Characterization of a novel Na+-independent amino acid transporter in horse erythrocytes.
Ontology highlight
ABSTRACT:
SUBMITTER: Fincham DA
PROVIDER: S-EPMC1144803 | biostudies-other | 1985 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.
| S-EPMC2851542 | biostudies-literature
Project description:The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na(+) ions and one proton, and the counter-transport of one K(+) ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K(+) ions and the number of Na(+) ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na(+) ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na(+) binding. Mutations to all three proposed Na(+) sites influence the binding of substrate and/or Na(+), or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na(+) affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na(+) and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na(+) and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange.
| S-EPMC4067185 | biostudies-literature
Project description:We recently cloned the human Na(+)-independent system L neutral amino acid transporter LAT3. The aim of the present study was to characterize the molecular nature of mouse LAT3 at the protein level. Isolated mouse LAT3 showed 83% identity to human LAT3. Xenopus oocytes injected with mouse LAT3 cRNA showed the same functional property as human LAT3. Reverse transcriptase-polymerase chain reaction revealed apparent transcripts of mouse LAT3 in the liver, skeletal muscle, and pancreas, an expression pattern identical to that found in humans. Antibody generated against mouse LAT3 detected both approximately 58-kd and 48-kd bands in the sample from liver and only a 48-kd band in skeletal muscle and pancreas. Immunohistochemical study showed its clear localization in the plasma membrane of liver and skeletal muscle, whereas it was only detectable in the endoplasmic reticulum and in crystalline inclusions in pancreatic acinar cells. Starvation induced up-regulation of mouse LAT3 protein and mRNA in both liver and skeletal muscle but not in pancreas. These results suggest that LAT3 may indeed function as an amino acid transporter, transporting branched-chain amino acids from liver and skeletal muscle to the bloodstream and thereby participating in the regulatory system of interorgan amino acid nutrition.
| S-EPMC1864869 | biostudies-literature
Project description:Uptake of long-chain and aromatic neutral amino acids into cells is known to be catalyzed by the Na(+)-independent system L transporter, which is ubiquitous in animal cells and tissues. We have used a Xenopus oocyte expression system to clone the cDNA of a system L transporter from a rat kidney cDNA library. The 2.3-kilobase cDNA codes for a protein of 683 amino acids. The transporter has four putative membrane-spanning domains and bears no sequence or structural homology to any known animal or bacterial transporter. When transcribed and expressed in Xenopus oocytes, the transporter exhibits many, but not all, of the characteristics of L-system transporters, suggesting that this represents one of several related L-system transporters.
| S-EPMC48162 | biostudies-other
Project description:Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B(0)AT1 (slc6a19) and was hence named B(0)AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K(0.5) (half-saturation constant) ranging from 40 to 200 microM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K(0.5) values in the millimolar range. Transport of neutral amino acids via B(0)AT2 was Na+-dependent, Cl--independent and electrogenic. Superfusion of mouse B(0)AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na+-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na+/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K(0.5) values, suggesting that they share the same binding site. A mouse B(0)AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B(0)AT2 in transporting neurotransmitter precursors and neuromodulators is proposed.
| S-EPMC1383701 | biostudies-literature
Project description:The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and alpha-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)). We sought to identify the mechanism by which amino acids induce Ca(2+) signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na(+)-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca(2+) signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca(2+) signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca(2+) signaling in STC-1 cells was suppressed by removing Na(+) from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca(2+)](i); 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca(2+) signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca(2+)](i) was associated with membrane depolarization and mediated by Ca(2+) influx, since it depended on extracellular Ca(2+); 6) the increase in [Ca(2+)](i) in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1-10 muM) or nitrendipine (1 muM), which block L-type voltage-sensitive Ca(2+) channels. We hypothesize that the inward current of Na(+) associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca(2+) channels that mediate Ca(2+) influx, thereby leading to an increase in the [Ca(2+)](i) in enteroendocrine STC-1 cells.
| S-EPMC2889636 | biostudies-literature
Project description:Excitotoxicity in the brain is a causal factor in several neurological and neurodegenerative disorders. Excitatory amino acid transporter 2 (EAAT2), an astrocytic glutamate transporter involved in the clearance of >80% of synaptic glutamate, is considered a therapeutically relevant target for excitotoxicity. We have previously designed GT951, an activator of EAAT2 with nanomolar efficacy but limited in vivo bioavailability. In this study, a pharmacophore-based screening and optimization resulted in the design of GTS467 and GTS511. GTS467 and GTS511 have low nanomolar efficacy in the glutamate uptake assay. Pharmacokinetic profiles (PK) of GTS511 show a >6 h half-life and higher bioavailability in plasma and the brain under all three routes of administration in rats. Similarly, GTS467 has high oral bioavailability (80-85%) in the brain and plasma with a >1 h half-life under all three dosing routes. These encouraging efficacy and PK profiles suggest that GTS511 and GTS467 can be further developed to treat neurological disorders caused by excitotoxicity.
| S-EPMC9575181 | biostudies-literature
Project description:We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.
| S-EPMC1222103 | biostudies-other
Project description:Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na+ or H+ gradients. The present work extends the Na+ and H+ dogma by including the H+ and K+ paradigm. Lepidopteran insect larvae have a high K+ and a low Na+ content, and their midgut cells lack Na+/K+ ATPase. Instead, an H+ translocating, vacuolar-type ATPase generates a voltage of approximately -240 mV across the apical plasma membrane of so-called goblet cells, which drives H+ back into the cells in exchange for K+, resulting in net K+ secretion into the lumen. The resulting inwardly directed K+ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K+-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes alpha-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K+, Na+, and to a lesser extent Li+ were accepted as cotransported ions, but K+ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl(-)-dependent, and the K+/Na+ selectivity increased with hyperpolarization of oocytes, reflecting the increased K+/Na+ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na+ and Cl(-)-coupled neurotransmitter transporter family.
| S-EPMC20272 | biostudies-literature
Project description:Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacological properties were characterized at the EAAT1-4 subtypes. The l- threo-3-substituted Asp derivatives 13a-e and 13g-k were nonsubstrate inhibitors, exhibiting pan activity at EAAT1-4 with IC50 values ranging from 0.49 to 15 ?M. Comparisons between (dl- threo)-19a-c and (dl- erythro)-19a-c Asp analogues confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogues (3b-e) of l-TFB-TBOA (3a) were shown to be potent EAAT1-4 inhibitors, with IC50 values ranging from 5 to 530 nM. Hybridization of the nonselective EAAT inhibitor l-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compounds 8 and 9, respectively, which were nonselective EAAT inhibitors displaying considerably lower IC50 values at EAAT1-4 (11-140 nM) than those displayed by the respective parent molecules.
| S-EPMC6139576 | biostudies-literature