Ions play key mechanistic roles in the gating dynamics of neurotransmitter:sodium symporters (NSSs). In recent microsecond scale molecular dynamics simulations of a complete model of the dopamine transporter, a NSS protein, we observed a partitioning of K+ ions from the intracellular side toward the unoccupied Na2 site of dopamine transporter following the release of the Na2-bound Na+. Here we evaluate with computational simulations and experimental measurements of ion affinities under corresponding conditions, the consequences of K+ binding in the Na2 site of LeuT, a bacterial homolog of NSS, when both Na+ ions and substrate have left, and the transporter prepares for a new cycle. We compare the results with the consequences of binding Na+ in the same apo system. Analysis of >50-μs atomistic molecular dynamics and enhanced sampling trajectories of constructs with Glu290, either charged or neutral, point to the Glu290 protonation state as a main determinant in the structural reconfiguration of the extracellular vestibule of LeuT in which a “water gate” opens through coordinated motions of residues Leu25, Tyr108, and Phe253. The resulting water channel enables the binding/dissociation of the Na+ and K+ ions that are prevalent, respectively, in the extracellular and intracellular environments.