(A) Representative current traces for hEAAT1, M128K, M128R, P290R, and uninjected cells recorded upon voltage jumps (from –30 mV to +60 mV) in 96 mM NaCl buffer are shown. (B) The relaxation time (t) for the pre–steady state of each recorded current trace was measured. One-way ANOVA tests (Brown-Forsythe) were performed F(4, 58.1) = 75.91, ****P < 0.0001. The exact numbers of cells (n) used are indicated. (C) A representative time evolution of one Na⁺-hopping trajectory observed during molecular dynamic simulations of trimeric hEAAT1, in a glial membrane. The graph shows the distance between a Na⁺ ion and D400 in the Na3 site. (D and E) For WT hEAAT1, the Na⁺ ion position at Na1 moves toward the Na3 site (indicated by black arrow) where it remained stably bound until the end of the 500-ns simulation. (F) For M128R, the Na⁺ ion remains in Na1 during the 500-ns simulation. Major residues involved in Na⁺ coordination in Na1 (D487) and in Na3 (D400) are highlighted in stick representation and only the Na⁺ ion in Na1 is shown for clarity. (G–I) The distance between the Na⁺ ion and D400 (Na3) was used to monitor movements of the ion from the Na1 to the Na3 site. Distance distribution plots show 4 independent 500-ns simulations. (G) In WT hEAAT1, we observed a large population with smaller distances (<3 Å) in all 3 monomers (M1–M3), suggesting movement of the Na⁺ ion, initially in Na1, to Na3. (H) This population is absent in the M128R system, as highlighted by larger distance distributions (>3 Å). (I) In the M128K system, a small population corresponding to the movement of a Na⁺ ion moving from Na1 to Na3 (<3 Å) is observed.