New inhibitors for the neutral amino acid transporter ASCT2 reveal its Na+-dependent anion leak.
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The neutral amino acid transporter ASCT2 catalyses uncoupled anion flux across the cell membrane in the presence of transported substrates, such as alanine. Here, we report that ASCT2 conducts anions already in the absence of transported substrates through a leak anion-conducting pathway. The properties of this leak anion conductance were studied by electrophysiological recording from ASCT2-expressing HEK293 cells. We found that the leak anion conductance was inhibited by the binding of the newly characterized inhibitors benzylserine and benzylcysteine to ASCT2. These inhibitors competitively prevent binding of transported substrates to ASCT2, suggesting that they bind to the ASCT2 binding site for neutral amino acid substrates. The leak anion conductance exhibits permeation properties that are similar to the substrate-activated anion conductance of ASCT2, preferring hydrophobic anions such as thiocyanate. Inhibition of the leak anion conductance by benzylserine requires the presence of extracellular, but not intracellular Na(+). The apparent affinity of ASCT2 for extracellular Na(+) was determined as 0.3 mm. Interestingly, a Na(+)-dependent leak anion conductance with similar properties was previously reported for the related excitatory amino acid transporters (EAATs), suggesting that this leak anion conductance is highly conserved within the EAAT protein family. (+info)
Glutamine availability up-regulates expression of the amino acid transporter protein ASCT2 in HepG2 cells and stimulates the ASCT2 promoter.
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Glutamine transport into the human hepatoma cell line HepG2 is catalysed primarily by an ASCT2-type transporter identical in sequence with that cloned previously from JAR cells. An antibody raised against the C-terminus of the ASCT2 protein was shown to recognize ASCT2 on Western blots. Using this antibody, it was found that variation in cell growth rate did not affect ASCT2 expression, but both growth rate and ASCT2 expression were significantly reduced by glutamine deprivation. Expression of a number of other proteins was shown to be unaffected under these conditions. The sequence of the 5'-flanking region of the ASCT2 gene was derived from the human genome database. A 907 bp fragment of this sequence was directionally ligated into a luciferase reporter vector and was shown to exhibit promoter activity when transfected into HepG2 cells. Promoter activity was greatly reduced when transfection was performed in glutamine-free medium and was restored when glutamine was added post-transfection. The absence of other essential amino acids did not affect promoter activity, and glutamine deprivation did not affect the MCT1 (monocarboxylate transporter 1) promoter. These results indicate that both ASCT2 promoter activity and ASCT2 protein expression in these cells are dependent on glutamine availability. (+info)
Endogenous retroviral syncytin: compilation of experimental research on syncytin and its possible role in normal and disturbed human placentogenesis.
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Placental syncytin was first described in the year 2000 as a fusogenic glycoprotein originally derived from a human endogenous retroviral envelope gene. Although the presence of stable integrated retroviral elements within the human genome has been known for many years, their biological significance is still obscure and has usually been designated as irrelevant or even harmful. Syncytin, however, demonstrates tissue-specific expression and distinctive receptor interaction during trophoblast cell differentiation and syncytium formation. These findings indicate an involvement of syncytin in the development of the human placenta. Disturbances in placental architecture leading to severe placental dysfunction, such as pre-eclampsia, may therefore be discussed as a consequence of an altered syncytin system. We evaluate the hypothesis that syncytin is essential for human placenta formation and may also have played an important role in human placental evolution. (+info)
Syncytin: the major regulator of trophoblast fusion? Recent developments and hypotheses on its action.
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Syncytin is a membrane protein derived from the envelope gene of an endogenous retrovirus of the HERV-W family. The gene appears to be almost exclusively expressed in placenta; the protein was found in particular in syncytiotrophoblast. After transfection into various cell types it has proven to be a very fusogenic protein, inducing the formation of syncytia. Therefore, the question rises as to whether syncytin is responsible for the fusion process of villous cytotrophoblast into syncytiotrophoblast in vivo. If so, how is this fusion process regulated if syncytin is found all over the syncytiotrophoblast? Can this process be regulated through local or temporal changes in syncytin expression, or is syncytin merely one factor in a cascade of events leading to fusion limited at some other level? This review will try to summarize the published data on the regulation of fusion in trophoblast models as well as on the localization and regulation of syncytin expression and of its presumed receptors. Assuming that syncytin is the key factor inducing trophoblast fusion, a number of models will be presented by which syncytin and/or its receptors might regulate this process. In some of the hypotheses proposed, local coexpression of syncytin and receptor, leading to blocking of one factor by the other, is of functional relevance. (+info)
ATB0/ASCT2 expression in residual rabbit bowel is decreased after massive enterectomy and is restored by growth hormone treatment.
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Two weeks after 70% enterectomy, glutamine (Gln) transport is downregulated in rabbit residual bowel due to a decrease in system B(0) activity. Providing epidermal growth factor (EGF) and growth hormone (GH) restores Gln transport by increasing systems A and B(0,+) activities. We hypothesized that changes in Na(+)-dependent broad-spectrum neutral amino acid transporter (ATB(0)/ASCT2) protein and mRNA expression correlate with system B(0) activity. New Zealand White rabbits underwent 70% jejunoileal resection or no resection. Resected rabbits immediately received parenteral EGF, GH, both, or neither agent for 2 wk. Tissues harvested from jejunum, ileum, and colon were subjected to Western and Northern blot analyses for ATB(0)/ASCT2 protein and mRNA. In all tissues, ATB(0)/ASCT2 mRNA was reduced by approximately 50% in resected rabbits compared with nonresected controls. Similar reductions in protein amount occurred in the ileum and cecum. None of the growth factor treatments restored ATB(0)/ASCT2 protein, but GH treatment increased ATB(0)/ASCT2 mRNA abundance 250% in the residual ileum. Because changes in the ATB(0)/ASCT2 protein amount paralleled those in the system B(0) activity in this model, it is likely that this is the protein responsible for this transport system. The increase in mRNA abundance in rabbits treated with GH for 2 wk may be a harbinger of subsequent increases in transporter protein and activity. Unlike reported upregulation of transporters in human colon after small bowel resection, ATB(0)/ASCT2 protein and mRNA expression in rabbit colon are decreased, suggesting different regulatory pathways. (+info)
Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides.
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The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na+. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl2, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The Km for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein. (+info)
Improved transduction of human sheep repopulating cells by retrovirus vectors pseudotyped with feline leukemia virus type C or RD114 envelopes.
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Gene therapy for hematopoietic diseases has been hampered by the low frequency of transduction of human hematopoietic stem cells (HSCs) with retroviral vectors pseudotyped with amphotropic envelopes. We hypothesized that transduction could be increased by the use of retroviral vectors pseudotyped with envelopes that recognize more abundant cellular receptors. The levels of mRNA encoding the receptors of the feline retroviruses, RD114 and feline leukemia virus type C (FeLV-C), were significantly higher than the level of gibbon ape leukemia virus (GaLV) receptor mRNA in cells enriched for human HSCs (Lin- CD34+ CD38-). We cotransduced human peripheral blood CD34+ cells with equivalent numbers of FeLV-C and GALV or RD114 and GALV-pseudotyped retroviruses for injection into fetal sheep. Analysis of DNA from peripheral blood and bone marrow from recipient sheep demonstrated that FeLV-C- or RD114-pseudotyped vectors were present at significantly higher levels than GALV-pseudotyped vectors. Analysis of individual myeloid colonies demonstrated that retrovirus vectors with FeLV-C and RD114 pseudotypes were present at 1.5 to 1.6 copies per cell and were preferentially integrated near known genes We conclude that the more efficient transduction of human HSCs with either FeLV-C- or RD114-pseudotyped retroviral particles may improve gene transfer in human clinical trials. (+info)
Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells.
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We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis. (+info)