Structure/Function Studies of system x c − : identification of critical residues involved in Cl − binding

System x c − is a Na + ‐independent, anionic amino acid transporter that mediates the direct exchange of extracellular cystine for intracellular glutamate. This transporter has been demonstrated to play a significant role in 1) supplying cells with precursors for glutathione synthesis (Bannai and Tateishi, 1986) and 2) the regulation of extracellular levels of glutamate and dopamine in the rat striatum (Baker et al., 2002). Previous work in our lab has demonstrated that System x c − is inhibited in the complete absence of extracellular chloride, or in the presence of anion site inhibitors, e.g. 9‐anthracene carboxylic acid. Here we report that the Km, but not the Vmax, for cystine for transport is dependent on the concentration of extracellular Cl − , suggesting that Cl − binds to the transporter first and modulates the binding of cystine to the transporter. In addition, we have employed a site‐directed mutagenesis approach to identify amino acids within xCT that interact with Cl − . Specifically, we have developed native and mutant constructs of xCT in the vector, pSP6 (Promega), transcribed them in vitro , and injected the mRNA encoding these proteins into Xenopus oocytes. Radioligand uptake assays have been used to measure the activity of the mutant and native transporter. From this analysis we have been able to assess the importance of multiple residues, located near the putative substrate binding site, in the Cl − binding step of the transport cycle.