Lysinuric protein intolerance: mechanisms of pathophysiology

Heteromeric amino acid transporters (HATs) are composed of two subunits, a polytopic membrane protein (the light subunit) and a disulfide-linked type II membrane glycoprotein (the heavy subunit). HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, xc-, and b(0,+)). The light subunits confer the amino acid transport specificity to the HAT. Two transporters of this family are relevant for inherited aminoacidurias. Mutations in any of the two genes coding for the subunits of system b(0,+) (rBAT and b(0,+)AT) lead to cystinuria (MIM 220100). Transport defects in a system y(+)L isoform, composed of 4F2hc and y(+)LAT-1, result in lysinuric protein intolerance (LPI) (MIM 222700). In this case, only mutations in the light subunit y(+)LAT-1, but not in the heavy chain 4F2hc, cause the disease. LPI, in addition to affecting intestinal and renal reabsorption of amino acids, is a multisystemic disease affecting the urea cycle and presents also with symptoms related to the immune system. The pathogenesis of these alterations is less well, or not understood at all.     

Identification and characterisation of human xCT that co-expresses, with 4F2 heavy chain, the amino acid transport activity system xc –

We have identified a new human complementary deoxyribonucleic acid (cDNA), for the xc- amino acid transporter (HGMW-approved name SLC7A11; also known as human xCT), that, when co-expressed with the heavy chain of surface antigen 4F2 (4F2hc, also termed CD98), but not with rBAT, (related to the bo,+ amino acid transporter), induces system xc- transport activity in Xenopus oocytes. Human xCT is the seventh human member of the family of amino acid transporters that are subunits of 4F2hc or rBAT and, inview of its amino acid sequence identity (89%) with mouse xCT, is most probably the human orthologue thereof. The amino acid transport activity induced by the co-expression of human 4F2hc and xCT in Xenopus oocytes was sodium independent and specific for L-cystine, L-glutamate and L-aspartate. This activity also functioned in an exchange mode (e.g. cystine/glutamate) with a substrate stoichiometry of 1:1. Expression of human xCT alone in oocytes did not induce amino acid transport activity and the expressed xCT protein was localised intracellularly. When human xCT was co-expressed with 4F2hc, the former localised to the oocyte plasma membrane. Tissue-expression studies showed that human SLC7A11 mRNA is expressed mainly in the brain, but also in pancreas and in cultured cell lines. The transport characteristics of human xCT and the distribution of its tissue expression strongly suggest that it corresponds to the human amino acid transporter system xc-.     

Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype

Most mutations in the rBAT subunit of the heterodimeric cystine transporter rBAT-b(0,+)AT cause type I cystinuria. Trafficking of the transporter requires the intracellular assembly of the two subunits. Without its partner, rBAT, but not b(0,+)AT, is rapidly degraded. We analyzed the initial biogenesis of wild-type rBAT and type I cystinuria rBAT mutants. rBAT was degraded, at least in part, via the ERAD pathway. Assembly with b(0,+)AT within the endoplasmic reticulum (ER) blocked rBAT degradation and could be independent of the calnexin chaperone system. This system was, however, necessary for post-assembly maturation of the heterodimer. Without b(0,+)AT, wild-type and rBAT mutants were degraded with similar kinetics. In its presence, rBAT mutants showed strongly reduced (L89P) or no transport activity, failed to acquire complex N-glycosylation and to oligomerize, suggesting assembly and/or folding defects. Most of the transmembrane domain mutant L89P did not heterodimerize with b(0,+)AT and was degraded. However, the few [L89P]rBAT-b(0,+)AT heterodimers were stable, consistent with assembly, but not folding, defects. Mutants of the rBAT extracellular domain (T216M, R365W, M467K and M467T) efficiently assembled with b(0,+)AT but were subsequently degraded. Together with earlier results, the data suggest a two-step biogenesis model, with the early assembly of the subunits followed by folding of the rBAT extracellular domain. Defects on either of these steps lead to the type I cystinuria phenotype.     

Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity

Members of the newly discovered glycoprotein-associated amino acid transporter family (gpaAT-family) share a similar primary structure with >40% identity, a predicted 12-transmembrane segment topology and the requirement for association with a glycoprotein (heavy chain) for functional surface expression. Five of the six identified gpaATs (light chains) associate with the surface antigen 4F2 heavy chain (4F2hc = CD98), a ubiquitous plasma membrane protein induced in cell proliferation, and which is also highly expressed at the basolateral surface of amino acid transporting epithelia. The differing tissue localizations of the 4F2hc-associated gpaATs appear to complement each other. As yet, a single gpaAT (b(0,+)AT) has been shown to associate with rBAT, a 4F2hc-related glycoprotein mainly localized in intestine and kidney luminal brush-border membranes. The transport characteristics of gpaATs have been shown, by expression in heterologous systems, to correspond to the previously described transport systems L, y+L, xc- and b(o,+). These (obligatory) exchangers of broad substrate specificity (with the exception of xCT) are expected to equilibrate the concentrations of their substrate amino acids across membranes. Thus, the driving force provided by a transmembrane gradient of one substrate amino acid, such as that generated by a parallel functioning unidirectional transporter, can be used by a gpaAT to fuel the secondary active vectorial transport of other exchangeable species. Vectorial transport of specific amino acids is also promoted by the intrinsic asymmetry of these exchangers. The fact that genetic defects of the epithelial gpaATs b(0,+)AT and y+LAT1 cause non-type I cystinuria and lysinuric protein intolerance, respectively, demonstrates that these gpaATs perform vectorial secondary and/or tertiary active transport of specific amino acids in vivo.     

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.     

CATs and HATs: the SLC7 family of amino acid transporters

The SLC7 family is divided into two subgroups, the cationic amino acid transporters (the CAT family, SLC7A1–4) and the glycoprotein-associated amino acid transporters (the gpaAT family, SLC7A5–11), also called light chains or catalytic chains of the hetero(di)meric amino acid transporters (HAT). The associated glycoproteins (heavy chains) 4F2hc (CD98) or rBAT (D2, NBAT) form the SLC3 family. Members of the CAT family transport essentially cationic amino acids by facilitated diffusion with differential trans-stimulation by intracellular substrates. In some cells, they may regulate the rate of NO synthesis by controlling the uptake of l-arginine as the substrate for nitric oxide synthase (NOS). The heterodimeric amino acid transporters are, in contrast, quite diverse in terms of substrate selectivity and function (mostly) as obligatory exchangers. Their selectivity ranges from large neutral amino acids (system L) to small neutral amino acids (ala, ser, cys-preferring, system asc), negatively charged amino acid (system x_c^–) and cationic amino acids plus neutral amino acids (system y⁺L and b^0,+-like). Cotransport of Na⁺ is observed only for the y⁺L transporters when they carry neutral amino acids. Mutations in b^0,+-like and y⁺L transporters lead to the hereditary diseases cystinuria and lysinuric protein intolerance (LPI), respectively.     

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.     

A y(+)LAT-1 mutant protein interferes with y(+)LAT-2 activity: implications for the molecular pathogenesis of lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. The SLC7A7 gene, mutated in LPI, encodes the y(+)LAT-1 protein, which is the light subunit of the heterodimeric CAA transporter in which 4F2hc is the heavy chain subunit. Co-expression of 4F2hc and y(+)LAT-1 induces the y(+)L activity. This activity is also exerted by another complex composed of 4F2hc and y(+)LAT-2, the latter encoded by the SLC7A6 gene and more ubiquitously expressed than SLC7A7. On the basis of both the pattern of expression and the transport activity, y(+)LAT-2 might compensate for CAA transport when y(+)LAT-1 is defective. By expression in Xenopus laevis oocytes and mammalian cells, we functionally analysed two SLC7A7 mutants, E36del and F152L, respectively, the former displaying a partial dominant-negative effect. The results of the present study provide further insight into the molecular pathogenesis of LPI: a putative multiheteromeric structure of both [4F2hc/y(+)LAT-1] and [4F2hc/y(+)LAT-2], and the interference between y(+)LAT-1 and y(+)LAT-2 proteins. This interference can explain why the compensatory mechanism, that is, an increased expression of SLC7A6 as seen in lymphoblasts from LPI patients, may not be sufficient to restore the y(+)L system activity.     

Cystinuria: from diagnosis to follow-up

Cystinuria is an autosomal recessive disorder characterized by an impaired transport of cystine and dibasic aminoacids, lysine, arginine and ornithine in the proximal renal tubule and in the epithelial cells of the gastrointestinal tract. Recurrent cystine nephrolithiasis is the main clinical feature. Mutations in SLC3A1 and/or SLC7A9 genes, which are encoding respectively the rBAT and the b(0,+)AT proteins of the amino acid transport system, are responsible of this disorder thus inducing a high dibasic amino acid excretion. Diagnostic is based on stone analysis by infrared spectroscopy or microscopic examination of urine which may reveal typical cystine crystals. Quantitative cystine excretion, which may be assessed by aminoacid chromatography, is higher in cystinic patients. Molecular approach can identify mutations which are responsible of this pathology. Medical treatment is mainly based on hydratation and urine alkalinisation, with the addition of thiol derivative only in refractory cases. Follow-up based on pH and specific gravity determination in urine samples and cystine crystal volume measurement are used to optimally monitor the medical treatment of cystinuric patients. Even with medical management, long-term outcome is poor due to insufficient efficacy and low patient compliance. Many patients suffer from renal insufficiency as a result of recurrent stone formation and repeated surgical procedures.     

Kidney amino acid transport

Near complete reabsorption of filtered amino acids is a main specialized transport function of the kidney proximal tubule. This evolutionary conserved task is carried out by a subset of luminal and basolateral transporters that together form the transcellular amino acid transport machinery similar to that of small intestine. A number of other amino acid transporters expressed in the basolateral membrane of proximal kidney tubule cells subserve either specialized metabolic functions, such as the production of ammonium, or are part of the cellular housekeeping equipment. A new finding is that the luminal Na⁺-dependent neutral amino acid transporters of the SLC6 family require an associated protein for their surface expression as shown for the Hartnup transporter B⁰AT1 (SLC6A19) and suggested for the l-proline transporter SIT1 (IMINO^B, SLC6A20) and for B⁰AT3 (XT2, SLC6A18). This accessory subunit called collectrin (TMEM27) is homologous to the transmembrane anchor region of the renin–angiotensin system enzyme ACE2 that we have shown to function in small intestine as associated subunit of the luminal SLC6 transporters B⁰AT1 and SIT1. Some mutations of B⁰AT1 differentially interact with these accessory subunits, providing an explanation for differential intestinal phenotypes among Hartnup patients. The basolateral efflux of numerous amino acids from kidney tubular cells is mediated by heteromeric amino acid transporters that function as obligatory exchangers. Thus, other transporters within the same membrane need to mediate the net efflux of exchange substrates, controlling thereby the net basolateral amino transport and thus the intracellular amino acid concentration.     

Slc7a9-deficient mice develop cystinuria non-I and cystine urolithiasis

Cystinuria is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in urolithiasis of cystine. Cystinuria is caused by defects in the amino acid transport system b0,+ (i.e. the rBAT/b0,+AT heteromeric complex). Mutations in SLC3A1, encoding rBAT, cause cystinuria type A, characterized by a silent phenotype in heterozygotes (phenotype I). Mutations in SLC7A9, encoding b0,+AT, cause cystinuria type B, in which heterozygotes in most cases hyperexcrete cystine and dibasic amino acids (phenotype non-I). To facilitate in vivo investigation of b0,+AT in cystinuria, Slc7a9 knockout mice have been generated. Expression of b0,+AT protein is completely abolished in the kidney of Slc7a9-/- mice ('Stones'). In contrast, Stones expressed significant amounts of rBAT protein, which is covalently linked to unidentified light subunit(s). Stones mice present a dramatic hyperexcretion of cystine and dibasic amino acids, while Slc7a9+/- mice show moderate but significant hyperexcretion of these amino acids (phenotype non-I). Forty-two per cent of Stones mice develop cystine calculi in the urinary system. Calculi develop during the first month of life and grow throughout the life span of the animals. Histopathology in kidney reveals typical changes for urolithiasis (tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis). The fact that some Stones mice, generated in a mixed genetic background, develop cystine calculi from an early age, while others do not develop them in their first year of life, suggests the involvement of modifier genes in the lithiasis phenotype. Thus, Stones provide a valid model of cystinuria which can be used in the study of genetic, pharmacological and environmental factors involved in cystine urolithiasis.     

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y⁺), while no information exists on the role of the heterodimeric transporters of system y⁺L (4F2hc/y⁺LAT1 and 4F2hc/y⁺LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y⁺L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y⁺), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y⁺LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y⁺LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y⁺ (CAT1 transporter) and system y⁺L (4F2hc/y⁺LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.     

Recycling of aromatic amino acids via TAT1 allows efflux of neutral amino acids via LAT2-4F2hc exchanger

The rate of amino acid efflux from individual cells needs to be adapted to cellular demands and plays a central role for the control of extracellular amino acid homeostasis. A particular example of such an outward amino acid transport is the basolateral efflux from transporting epithelial cells located in the small intestine and kidney proximal tubule. Because LAT2-4F2hc (Slc7a8–Slc3a2), the best known basolateral neutral amino acid transporter of these epithelial cells, functions as an obligatory exchanger, we tested whether TAT1 (Slc16a10), the aromatic amino-acid facilitated diffusion transporter, might allow amino acid efflux via this exchanger by recycling its influx substrates. In this study, we show by immunofluorescence that TAT1 and LAT2 indeed colocalize in the early kidney proximal tubule. Using the Xenopus laevis oocytes expression system, we show that l-glutamine is released from oocytes into an amino-acid-free medium only when both transporters are coexpressed. High-performance liquid chromatography analysis reveals that several other neutral amino acids are released as well. The transport function of both TAT1 and LAT2-4F2hc is necessary for this efflux, as coexpression of functionally inactive but surface-expressed mutants is ineffective. Based on negative results of coimmunoprecipitation and crosslinking experiments, the physical interaction of these transporters does not appear to be required. Furthermore, replacement of TAT1 or LAT2-4F2hc by the facilitated diffusion transporter LAT4 or the obligatory exchanger LAT1, respectively, supports similar functional cooperation. Taken together, the results suggest that the aromatic amino acid diffusion pathway TAT1 can control neutral amino acid efflux via neighboring exchanger LAT2-4F2hc, by recycling its aromatic influx substrates.     

Lysinuric protein intolerance: update and extended mutation analysis of the SLC7A7 gene

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is caused by mutations in the SLC7A7 gene, which encodes the y(+)LAT-1 protein, the catalytic light chain subunit of a complex belonging to the heterodimeric amino acid transporter family. Coexpression of 4F2hc (the heavy chain subunit) and y(+)LAT-1 induces y(+)L activity (CAA transport). So far a total of 43 different mutations of the SLC7A7 gene, nine of which newly reported here, have been identified in a group of 130 patients belonging to at least 98 independent families. The mutations are distributed along the entire gene and include all different types of mutations. Five polymorphisms within the SLC7A7 coding region and two variants found in the 5'UTR have been identified. A genuine founder effect mutation has been demonstrated only in Finland, where LPI patients share the same homozygous mutation, c.895-2A>T. LPI patients show extreme variability in clinical presentation, and no genotype-phenotype correlations have been defined. This phenotypic variability and the lack of a specific clinical presentation have caused various misdiagnoses. At the biochemical level, the elucidation of SLC7A7 function will be necessary to understand precise disease mechanisms and develop more specific and effective therapies. In this review, we summarize the current knowledge of SLC7A7 mutations and their role in LPI pathogenesis.     

System L: heteromeric exchangers of large,neutral amino acids involved in directional transport

The plasma membrane transport system L is in many cells the only (efficient) pathway for the import of large branched and aromatic neutral amino acids. The corresponding transporters are hetero(di)mers composed of a catalytic subunit (LAT1 or LAT2=light chain=glycoprotein-associated amino acid transporter) associated covalently with the glycoprotein 4F2hc/CD98 (heavy chain). The tissue distribution of LAT1 suggests that it is involved mainly in transporting amino acids into growing cells and across some endothelial/epithelial secretory barriers, whereas the localization of LAT2 indicates that it is mainly involved in the basolateral efflux step of transepithelial (re)absorptive amino acid transport. However, system L transporters are obligatory amino acid exchangers with 1:1 stoichiometry, with similar (but not identical) intra- and extracellular substrate selectivities and with highly asymmetrical apparent affinities (low affinity inside). Therefore, net directional transport of large, neutral amino acids by system L depends on the parallel expression of a unidirectional transporter with overlapping selectivity (for instance systems A or N) that provides/recycles amino acids that drive system L exchange function. By mediating the regulated flux of these exchange substrates, unidirectional transporters control the activity of system L.     

The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology

Mammalian members of the SLC15 family are electrogenic transporters that utilize the proton-motive force for uphill transport of short chain peptides and peptido-mimetics into a variety of cells. The prototype transporters of this family are PEPT1 (SLC15A1) and PEPT2 (SLC15A2), which mediate the uptake of peptide substrates into intestinal and renal epithelial cells. More recently, other sites of functional expression of the two proteins have been identified such as bile duct epithelium (PEPT1), glia cells and epithelia of the choroid plexus, lung and mammary gland (PEPT2). Both proteins can transport essentially every possible di- and tripeptide regardless of the substrate's net charge, but operate stereoselectively. Based on peptide-like structures, various drugs and prodrugs are transported as well, allowing efficient intestinal absorption of the compounds via PEPT1. In kidney tubules both peptide transporters can mediate the renal reabsorption of the filtered compounds thus affecting their pharmacokinetics. Recently, two new peptide transporters, PHT1 (SLC15A4) and PHT2 (SLC15A3), were identified in mammals. They possess an overall amino acid identity with the PEPT-series of 20% to 25%. PHT1 and PHT2 were shown to transport free histidine and certain di- and tripeptides, but it is not yet clear whether they are located on the plasma membrane or represent lysosomal transporters for the proton-dependent export of histidine and dipeptides from lysosomal protein degradation into the cytosol.     

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.     

Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6

The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins.     

An amino acid transporter involved in gastric acid secretion

Gastric acid secretion is regulated by a variety of stimuli, in particular histamine and acetyl choline. In addition, dietary factors such as the acute intake of a protein-rich diet and the subsequent increase in serum amino acids can stimulate gastric acid secretion only through partially characterized pathways. Recently, we described in mouse stomach parietal cells the expression of the system L heteromeric amino acid transporter comprised of the LAT2-4F2hc dimer. Here we address the potential role of the system L amino acid transporter in gastric acid secretion by parietal cells in freshly isolated rat gastric glands. RT-PCR, western blotting and immunohistochemistry confirmed the expression of 4F2-LAT2 amino acid transporters in rat parietal cells. In addition, mRNA was detected for the B⁰AT1, ASCT2, and ATB(0+) amino acid transporters. Intracellular pH measurements in parietal cells showed histamine-induced and omeprazole-sensitive H⁺-extrusion which was enhanced by about 50% in the presence of glutamine or cysteine (1 mM), two substrates of system L amino acid transporters. BCH, a non-metabolizable substrate and a competitive inhibitor of system L amino acid transport, abolished the stimulation of acid secretion by glutamine or cysteine suggesting that this stimulation required the uptake of amino acids by system L. In the absence of histamine glutamine also stimulated H⁺-extrusion, whereas glutamate did not. Also, phenylalanine was effective in stimulating H⁺/K⁺-ATPase activity. Glutamine did not increase intracellular Ca²⁺ levels indicating that it did not act via the recently described amino acid modulated Ca²⁺-sensing receptor. These data suggest a novel role for heterodimeric amino acid transporters and may elucidate a pathway by which protein-rich diets stimulate gastric acid secretion.P. Kirchhoff and M.H. Dave contributed equally to this study and therefore share first authorship     

The SLC38 family of sodium–amino acid co-transporters

Transporters of the SLC38 family are found in all cell types of the body. They mediate Na⁺-dependent net uptake and efflux of small neutral amino acids. As a result they are particularly expressed in cells that grow actively, or in cells that carry out significant amino acid metabolism, such as liver, kidney and brain. SLC38 transporters occur in membranes that face intercellular space or blood vessels, but do not occur in the apical membrane of absorptive epithelia. In the placenta, they play a significant role in the transfer of amino acids to the foetus. Members of the SLC38 family are highly regulated in response to amino acid depletion, hypertonicity and hormonal stimuli. SLC38 transporters play an important role in amino acid signalling and have been proposed to act as transceptors independent of their transport function. The structure of SLC38 transporters is characterised by the 5?+?5 inverted repeat fold, which is observed in a wide variety of transport proteins.