The molecular strategy for alkali cation selectivity by a bacterial sodium channel resembles those of eukaryotic calcium and potassium channels, rather than those of eukaryotic sodium channels.

Neurotransmitter:sodium symporter (NSS) proteins are secondary Na(+)-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs-Leucine Transporter (LeuT)-and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na(+) binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na(+), but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na(+) affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na(+) over K(+) in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na(+)-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.

Background: The intramolecular pathways propagating the impact of Na+ binding in neurotransmitter:sodium symporters (NSSs) are not sufficiently understood.Results: We identified computationally and verified experimentally an interaction network connecting Na+ binding with the intracellular gate.Conclusion: The identified pathways are conserved between bacterial LeuT and eukaryotic hDAT.Significance: We gain a new understanding of the structural basis for the functional role of Na+ binding in NSSs.

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.