Effects of K-site mutations on homoexchange and apparent Na⁺ and glutamate affinities. Whole-cell current recordings were performed to test the apparent affinity of Na⁺/glutamate for all mutant transporters. (A) Typical Na⁺/Glu⁻-induced EAAC1_D454N anion current, tested under forward transport conditions. Glutamate concentration was saturating (2.5 mM) outside, with 130 mM KSCN inside. Duration of Na⁺/glutamate application is indicated by the gray bar. (B) Transient anion current was observed in EAAC1_D454N when switching the external solution from NMG⁺ to 140 mM Na⁺/5 mM glutamate (gray bar) in the forward transport mode. The internal solution contained KSCN. (C) Anion currents in homoexchange mode are compared for mutant transporters in the predicted K⁺-binding sites. (D–F) The Na⁺ apparent K_m was determined as 12.9 ± 2.5 mM for EAAC1_WT (D) and 27.6 ± 3.8 mM for EAAC1_D454N (E); Na⁺ apparent affinity for the K⁺ binding site mutant transporters is shown in F. (G and H) The K_m for glutamate under homoexchange conditions was determined from dose–response curves as 3.4 ± 0.6 µM for EAAC1_WT (G) and 20.5 ± 2.5 µM for EAAC1_D454N (H). (I) Glutamate K_m summary for all mutant transporters. In all experiments, the transmembrane potential was 0 mV, and error bars represent SD.