CYSTEINE SCANNING OF THE SURROUNDINGS OF AN ALKALI-ION BINDING-SITE OF THE GLUTAMATE TRANSPORTER GLT-1 REVEALS A CONFORMATIONALLY SENSITIVERESIDUE

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 directi on. Recently we have identified two adjacent amino acid residues of th e glutamate transporter GLT-1 that influence potassium coupling. Using the scanning cysteine accessibility method we have now explored the h ighly conserved region surrounding them. Replacement of each of the fi ve consecutive residues 396-400 by cysteine abolished transport activi ty but at several other positions the substitution is tolerated. One r esidue, tyrosine 403, was identified where cysteine substitution rende rs the transporter sensitive to modification by positively charged met hanethiosulfonate derivates in a sodium-protectable fashion. In the pr esence of sodium, the nontransported glutamate analogue dihydrokainate potentiated the covalent modification, presumably by binding to the g lutamate site and locking the protein in a conformation in which tyros ine 403 is accessible from the external bulk medium. In contrast, tran sported 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 transport ers against N-ethylmaleimide, which is highly permeant but unable to m odify 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 o f the potassium binding site.