Mutants blocking the translocation step but not binding steps. Anion currents were activated under homoexchange conditions, extracellular 10 mM Na⁺ and 5 mM glutamate, intracellular solutions contained 130 mM NaSCN and 5 mM glutamate. Subsequently, we applied K⁺ through solution exchange to inhibit steady-state anion current. (A) Illustrated concept of the method. (B) Location of the tested amino acid residues shown in three different according to contribution to K1, K2, and Na3 sites. (C) EAAC1_WT and mutants’ steady-state anion current as a function of K⁺ concentration. (D) K⁺ apparent affinity for mutant transporters determined from the dose–response curves shown in C. In C and D, error bars represent SD.