To explore rearrangements of the reentrant loop HP2 relative to transmembrane domains (TMs) 7 and 8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. The pairs were introduced around 10-15 A "above" the residues, which make contact with substrate in the related archaeal homologue Glt(Ph). Transport by the double mutants M449C/L466C (HP2/TM 8), L453C/I463C (HP2/TM 8), and I411C/I463C (TM 7/TM 8) was inhibited by copper(II)(1,10-phenanthroline)(3) (CuPh) and by Cd(2+). Inhibition was only observed when the two cysteines were present in the same construct, but not with the respective single cysteine mutants or when only one cysteine was paired with a mutation to another residue. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of M449C/L466C by CuPh. The non-transportable analogues kainate and d, l-threo-beta-benzyloxyaspartate are expected to stabilize an outward-facing conformation, but only the latter potentiated the effect of CuPh on M449C/L466C. However, both analogues increased the aqueous accessibility of the cysteines introduced at positions 449 and 466 to a membrane-impermeant sulfhydryl reagent. Inhibition of L453C/I463C by CuPh was protected not only by glutamate but also by the two analogues. In contrast, these ligands had very little effect on the inhibition of I411C/I463C by CuPh. Our results are consistent with the proposal that HP2 serves as the extracellular gate of the transporter and indicate that glutamate and the two analogues induce distinct conformations of HP2.