The concentration of glutamate within the synapse is regulated by excitatory amino acid transporters (EAATs). EAATs function not only as glutamate transporters, but also as substrate-activated chloride (Cl^-) channels. In a recent structure of the EAAT1 homologue Glt_Ph, a cavity lined by polarisable residues, has been identified (Verdon et al., 2012). In this study, site directed mutagenesis of EAAT1 and electrophysiology were used to determine if this cavity is a partially open Cl^- channel. Furthermore, double-cysteine mutants were generated in a cysteine-less EAAT1 background in an attempt to trap the EAAT1 Cl^- channel in the open state.

When WT and mutant EAAT1 transporters are expressed in Xenopus laevis oocytes, currents observed at +60 mV are primarily attributed to Cl⁻ conductance. For this reason, currents at +60 mV are indicative of Cl⁻ channel function. When residues within this region were mutated to alanine or valine, both increases and decreases in Cl^- currents were observed. For example, in P392A the normalised current at +60 mV was 5.8 ± 0.6, whereas that of WT EAAT1 was 2.00 ± 0.07, suggesting a role for these residues in Cl^- conductance.

In the double-cysteine mutant M89C/I469C, incubation with the reducing agent dithiothreitol resulted in a significant decrease in currents at +60 mV, and subsequent treatment with the oxidising reagent copper phenanthroline resulted in the recovery of these currents. These results suggest that these two cysteines spontaneously crosslink to trap EAAT1 in a Cl^- conducting state, a result which will assist in future structural studies of Glt_Ph.