Cysteine scanning of the surroundings of an alkali-ion binding site of the glutamate transporter GLT-1 reveals a conformationally sensitive residue.

Glutamate transporters remove this transmitter from the extracellular space by cotransport with three sodium ions and a proton. The cycle is completed by translocation of a potassium ion in the opposite direction. Recently we have identified two adjacent amino acid residues of the glutamate transporter GLT-1 that influence potassium coupling. Using the scanning cysteine accessibility method we have now explored the highly conserved region surrounding them. Replacement of each of the five consecutive residues 396-400 by cysteine abolished transport activity but at several other positions the substitution is tolerated. One residue, tyrosine 403, was identified where cysteine substitution renders the transporter sensitive to modification by positively charged methanethiosulfonate derivates in a sodium-protectable fashion. In the presence of sodium, the nontransported glutamate analogue dihydrokainate potentiated the covalent modification, presumably by binding to the glutamate site and locking the protein in a conformation in which tyrosine 403 is accessible from the external bulk medium. In contrast, transported substrates significantly slowed the reaction, suggesting that during the transport cycle residue 403 becomes occluded. On the other hand, transportable substrates are not able to protect Y403C transporters against N-ethylmaleimide, which is highly permeant but unable to modify cysteine residues buried within membrane proteins. These results indicate that tyrosine 403 is alternately accessible from either side of the membrane, consistent with its role as structural determinant of the potassium binding site.