturbed brain activity. With concurrent LNAA supplementation, Phe influx was completely blocked and there was no slowing of EEG activity. These results are relevant for further characterization of the LNAA carrier and of the pathophysiology underlying brain dysfunction in PKU and for treatment of patients with PKU, as brain function might be improved by continued LNAA supplementation.

Adult↗
PubMed1978

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.

Alanine↗