Removal of glutamate from the synaptic cleft by (Na ؉ ؉ K ؉ )-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30 -100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.Glutamate transporters in the brain ensure that the synaptic levels of the transmitter are kept below the neurotoxic levels (1-6). These transporters, located in the plasma membrane of nerve and glial cells, play an important role in limiting the duration of synaptic excitation (7-10). The uptake process is electrogenic (11-13), involving co-transport of three sodium ions, a proton, and a glutamate molecule and countertransport of a potassium ion (14 -16). In addition to the coupled flux, glutamate transporters mediate a thermodynamically uncoupled chloride flux activated by two of the molecules they transport, sodium and glutamate (17, 18).The five known eukaryotic glutamate transporters, GLT-1 (19), GLAST-1 (20), EAAC-1 (21), , and EAAT-5 (22), have an overall amino acid identity of ϳ50%. The homology is significantly higher in the carboxyl-terminal half of the transporters. Topology studies suggest that this region of the protein has an intriguing arrangement containing two oppositely oriented reentrant loops, the two transmembrane domains (TMs) 1 7 and 8, and an outward-facing hydrophobic region (Refs. 23-25, and see Fig. 1). Some of the features of the membrane topology remain under debate (26).Several amino acid residues critical for the function of glutamate transporters are located in the carboxyl-terminal half. Two adjacent amino acid residues of GLT-1, Tyr-403 and Glu-404, are located in TM7 (Fig. 1) and are conserved in all other glutamate transporters. Both were implicated in the binding of potassium ions (16,27) and appear to be close to one of the sodium binding sites (27). A conserved arginine residue, Arg...