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Mapping the chloride permeation pathway of a human glutamate transporter

R.J. Cater, R.J. Vandenberg and R.M. Ryan, Transport Biology Group, Discipline of Pharmacology and Bosch Institute, University of Sydney, NSW 2006, Australia.

The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). EAATs function not only as glutamate transporters, but also as substrate activated chloride (Cl) channels. Several crystal structures of the EAAT homologue, GltPh, at different stages of the transport cycle have been solved (Boudker et al., 2007; Reyes et al., 2009; Verdon et al., 2012; Yernool et al., 2004). In the most recent structure to be solved (Verdon et al., 2012) a small cavity lined by polarizable residues, several of which have been implicated in Cl permeation (Ryan et al., 2004), has been identified. We hypothesize that throughout the transport cycle this cavity opens up to form the Cl channel. In this study, site directed mutagenesis of EAAT1 and electrophysiology have been utilized to determine if this cavity forms part of the Cl permeation pathway. Additionally, double cysteine mutants were generated in a cys-less EAAT1 background and analysed in an attempt to trap the protein in a purely Cl conducting state to assist with further structural studies of GltPh. When WT and mutant EAAT1 transporters are expressed in Xenopus laevis oocytes, the current observed at +60 mV is primarily attributed to Cl conductance. For this reason, current at +60 mV is indicative of Cl channel function. When residues T396, S366 and P392 are mutated to valine, alterations in Cl channel function occur without effecting glutamate transport (See table for K0.5 values and currents at +60 mV) thus indicating a role for these residues in the formation of the Cl permeation pathway.

Figure 1

After treatment of the double cysteine mutant M89C/I469C with the reducing agent dithiothreitol (DTT), the current at +60 mV decreases. Subsequent treatment with the oxidizing reagent copper phenanthroline (CuPhe) leads to a recovery of the current at +60 mV (see Figure). These results suggest that these two cysteine residues can form a spontaneous crosslink and trap the transporter in a Cl conducting state.

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