Conformational Dynamics on the Extracellular Side of LeuT Controlled by Na+ and K+ Ions and the Protonation State of Glu290.

Abstract Ions play key mechanistic roles in the gating dynamics of Neurotransmitter:sodium symporters (NSS). In recent microsecond scale molecular dynamics (MD) simulations of a complete model of the dopamine transporter (DAT), a NSS protein, we observed a partitioning of K+ ions from the intracellular side towards the unoccupied Na2 site of DAT 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 to the consequences of binding Na+ in the same apo system. Analysis of >50 µs atomistic MD and enhanced sampling trajectories of constructs with E290 either charged or neutral, point to the E290 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 L25, Y108, and F253. The resulting water channel enables the binding/dissociation of the Na+ and K+ ions that are prevalent, respectively, in the extracellular and intracellular environments.