Ion-coupled transport of
neurotransmitter molecules by
neurotransmitter:
sodium symporters (NSS) plays an important role in the regulation of
neuronal signaling. One of the ma jor events in the trans- port cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound
ions and
substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high- resolution structure for a
bacterial homologue of Na+ /
Cl- -dependent
neurotransmitter transporters, LeuT, o ff ers a unique opportunity to analyze the functional roles of the multi-ion
binding sites within the binding pocket. The binding pocket of LeuT contains two
metal binding sites. The fi rst
ion in site NA1 is directly coupled to the bound
substrate (Leu) with the second
ion in the neighboring site (NA2) only about 6 A away. Extensive, fully atomistic, MD and free energy simulations of LeuT in an explicit
lipid bilayer are performed to evaluate
substrate binding a ffi nity as a function of the
ion load (single vs double occupancy) and occupancy by speci fi c monovalent
cations. It was shown that
double ion occupancy of the binding pocket is required to ensure
substrate coupling to Na+ and not to Li+ or K+
cations. Furthermore, it was found that presence of the
ion in site NA2 is required for structural stability of the binding pocket as well as "ampli fi ed" selectivity for Na+ in the case of
double ion occupancy.
