Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co-and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, Glt Ph , only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of Glt Ph as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in Glt Ph ) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.excitatory amino acid transporters | fluorescence | the sodium/aspartate symporter from Pyrococcus horikoshii (Glt Ph ) | simulations G lutamate, the main excitatory neurotransmitter in the central nervous system, is removed from the extracellular synaptic space by the glutamate excitatory amino acid transporters EAAT1-5 (1, 2). These transporters thereby maintain an efficient synaptic communication between neurons and prevent extracellular glutamate from reaching neurotoxic levels (1, 2). EAATs are trimeric proteins in which each subunit functions as an independent transporter (3, 4). Each subunit has eight transmembrane domains and two membrane inserted hair-pin (HP) loops (5-7). EAATs use the Na + and K + gradients in driving the uptake of glutamate against a concentration gradient (8). The uptake of one glutamate is coupled to the cotransport of three Na + ions and one H + ion and the countertransport of one K + ion (9). How the thermodynamically coupled transport of glutamate, H + , Na + , and K + ions is accomplished by glutamate transporters is not known. Here we present evidence for a binding site for Na + ions that suggests a mechanism for the coupling of sodium and glutamate transport.At least one extracellular Na + ion appears to bind before glutamate can bind, and at least one extracellular Na + ion appears to bind after glutamate has bound to the transporter (10-12). For example, in the absence of glutamate, a fluorophore attached to a cysteine at position A430 on HP2 in EAAT3 reported voltageand Na + -dependent fluorescence changes, consistent with Na + binding to the glutamate-free transporter and inducing a conformational change in HP2. Li + also supports glutamat...

Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport 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 homolog of Na(+)/Cl(-)-dependent neurotransmitter transporters, LeuT, offers 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 first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific 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 amplified selectivity for Na(+) in the case of double ion occupancy.

The binding of ligands to protein receptors with high affinity and specificity is central to many cellular processes. The quest for the development of computational models capable of accurately evaluating binding affinity remains one of the main goals of modern computational biophysics. In this work, free energy perturbation/molecular dynamics simulations were used to evaluate absolute and relative binding affinity for three different antidepressants to a sodium-dependent membrane transporter, LeuT, a bacterial homologue of human serotonin and dopamine transporters. Dysfunction of these membrane transporters in mammals has been implicated in multiple diseases of the nervous system, including bipolar disorder and depression. Furthermore, these proteins are key targets for antidepressants including fluoxetine (aka Prozac) and tricyclic antidepressants known to block transport activity. In addition to being clinically relevant, this system, where multiple crystal structures are readily available, represents an ideal testing ground for methods used to study the molecular mechanisms of ligand binding to membrane proteins. We discuss possible pitfalls and different levels of approximation required to evaluate binding affinity, such as the dependence of the computed affinities on the strength of constraints and the sensitivity of the computed affinities to the particular partial charges derived from restrained electrostatic potential fitting of quantum mechanics electrostatic potential. Finally, we compare the effects of different constraint schemes on the absolute and relative binding affinities obtained from free energy simulations.