Characterizing unexpected interactions of a glutamine transporter inhibitor with members of the SLC1A transporter family

The solute carrier 1A family comprises a group of membrane proteins that act as dual-function amino acid transporters and chloride (Cl(−)) channels and includes the alanine serine cysteine transporters (ASCTs) as well as the excitatory amino acid transporters. ASCT2 is regarded as a promising target for cancer therapy, as it can transport glutamine and other neutral amino acids into cells and is upregulated in a range of solid tumors. The compound L-γ-glutamyl-p-nitroanilide (GPNA) is widely used in studies probing the role of ASCT2 in cancer biology; however, the mechanism by which GPNA inhibits ASCT2 is not entirely clear. Here, we used electrophysiology and radiolabelled flux assays to demonstrate that GPNA activates the Cl(−) conductance of ASCT2 to the same extent as a transported substrate, whilst not undergoing the full transport cycle. This is a previously unreported phenomenon for inhibitors of the solute carrier 1A family but corroborates a body of literature suggesting that the structural requirements for transport are distinct from those for Cl(−) channel formation. We also show that in addition to its currently known targets, GPNA inhibits several of the excitatory amino acid transporters. Together, these findings raise questions about the true mechanisms of its anticancer effects.