Enhanced small neutral but not branched chain amino acid transport after epigenetic sodium coupled neutral amino acid transporter‐2 (SNAT2) cDNA expression in myoblasts

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 α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ 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 Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ 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 [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

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Identification and Functional Characterization of the Lactococcus lactis CodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

Journal of Bacteriology ◽

10.1128/jb.188.9.3280-3289.2006 ◽

2006 ◽

Vol 188 (9) ◽

pp. 3280-3289 ◽

Cited By ~ 42

Author(s):

Chris D. den Hengst ◽

Maarten Groeneveld ◽

Oscar P. Kuipers ◽

Jan Kok

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Lactococcus Lactis ◽

Amino Acid Transport ◽

Amino Acid Transporter ◽

Acid Transport ◽

Amino Acid Permease ◽

Branched Chain Amino Acid ◽

Acid Transporter ◽

Branched Chain

ABSTRACT Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

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The C-terminal domain of the neutral amino acid transporter SNAT2 regulates transport activity through voltage-dependent processes

Biochemical Journal ◽

10.1042/bj20100507 ◽

2011 ◽

Vol 434 (2) ◽

pp. 287-296 ◽

Cited By ~ 12

Author(s):

Zhou Zhang ◽

Catherine B. Zander ◽

Christof Grewer

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Amino Acid Transporter ◽

Membrane Potentials ◽

Neutral Amino Acid ◽

Acid Transport ◽

Membrane Expression ◽

Terminal Domain ◽

Neutral Amino Acid Transporter ◽

C Terminus

SNAT (sodium-coupled neutral amino acid transporter) 2 belongs to the SLC38 (solute carrier 38) family of solute transporters. Transport of one amino acid molecule into the cell is driven by the co-transport of one Na+ ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. In the present paper, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of this deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (<0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were normal in the truncated transporter. SNAT2Del C-ter (13 residues deleted from the C-terminus) showed 3-fold higher apparent affinity for alanine, and 2-fold higher Na+ affinity compared with wild-type SNAT2, suggesting that the C-terminus is not required for high-affinity substrate and Na+ interaction with SNAT2. The pH sensitivity of amino acid transport was retained partially after the truncation. In contrast with the truncation after TM (transmembrane domain) 11, the deletion of TM11 resulted in an inactive transporter, most probably due to a defect in cell surface expression. Taken together, the results demonstrate that the C-terminal domain of SNAT2 is an important voltage regulator that is required for a normal amino acid translocation process at physiological membrane potentials. However, the C-terminus appears not to be involved in the regulation of membrane expression.

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Molecular Cloning of Mouse Amino Acid Transport System B0, a Neutral Amino Acid Transporter Related to Hartnup Disorder

Journal of Biological Chemistry ◽

10.1074/jbc.m400904200 ◽

2004 ◽

Vol 279 (23) ◽

pp. 24467-24476 ◽

Cited By ~ 161

Author(s):

Angelika Bröer ◽

Karin Klingel ◽

Sonja Kowalczuk ◽

John E. J. Rasko ◽

Juleen Cavanaugh ◽

...

Keyword(s):

Amino Acid ◽

Molecular Cloning ◽

Transport System ◽

Amino Acid Transport ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Acid Transport ◽

Amino Acid Transport System ◽

Neutral Amino Acid Transporter ◽

Hartnup Disorder

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Mutations of the basic amino acid transporter gene associated with cystinuria

Biochemical Journal ◽

10.1042/bj3100951 ◽

1995 ◽

Vol 310 (3) ◽

pp. 951-955 ◽

Cited By ~ 30

Author(s):

K Miyamoto ◽

K Katai ◽

S Tatsumi ◽

K Sone ◽

H Segawa ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Basic Amino Acid ◽

Amino Acid Transporter ◽

Regulatory Subunit ◽

Acid Transport ◽

Transport Activity ◽

Wild Type ◽

Neutral Amino Acid Transporter ◽

Acid Transporter

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

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Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218794627 ◽

2018 ◽

Vol 24 (2) ◽

pp. 111-120 ◽

Cited By ~ 6

Author(s):

Sanjay J. Danthi ◽

Beirong Liang ◽

Oanh Smicker ◽

Benjamin Coupland ◽

Jill Gregory ◽

...

Keyword(s):

Amino Acid ◽

High Throughput Screening ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Imaging Plate ◽

Acid Uptake ◽

Functional Assays ◽

Neutral Amino Acid Transporter ◽

Acid Transporter ◽

Pharmacologically Active

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

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Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

Biochemical Journal ◽

10.1042/bj3460705 ◽

2000 ◽

Vol 346 (3) ◽

pp. 705-710 ◽

Cited By ~ 58

Author(s):

Angelika BRÖER ◽

Carsten WAGNER ◽

Florian LANG ◽

Stefan BRÖER

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Anion Channel ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Xenopus Laevis Oocytes ◽

Anion Conductance ◽

Neutral Amino Acid Transporter ◽

Inward Currents ◽

Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

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