Fig. 1.

Transport cycle and transport activity of D455 mutants. (A) Transport cycle of GLT-1 and other eukaryotic glutamate transporters (full lines). After binding of sodium, acidic amino acid (AAA⁻), and a proton from the extracellular medium (left), the outward-facing substrate-loaded translocation complex is formed. After the closing of the external gate and the opening of the internal one, the substrate and cotransported ions dissociate into the cytoplasm (right). Subsequently, intracellular potassium enters the binding pocket. After the internal gate closes, the external gate opens and potassium is released into the extracellular medium, completing net influx. Net efflux, observed at elevated extracellular potassium concentrations, proceeds via reversal of the above steps. Exchange involves reversible translocation via the glutamate half-cycle and does not require potassium. When a bulky nontransportable analog (AAA⁻-X) binds from the extracellular medium together with sodium [the stoichiometry (n) is unknown], the transporter remains locked in the outward-facing conformation (dashed line) because translocation cannot proceed. (B) The Cys-less GLT-1 and the indicated mutants in the Cys-less GLT-1 background were expressed in HeLa cells, and transport was measured as described in the Materials and Methods. The data are given as percent of activity of Cys-less GLT-1.