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PubMed2003

[Cystinuria update: clinical, biochemical and genetic aspects].

Cystinuria is an autosomal recessive disorder with an estimated incidence of 1 case in 7000 live births, that results in elevated urinary excretion of cystine and dibasic aminoacids: ornithine, lysine and arginine. Discussed by Sir Archibald Edward Garrod, in 1908, as one of the four first known inborn errors of metabolism, it is characterized by a defect in transport of cystine and dibasic aminoacids, that affects their reabsortion in both renal tubule and gastrointestinal tract. To date, according to the recent molecular findings, two genes have been identified as responsible for this disease: SLC3A1 and SLC7A9. A more accurate pheno/genotyping identification of cystinuric patients will allow to improve prophilaxis and therapy for this illness. Cystinuria only causes recurrent urolithiasis (about 1-2 / of renal calculi in adults) and its associated complications as clinical feature because of poor cystine solubility at low pH. An accurate control over prohylaxis (based on high water intake and potassium citrate treatment, on first line, and tiol-derivatives treatment, on second line) must be taken in patients -like homozygous type I- with high lithiasis risk. However, approximately one half of patients under prophylaxis control will develop recurrent lithiasis; in this case, only urology or surgical approaches would be possible. 474 Updated knowledge about biochemical, genetic, clinical, diagnosis, prevention, treatment and prognosis aspects of this, relatively unusual, disease has been reviewed in this article.

Amino Acid Transport Systems, Basic↗
PubMed1994

Electrogenic amino acid exchange via the rBAT transporter.

A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Alanine↗
PubMed1996

Effects of truncation of the COOH-terminal region of a Na+-independent neutral and basic amino acid transporter on amino acid transport in Xenopus oocytes.

To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Delta511-685 mutant, stimulated the uptake of these amino acids. The Delta511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Delta511-685 mutant also increased the uptake of leucine. The Delta511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Delta511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Delta511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Delta511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Delta511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.

Amino Acid Sequence↗
PubMedDate not supplied

Co-localization and interaction of b0,+-type amino acid transporter 1 (BAT1) with caveolin-1 in rat kidney.

BACKGROUND: Cystinuria has been proposed as an inherited disease causing disorders in renal cystine and basic amino acid transport in the proximal tubules. Although cystinuria-related amino acid transporter gene related to b0,+-type amino acid transporter (rBAT1) and its substrate transport properties have been reported, the functional regulatory mechanisms remain to be elucidated. In this study, protein-protein interaction between rBAT1 and caveolin (Cav)-1 was investigated. METHODS: The renal distribution of rBAT1, rBAT and Cav-1 were demonstrated by employing reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Co-localization of rBAT1 and Cav-1 was observed by immunocytochemistry in primary cultured renal proximal tubule-derived cells using a confocal microscope. This result was confirmed by Western blot analysis of isolated caveolae-rich membrane fraction and immunoprecipitation experiments using respective antibodies. RESULTS: In the separated rat kidney tissues following the corticomedullary axis, Cav-1 mRNA and protein expressions were increased from the cortex to the inner medulla. rBAT1 mRNA and protein expression were detected mainly in the outer medulla. Confocal microscopic results showed rBAT1 and Cav-1 co-localization in the plasma membrane. This result was confirmed by Western blot analysis of caveolae-rich membrane fraction and immunoprecipitates by respective antibodies. The effect of Cav-1 on rBAT1 function was evaluated using Cav-1 antisense oligodeoxynucleotide (ODN). The [14C] arginine uptake by rBAT1 was unchanged by the treatment with antisense ODN. CONCLUSIONS: From these results, rBAT1 and Cav-1 share a cellular expression in the segregated caveolae structure. As caveolae are rich in signaling molecules, BAT1 could play a role in diverse pathophysiological processes.

Amino Acid Transport Systems, Basic↗
PubMed2002

Lysine uptake by cloned hCAT-2B: comparison with hCAT-1 and with trophoblast surface membranes.

To study the cationic amino-acid transporter hCAT-2B of human placenta, total RNA was harvested from primary cultured trophoblast and from the BeWo choriocarcinoma cell line (b30 clone) and used for reverse transcription (RT) and polymerase chain reaction (PCR). Primers based on published sequences identified expression of mRNA for hCAT-2B. RT-PCR yielded a 2.06 kb hCAT-2B cDNA, which was cloned. hCAT-2B cRNA injection into Xenopus laevis oocytes stimulated saturable lysine uptake (Km approximately 125 mM). In the presence of Na+, uptake was completely inhibited by L-arginine but only partially by neutral amino acids. To compare directly the interaction of hCAT-1 and hCAT-2B with neutral amino acids and sodium, we examined the inhibition of these transporters by L-leucine and L-alanine over a wide concentration range. L-Alanine and L-leucine inhibit uptake by hCAT-2B substantially less completely than uptake by hCAT-1. The interaction of hCAT-2B resembles that of system y+ in the microvillous membrane of human placenta, while that of hCAT-1 is more comparable to that of system y+ in basal membrane. The identification and characterization of the various cationic amino-acid transporters of the human placenta have the potential to increase the understanding of the cellular mechanism of transplacental transfer.

Alanine↗
PubMed1999

New glycoprotein-associated amino acid transporters.

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked "light chain" of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y(+)LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b(0,+)AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family).

Amino Acid Transport System y+↗
PubMed2003

The ancillary proteins of HATs: SLC3 family of amino acid transporters.

The heteromeric amino acid transporters (HATs) are composed of a light and a heavy subunit linked by a disulfide bridge. The heavy subunits are the SLC3 members (rBAT and 4F2hc), whereas the light subunits are members of the SLC7 family of amino acid transporters. SLC3 proteins are type II membrane glycoproteins (i.e., one single transmembrane domain and the C-terminus located outside the cell) with a bulky extracellular domain that shows homology with alpha-glucosidases. rBAT heterodimerizes with b(0,+)AT (SLC7A9) constituting the amino acid transport b(0,+), the main system responsible for the apical reabsorption of cystine in kidney. The defect in this system causes cystinuria, the most common primary inherited aminoaciduria. 4F2hc subserves various amino acid transport systems by dimerization with different SLC7 proteins. The main role of SLC3 proteins is to help routing of the holotransporter to the plasma membrane. A working model for the biogenesis of HATs based on recent data on the rBAT/b(0,+)AT heterodimeric complex is presented. 4F2hc is a multifunctional protein, and in addition to its role in amino acid transport, it may be involved in other cellular functions. Studies on two SLC7 members (Asc-2 and AGT1) demonstrate heterodimerization with unknown heavy subunits.

Amino Acid Transport Systems, Basic↗
PubMed2000

Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA.

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.

Amino Acid Transport Systems, Basic↗
PubMed2003

Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT.

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.

3T3 Cells↗
PubMed1995

Heavy metal mediated inhibition of rBAT-induced amino acid transport.

rBAT, a protein which is located in the brush border membranes of intestine and renal proximal tubule cells, was recently shown to induce electrogenic countertransport of neutral and dibasic amino acids after its expression in Xenopus oocytes. Here, we studied the effects of heavy metals on rBAT induced amino acid transport in Xenopus oocytes to clarify a possible involvement of rBAT in heavy metal-induced aminoaciduria. The heavy metals Hg2+ and Pb2+ inhibited rBAT-induced amino acid transport with a different profile of action. The Pb2+ mediated inhibition occurred rapidly upon superfusion and was readily reversible upon washout. The maximal inhibition caused by Pb2+ was about 50% of the amino acid-induced currents at an apparent affinity (Km) of about 10 microM. In contrast, the Hg(2+)-mediated inhibition occurred rather slowly, depending on its concentration, and was not reversible during washout with control solution. However, the Hg(2+)-mediated amino acid transport inhibition could be reversed with Hg2+ chelating agents and reducing compounds. Other oxidative agents, such as the membrane permeable 2,2'-Dithio-bis(5-Nitropyridine) (DTNP), but not the membrane impermeable 5,5'-Dithio-bis (2-Nitrobenzoic acid) (DTNB), mimicked the effect of Hg2+, and their effect could similarly be reversed with 2,3-Dihydroxybutane-1,4-dithiol (DTE). In conclusion, Pb2+ and Hg2+ inhibit rBAT-induced amino acid transport in a noncompetitive, allosteric fashion. Blockade of rBAT-induced amino acid transport may be involved in aminoaciduria following mercury or lead intoxication.

Amino Acid Transport Systems, Basic↗
PubMed1996

Regulation of cationic amino acid transporter (CAT) gene expression.

The Cat family of genes have revealed unexpected complexity and regulation. The first 5 years of research have been productive, yet many important questions remain to be addressed. A major problem for the field of amino acid transport is a severe paucity of data on the structure of these proteins and their arrangement in membranes. There is a need for epitope-specific antisera to distinguish the mCAT2/2A proteins and reagents that will specifically block transport to each of these proteins. The question of gene redundancy will be answered by using genetic approaches such as targeted gene ablation, tissue-specific gene knockout and transgenic overexpression. From this information and more physiological analysis, it will be possible to determine the role and significance of these transport proteins.

Amino Acid Transport Systems, Basic↗
PubMed1998

Cationic amino acid transporter mRNA expression in rat kidney and liver.

Expression of rat cationic amino acid transporter 2 (r-CAT-2) mRNA was studied in kidney and liver using Northern blot analysis and nonradioactive in situ hybridization with a probe identifying both the r-CAT-2alpha and -2beta splice variants. Expression of r-CAT-2 mRNA was higher in the liver than in the kidney. Within the kidney, r-CAT-2 mRNA was more abundant in the outer and inner medulla than in the cortex. In the liver lobule, the intensity of the hybridization signal in hepatocytes decreased between the portal area and the central vein. In the kidney, hybridization signals were detected in parietal cells of Bowman's capsule, various tubule cells of outer and inner medulla, in endothelial and interstitial cells of inner medulla, and in papillary epithelial cells.

Amino Acid Transport Systems, Basic↗
PubMed2001

Identification and characterization of a novel member of the heterodimeric amino acid transporter family presumed to be associated with an unknown heavy chain.

We identified a novel amino acid transporter designated Asc-2 (for asc-type amino acid transporter 2). Asc-2 exhibited relatively low but significant sequence similarity to the members of the heterodimeric amino acid transporters. The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits in the heterodimeric amino acid transporters is conserved for Asc-2. Asc-2 is, however, not colocalized with the already known heavy chains such as 4F2 heavy chain (4F2hc) or related to b(0,+) amino acid transporter (rBAT) in mouse kidney. Because Asc-2 solely expressed or coexpressed with 4F2hc or rBAT did not induce functional activity, we generated fusion proteins in which Asc-2 is connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the function corresponding to the Na(+)-independent small neutral amino acid transport system asc. Distinct from the already identified system asc transporter Asc-1 which is associated with 4F2hc, Asc-2-mediated transport is less stereoselective and did not accept some of the high affinity substrates of Asc-1 such as alpha-aminoisobutyric acid and beta-alanine. Asc-2 message was detected in kidney, placenta, spleen, lung, and skeletal muscle. In kidney, Asc-2 protein was present in the epithelial cells lining collecting ducts. In the Western blot analysis on mouse erythrocytes and kidney, Asc-2 was detected as multiple bands in the nonreducing condition, whereas the bands shifted to a single band at lower molecular weight, suggesting the association of Asc-2 with other protein(s) via a disulfide bond. The finding of Asc-2 would lead to the establishment of a new subgroup of heterodimeric amino acid transporter family which includes transporters associated not with 4F2hc or rBAT but with other unknown heavy chains.

Amino Acid Sequence↗
PubMed2003

Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters.

Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.

Alanine↗
PubMed1999

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.

Amino Acid Sequence↗
PubMed2000

Functional analysis of novel mutations in y(+)LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI).

Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of the cationic amino acids lysine, arginine and ornithine at the basolateral membrane of the polar epithelial cells in the intestine and renal tubules, and by hyperammonemia after high-protein meals. LPI is caused by mutations in the SLC7A7 (solute carrier family 7, member 7) gene encoding y(+)LAT-1 (y(+)L amino acid transporter-1), which co-induces together with 4F2 heavy chain (4F2hc) system y(+)L in Xenopus oocytes. All Finnish LPI patients share the same founder mutation 1181-2A-->T (LPI(Fin)) not found in LPI patients elsewhere. Mutation screening of 20 non-Finnish LPI patients revealed 10 novel mutations: four deletions, two missense mutations, two nonsense mutations, a splice site mutation and a tandem duplication. Five LPI mutations (L334R, G54V, 1291delCTTT, 1548delC and LPI(Fin)) were studied functionally. All mutant proteins failed to co-induce amino acid transport activity when expressed with 4F2hc in Xenopus oocytes. Immunostaining experiments revealed that frameshift mutants 1291delCTTT, 1548delC and LPI(Fin)remained intracellular on expression with 4F2hc. In contrast, the missense mutants L334R and G54V reached the oocyte plasma membrane when co-expressed with 4F2hc, demonstrating that they are transport-inactivating mutations. This finding, together with the strong degree of conservation among all members of this family of amino acid transporters, indicates that residues L334 and G54 play a crucial role in the function of the y(+)LAT-1 transporter.

Amino Acid Metabolism, Inborn Errors↗