Spontaneous K⁺ binding in MD simulations reveals two potential K⁺ binding states. MD simulation results are based on an EAAT3 homology model (Glt_Ph template). Typical trajectories from microsecond-timescale simulations are selected and analyzed. (A–H) Binding state with K⁺ located close to the side chain of D454 (K1 site; A) and K⁺ located close to the side chain of E373 (K2 site; B). Trajectories from independent simulations demonstrating spontaneous K⁺ binding to K1 (C–E) and K2 (F–H) sites were analyzed. The colors represent different K⁺ ions. The binding of K⁺ to the K1 site was monitored by the distance between K⁺ and the C_γ atom of D454, while the binding of K⁺ to the K2 site was monitored by the distance between K⁺ and the C_δ atom of E373. The K⁺ concentration was 1 M.