N-Glycosylation influences transport, but not cellular trafficking, of a neuronal amino acid transporter SNAT1

SNAT1 is a system N/A neutral amino acid transporter that primarily expresses in neurons and mediates the transport of l-glutamine (Gln). Gln is an important amino acid involved in multiple cellular functions and also is a precursor for neurotransmitters, glutamate and GABA. In the present study, we demonstrated that SNAT1 is an N-glycoprotein expressed in neurons. We identified three glycosylation sites at asparagine residues 251, 257 and 310 in SNAT1 protein, and that the first two are the primary sites. The biotinylation and confocal immunofluorescence analysis showed that the glycosylation-impaired mutants and deglycosylated SNAT1 were equally capable of expressing on the cell surface. However, l-Gln and 3H-labeled methyl amino isobutyrate (MeAIB) was significantly compromised in N-glycosylation-impaired mutants and deglycosylated SNAT1 when compared with the wild-type control. Taken together, these results suggest that SNAT1 is an N-glycosylated protein with three de novo glycosylation sites and N-glycosylation of SNAT1 may play an important role in the transport of substrates across the cell membrane.

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

Journal of Cachexia Sarcopenia and Muscle ◽

10.1002/jcsm.12707 ◽

2021 ◽

Author(s):

Timothy Pearson ◽

Oskar Wendowski ◽

Penny P. Powell

Keyword(s):

Amino Acid ◽

Amino Acid Transport ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

Acid Transport ◽

Neutral Amino Acid Transporter ◽

Branched Chain Amino Acid ◽

Cdna Expression ◽

Acid Transporter ◽

Branched Chain

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Amino Acid Transporter LAT1 (SLC7A5) Mediates MeHg-Induced Oxidative Stress Defense in the Human Placental Cell Line HTR-8/SVneo

International Journal of Molecular Sciences ◽

10.3390/ijms22041707 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1707

Author(s):

Sebastian Granitzer ◽

Raimund Widhalm ◽

Martin Forsthuber ◽

Isabella Ellinger ◽

Gernot Desoye ◽

...

Keyword(s):

Oxidative Stress ◽

Amino Acid ◽

De Novo ◽

Structural Similarity ◽

Amino Acid Transporter ◽

Cell Number ◽

Mehg Exposure ◽

Acid Transporter ◽

And Oxidative Stress

The placental barrier can protect the fetus from contact with harmful substances. The potent neurotoxin methylmercury (MeHg), however, is very efficiently transported across the placenta. Our previous data suggested that L-type amino acid transporter (LAT)1 is involved in placental MeHg uptake, accepting MeHg-L-cysteine conjugates as substrate due to structural similarity to methionine. The aim of the present study was to investigate the antioxidant defense of placental cells to MeHg exposure and the role of LAT1 in this response. When trophoblast-derived HTR-8/SVneo cells were LAT1 depleted by siRNA-mediated knockdown, they accumulated less MeHg. However, they were more susceptible to MeHg-induced toxicity. This was evidenced in decreased cell viability at a usually noncytotoxic concentration of 0.03 µM MeHg (~6 µg/L). Treatment with ≥0.3 µM MeHg increased cytotoxicity, apoptosis rate, and oxidative stress of HTR-8/SVneo cells. These effects were enhanced under LAT1 knockdown. Reduced cell number was seen when MeHg-exposed cells were cultured in medium low in cysteine, a constituent of the tripeptide glutathione (GSH). Because LAT1-deficient HTR-8/SVneo cells have lower GSH levels than control cells (independent of MeHg treatment), we conclude that LAT1 is essential for de novo synthesis of GSH, required to counteract oxidative stress. Genetic predisposition to decreased LAT1 function combined with MeHg exposure could increase the risk of placental damage.

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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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