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

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 K(m) for glutamine derived from these experiments is in good agreement with the K(m) 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⁺.