Structural and biochemical characterization of the human neutral amino acid transporter ASCT2

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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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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