New inhibitors for the neutral amino acid transporter ASCT2 reveal its Na+-dependent anion leak

The neutral amino acid transporter ASCT2 catalyses uncoupled anion flux across the cell membrane in the presence of transported substrates, such as alanine. Here, we report that ASCT2 conducts anions already in the absence of transported substrates through a leak anionconducting pathway. The properties of this leak anion conductance were studied by electrophysiological recording from ASCT2-expressing HEK293 cells. We found that the leak anion conductance was inhibited by the binding of the newly characterized inhibitors benzylserine and benzylcysteine to ASCT2. These inhibitors competitively prevent binding of transported substrates to ASCT2, suggesting that they bind to the ASCT2 binding site for neutral amino acid substrates. The leak anion conductance exhibits permeation properties that are similar to the substrate-activated anion conductance of ASCT2, preferring hydrophobic anions such as thiocyanate. Inhibition of the leak anion conductance by benzylserine requires the presence of extracellular, but not intracellular Na+. The apparent affinity of ASCT2 for extracellular Na+ was determined as 0.3 mm. Interestingly, a Na+-dependent leak anion conductance with similar properties was previously reported for the related excitatory amino acid transporters (EAATs), suggesting that this leak anion conductance is highly conserved within the EAAT protein family.