Sodium Binding Research Articles

Markov State-Based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

The dopamine transporter (DAT) belongs to the neurotransmitter/sodium symporter (NSS) family of membrane proteins that are responsible for reuptake of neurotransmitters from the synaptic cleft to terminate a neuronal signal and enable subsequent neurotransmitter release from the presynaptic neuron. The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identified as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process are computed from the MSM, and transition path theory is used to identify the most probable sodium release pathways. An intermediate state is discovered on the sodium release pathway, and the results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.

Effect of Sodium and Chloride Binding on a Lecithin Bilayer. A Molecular Dynamics Study.

The effect of ion binding on the structural, mechanical, dynamic and electrostatic properties of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in a 0.5 M aqueous NaCl solution is investigated using classical atomistic molecular dynamics simulation with different force-field descriptions for ion-ion and ion-lipid interactions. Most importantly, the repulsive Lennard–Jones parameters for the latter were modified, such that approximately similar binding of cations and anions to the lipid membrane is achieved. This was done to qualitatively improve the apparent ion-lipid binding constants obtained from simulations with the original force field (Berger lipids and GROMOS87 ions in combination with the SPC water model) in comparison to experimental data. Furthermore, various parameters characterizing membrane structure, elasticity, order and dynamics are analyzed. It is found that ion binding as observed in simulations involving the modified in comparison to the original force-field description leads to: (i) a smaller salt-induced change in the area per lipid, which is in closer agreement with the experiment; (ii) a decrease in the area compressibility and bilayer thickness to values comparable to a bilayer in pure water; (iii) lipid deuterium order parameters and lipid diffusion coefficients on nanosecond timescales that are very similar to the values for a membrane in pure water. In general, salt effects on the structural properties of a POPC bilayer in an aqueous sodium-chloride solution appear to be reproduced reasonably well by the new force-field description. An analysis of membrane-membrane disjoining pressure suggests that the smaller salt-induced change in area per lipid induced by the new force-field description is not due to the alteration of membrane-associated net charge, but must rather be understood as a consequence of ion-specific effects on the arrangement of lipid molecules.

Molecular dynamic simulations of glycine amino acid association with potassium and sodium ions in explicit solvent

Salt solutions are the natural environment in which biological molecules act, and dissolved ions are actively involved in biochemical processes. With metal ions, the membrane potentials are maintained. Ions are crucial for the activity of many enzymes, and their ability to coordinate with chemical groups modulates protein-protein interactions. Here we present a comparative study of sodium and potassium coordination with zwitterionic glycine, by means of explicit solvent molecular dynamics. We demonstrated that contact ion pair of cations and carboxylate group splits into two distinct coordination states. Sodium binding is significantly stronger than for potassium. These results can shed light on the different roles of sodium and potassium ions in abiogenic peptide synthesis.

A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

The dopamine transporter (DAT) belongs to the neurotransmitter:sodium symporter (NSS) family of membrane proteins that are responsible for reuptake of neurotransmitters from the synaptic cleft to terminate a neuronal signal and enable subsequent neurotransmitter release from the presynaptic neuron. The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identified as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process are computed from the MSM, and transition path theory is used to identify the most probable sodium release pathways. An intermediate state is discovered on the sodium release pathway, and the results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.

Mechanism of Interaction Between Cefonicid Sodium and Trypsin by Spectroscopic and Molecular Docking Methods

The binding of cefonicid sodium (CFS) with trypsin was investigated by spectroscopic and molecular docking methods under different temperatures conditions (303, 310 and 318 K). The results demonstrated that the interaction between CFS and trypsin was taking place via static quenching with 1:1 binding ratio. The fluorescence datas were treated by using the double logarithmic equation, and the binding constants K a of the interaction of CFS-trypsin systems and the number of binding sites n were obtained. The thermodynamic parameters of CFS-trypsin systems under different temperatures were obtained by the thermodynamic equation. The experimental data show that the interactions between them were mainly hydrophobic interaction and hydrogen bonding interaction, and with the molecular docking results are consistent.

Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System.

A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [35S]GTPγS assays). The human H1R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH1R and insect cell Gαq. By contrast, functional expression of the hH2R required the generation of an hH2R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH2R to the long (GsαL) or short (GsαS) splice variant of Gαs resulted in comparable constitutive hH2R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH1R/hH2R and their guinea pig orthologues gpH1R/gpH2R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH3R with Gαi1, Gαi2, Gαi3, and Gαi/o in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li+, Na+, K+) and anions (Cl−, Br−, I−) revealed that anions with large radii most efficiently stabilize the inactive hH3R state. Potential sodium binding sites in the hH3R protein were analyzed by expressing specific hH3R mutants in Sf9 cells. In contrast to the hH3R, the hH4R preferentially couples to co-expressed Gαi2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH4R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH4R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH4R. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of HxR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP

Crystal structures of transporters with a LeuT-type structural fold assign core transmembrane domain 6 (TM6′) a central role in substrate binding and translocation. Here, the function of TM6′ in the sodium/proline symporter PutP, a member of the solute/sodium symporter family, was investigated. A complete scan of TM6′ identified eight amino acids as particularly important for PutP function. Of these residues, Tyr-248, His-253, and Arg-257 impact sodium binding, whereas Arg-257 and Ala-260 may participate in interactions leading to closure of the inner gate. Furthermore, the previous suggestion of an involvement of Trp-244, Tyr-248, and Pro-252 in proline binding is further supported. In addition, substitution of Gly-245, Gly-247, and Gly-250 affects the amount of PutP in the membrane. A Cys accessibility analysis suggests an involvement of the inner half of TM6′ in the formation of a hydrophilic pathway that is open to the inside in the absence of ligands and closed in the presence of sodium and proline. In conclusion, the results demonstrate that TM6′ plays a central role in substrate binding and release on the inner side of the membrane also in PutP and extend the knowledge on functionally relevant amino acids in transporters with a LeuT-type structural fold.

Sequence-dependent association of alginate with sodium and calcium counterions

Molecular dynamics simulation is used to study in detail the binding of sodium and calcium ions to alginate chains, and its dependence on the content of guluronic (G) acid residues. Our previous studies showed that chains with different G content associate through different structural mechanisms due to differing degrees of rigidity of the ion-alginate chain complexes. Polymannuronate and polyguluronate chains form highly ordered structures due to their more rigid nature. Heteropolymer chains are more flexible and hence associate at higher ion and alginate concentrations, however, with association behavior that has a clear dependence on G content. The nature of interactions of sodium and calcium ions are shown to differ for poly-M, poly-G, and the heteropolymer compositions. Scattering curves of Ca2+-alginate solutions where found to have good agreement with the broken-rod SAXS model with nontrivial but clear dependence on G content, which is explained by chain flexibility and its relation to ion condensation.

Studies on the binding ability of diclofenac sodium to cationic surfactants micelles in aqueous ethanol solutions

Binding of diclofenac sodium (DCF), a non-steroidal anti-inflammatory drug, to hexadecyltrimethylammonium bromide and tetradecyltrimethylammonium bromide micelles in water as well as in aqueous ethanol solutions (0.0, 1.0, 2.5, 5.0, 7.5, 10.0 %) has been studied using isothermal titration calorimetry (ITC), UV–visible spectroscopy, and dynamic light scattering (DLS) techniques. Calorimetric data indicate that DCF binds with both the surfactants in approx 1:1 stoichiometry with binding constant (K b; obtained from ITC measurements) of order of 104 M−1. For both surfactants, K b values increase and Gibbs free energy of binding (∆G 0) decreases with ethanol concentration. Although ethanol weakens the hydrophobic interactions between two reacting species, the electrostatic interactions being stronger result in an overall increase in binding constant. Calorimetric results have been compared with those obtained spectroscopically. Further size variation studies on micelles upon binding with DCF (using DLS) complement the results obtained from these two techniques.

Size‐Selective Binding of Sodium and Potassium Ions in Nanoporous Thin Films of Polymerized Liquid Crystals

The development of a nanoporous material from a columnar liquid crystalline complex between a polymerizable benzoic acid derivative and a 1,3,5‐tris(1H‐benzo[d]imidazol‐2‐yl)benzene template molecule is described. The morphology of the liquid crystalline complex is retained upon polymerization and quantitative removal of the template molecule affords a nanoporous material with the same lattice parameters. The nanoporous material selectively binds cations from aqueous solution, with selectivity for sodium and potassium ions over lithium and barium ions, as shown with FT‐IR. Binding is also quantified gravimetrically with a quartz crystal microbalance with dissipation monitoring, a technique that is used for this purpose for the first time here.

Effect of pH on the Binding of Sodium, Lysine, and Arginine Counterions to l‐Undecyl Leucinate Micelles

AbstractMicelle formation by the amino acid‐based surfactant undecylenyl l‐leucine was investigated as a function of solution pH with NMR, dynamic light scattering, and fluorescence spectroscopy. NMR and dynamic light scattering showed that 50 mM undecylenyl l‐leucine and 50 mM NaHCO3 solutions contained micelles approximately 20 Å in diameter and that micelle radius and the mole fraction of surfactant molecules associated with micelles changed very little with solution pH. The binding of the amino acids arginine and lysine to the anionic micelles was also investigated from pH 7.0 to 11.5. Below pH 9.0, the mole fraction of arginine cations bound to the micelles was approximately 0.4. Above pH 9.0, the arginine counterions became zwitterionic, and the mole fraction of bound arginine molecules decreased steadily to less than 0.1 at pH 11. When arginine dissociated from the micelles, their radii decreased from 14 to 10 Å. Similar behavior was observed with lysine; however, when lysine dissociated from the micelle surface, little change in micelle radius was observed. Two‐dimensional NMR experiments suggested that below pH 9.0, l‐arginine bound perpendicular to the micelle surface primarily though its side chain amine while l‐lysine bound parallel to the surface through both of its amine functional groups. Finally, the rate at which the amide protons on the surfactant headgoup exchanged with solvent was investigated with NMR spectroscopy. The exchange reaction was faster in solutions containing only surfactant monomers and slower when the surfactants were in micellar form and the headgoup amide protons were less exposed to solvent.

A spectroscopic and molecular docking approach on the binding of tinzaparin sodium with human serum albumin

Protein bound toxins are poorly removed by conventional extracorporeal therapies. Venous thromboembolism (VTE) is a major cause of morbidity and mortality in patients with cancer. The interaction between tinzaparin, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV–vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by tinzaparin (TP). The binding constants and binding stoichiometry can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG° reveals that the binding process is a spontaneous process. Thermodynamic analysis shows that the HSA-TP complex formation occurs via hydrogen bonds, hydrophobic interactions and undergoes slight structural changes as evident by far-UV CD. It indicated that the hydrophobic interactions play a main role in the binding of TP to human serum albumin. In addition, the distance between TP (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 2.21 nm according to the Förster's resonance energy transfer theory. For the deeper understanding of the interaction, thermodynamic, and molecular docking studies were performed as well. Our docking results suggest that TP forms stable complex with HSA (Kb ∼ 104) and its primary binding site is located in subdomain IIA (Sudlow Site I). The results obtained herein will be of biological significance in pharmacology and clinical medicine.

Characterization of sodium mobility, binding, and apparent viscosity in full-fat and reduced-fat model emulsion systems

Reduction of sodium in foods is a top priority in today’s food industry. However, sodium reduction, due to its vital importance as a tastant, as well as its contribution to many other food properties, is no simple task. This study investigated the mobility of sodium ions, the ionic binding of sodium ions to xanthan gum, a popular functional hydrocolloid used in many processed foods, and the effect of sodium ions on the apparent viscosity of full-fat and reduced-fat model emulsion systems with increasing concentrations of added sodium, ranging from 0 to 480 mg added sodium per 30 g serving. 23Na NMR spectroscopy was utilized to measure sodium ion mobility, determined by both longitudinal (T1) and transverse (T2) relaxation times, and sodium binding, expressed by the ratio of bound to total sodium peak areas as the sodium concentration in the system increased. It was found that apparent viscosity increased, then decreased with increasing sodium concentration, and the concentration at which apparent viscosity began to decrease was higher for the reduced-fat compared to the full-fat emulsion system. The sodium mobility and sodium binding did not seem to be directly affected by pH. The characterization of sodium mobility, binding, and apparent viscosity in full-fat and reduced-fat model emulsion systems provides a foundation upon which further effects of sodium binding and functionality, including saltiness perception, in food matrices can be investigated.

Interface Induced Growth and Transformation of Polymer-Conjugated Proto-Crystalline Phases in Aluminosilicate Hybrids: A Multiple-Quantum (23)Na-(23)Na MAS NMR Correlation Spectroscopy Study.

Nanostructured materials typically offer enhanced physicochemical properties because of their large interfacial area. In this contribution, we present a comprehensive structural characterization of aluminosilicate hybrids with polymer-conjugated nanosized zeolites specifically grown at the organic-inorganic interface. The inorganic amorphous Al-O-Si framework is formed by alkali-activated low-temperature transformation of metakaoline, whereas simultaneous copolymerization of organic comonomers creates a secondary epoxide network covalently bound to the aluminosilicate matrix. This secondary epoxide phase not only enhances the mechanical integrity of the resulting hybrids but also introduces additional binding sites accessible for compensating negative charge on the aluminosilicate framework. This way, the polymer network initiates growth and subsequent transformation of protocrystalline short-range ordered zeolite domains that are located at the organic-inorganic interface. By applying an experimental approach based on 2D (23)Na-(23)Na double-quantum (DQ) MAS NMR spectroscopy, we discovered multiple sodium binding sites in these protocrystalline domains, in which immobilized Na(+) ions form pairs or small clusters. It is further demonstrated that these sites, the local geometry of which allows for the pairing of sodium ions, are preferentially occupied by Pb(2+) ions during the ion exchange. The proposed synthesis protocol thus allows for the preparation of a novel type of geopolymer hybrids with polymer-conjugated zeolite phases suitable for capturing and storage of metal cations. The demonstrated (23)Na-(23)Na DQ MAS NMR combined with DFT calculations represents a suitable approach for understanding the role of Na(+) ions in aluminositicate solids and related inorganic-organic hybrids, particularly their specific arrangement and clustering at interfacial areas.

Comparative analysis the binding affinity of mycophenolic sodium and meprednisone with human serum albumin: Insight by NMR relaxation data and docking simulation

Mycophenolic sodium is an immunosuppressive agent that is always combined administration with corticosteroid in clinical practice. Considering the distribution and side-effect of the drug may change when co-administrated drug exist, this paper comparatively analyzed the binding ability of mycophenolic sodium and meprednisone toward human serum albumin by nuclear magnetic resonance relaxation data and docking simulation. The nuclear magnetic resonance approach was based on the analysis of proton selective and non-selective relaxation rate enhancement of the ligand in the absence and presence of macromolecules. The contribution of the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index. This approach allowed the comparison of the binding affinity of mycophenolic sodium and meprednisone. Molecular modeling was operated to simulate the binding model of ligand and albumin through Autodock 4.2.5. Competitive binding of mycophenolic sodium and meprednisone was further conducted through fluorescence spectroscopy.

An external sodium ion binding site controls allosteric gating in TRPV1 channels.

TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli.

Internal Sodium in GPCRs Strongly Responds to Transmembrane Voltage Changes

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in the human genome, mediating the propagation of extracellular ligand binding information into intracellular signal transduction cascades. Crystal structures have revealed a water-filled hydrophilic internal pocket within their transmembrane domain, extending from the orthosteric ligand-binding site to regions near the G protein binding site. Recent high-resolution structures have identified a sodium ion near the base of this pocket, coordinated by highly conserved residues (1,2). Owing to this conservation level, sodium binding may play a role for most rhodopsin-like GPCRs (3). Recently, functional or conformational effects have been shown to be elicited by physiological transmembrane voltage in several GPCRs, and voltage-clamp recordings have determined a gating charge of ∼0.85 e in the M1 and M2 muscarinic receptors (4). However, the nature of the voltage sensor has remained enigmatic. Here we show by MD and computational electrophysiology simulations (5) that the internal sodium ion in the delta-opioid and muscarinic receptors is highly mobile under the influence of transmembrane voltage changes. By calculating the energetics of ion movement along the pocket axis, we find that the free energy barriers to migration can be overcome by the physiological voltage range. Furthermore, we demonstrate that the motion along the axis creates a gating charge in excellent agreement with the experimentally measured gating currents. Thus, voltage-induced movements of Na+ in GPCRs may represent an attractive mechanism for voltage-regulation of GPCRs.1. Liu, W. et al., Science 337: 232-236, 2012.2. Fenalti, G. et al., Nature 506: 191-196, 2014.3.Katritch, V. et al., TiBS 39: 233-244, 2014.4. Ben-Chaim, Y. et al., Nature 444: 106-9, 2006.5. Kutzner, C. et al., Biophysical Journal 101: 809-817, 2011.

From Physics to Phenotype: New Insights into Allosteric Transport Mechanisms in LeuT

Proteins carrying out the many biological processes essential to cellular function act as biomolecular machines utilizing mechanisms that are most often allosteric in nature, i.e. the structural components involved in the mechanism, which are sometimes separated by large distances at the molecular scale, are coupled energetically. Although it is a ubiquitous mechanism, allostery is still poorly understood as an experimentally validated molecular mechanism underlying particular instances, and lacks a general theoretical approach to describe it quantitatively. We will present our efforts to address both of these inadequacies using the secondary active transporter LeuT as an example. First, we will introduce a model for secondary active transport (LeVine, Cuendet, Khelashvili, and Weinstein. In review) that implicitly includes the allosteric coupling between two separate processes: i) sodium and substrate binding, and ii) substrate binding and intracellular opening of the transporter to allow release. Next, a general theoretical approach for allostery will be addressed with a statistical mechanical model based on the Ising Model (LeVine and Weinstein, Entropy, 2015). This will be shown to provide analytical insight into how long-distance allosteric couplings, such as that occurring between substrate binding and intracellular opening in LeuT, can arise in a complex system of interacting structural components. Finally, we will address the lack of experimentally validated molecular mechanisms underlying allostery. We will discuss a proposed model for substrate binding/intracellular opening coupling in LeuT, discovered using a novel information theory-based analysis framework, N-body Information Theory (NbIT) (LeVine and Weinstein, PLoS Comp Biol, 2014), and will present a recent experimental validation of this model. This presentation of combined theoretical, computational, and experimental work will demonstrate how deeper mechanistic understanding of biomolecular machines can be achieved through improved models of allosteric behavior.

Is Sodium Monocarboxylate Transporter (SMCT1) A Protein Involved in the Apical Iodide Transport?

SMCT1 is a sodium-coupled cotransporter of monocarboxylates and short-chain fatty acids, which has been found in the apical membrane of diverse epithelia such as colon, kidney, brain and thyroid. In thyroid follicular cells, SMCT1 originally was associated with passive apical iodide (I-) transport. However, this evidence was subsequently rejected, as Xenopus oocytes expression studies of SMCT1 demonstrated that this protein does not cotransport iodide or other inorganic anions. Interestingly, the role of SMCT1 in I- transport is now controversial, because an I- transport mode occurs at low extracellular sodium concentrations, such as that found in thyroid follicular lumen. Considering this discrepancy, here we have performed a comparative study between SMCT1 and the sodium iodide symporter (NIS) in order to find conserved sodium/iodide binding motifs between both proteins. Specifically, homology models of hSMCT1 and hNIS were built based on the crystal structure of vSGLT, a sodium/galactose transporter from Vibrio parahaemolyticus. The hNIS model predicts an iodide-binding pocket between the transmembrane TM3 and TM7 segments, where mutated residues previously were identified in patients with hypothyroidism. A similar iodide-biding pocket was identified in the hSMCT1 model involving some conserved residues in homologs of these proteins. Putative sodium binding sites were also identified in the two models, which comprise characteristic sequence motifs of sodium-dependent transporters. Finally, molecular dynamics simulations of the iodide-bound models were performed, demonstrating that conserved aromatic residues may be key in iodide coordination. Our results provide further evidence of homology between SMCT1 and NIS transporters and predict residues involved in binding sodium and iodide. Future work will be essential for determining the precise transport mechanism in these proteins.

Localization of a Sodium Binding Site in the Sodium Translocating NADH: Ubiquinone Oxidoreductase

Many marine, soil and pathogenic bacteria, including Vibrio cholerae, Pseudomonas aeruginosa, Yersinia pestis and Chlamydia trachomatis, possess a unique respiratory enzyme called Na+-translocating NADH:quinone oxidoreductase (NQR). This enzyme is capable of pumping Na+ across the cell membrane, thus creating an electrochemical gradient that the cell can use for metabolic functions. NQR pumps Na+ by using the energy released by the oxidation-reduction (redox) reaction between NADH and ubiquinone. While the electron transfer reactions have been well characterized in this enzyme, the Na+ translocation mechanism is not yet well known. Several acid groups oriented towards the cytoplasmic surface participate in Na+ uptake, while another group of acidic residues are required for Na+ release; however, there is no clear pathway for Na+ to bind through NQR. A further investigation into one of the residues required for Na+ uptake, NqrE-E95, has led to the discovery of one of the two predicted Na+ binding sites. After determining that this residue is essential for enzyme function, a closer look into the crystal structure revealed that NqrE-E95 is in the proper orientation to form a binding site with NqrE-D99 and NqrE-Y106. It was then found that these residues are also important for the utilization of Na+ by NQR. Further studies are being conducted to elucidate the exact role of NqrE-D99 and NqrE-Y106 in binding and transport of Na+. Locating binding sites in NQR is the first step in understanding how Na+ is translocated, and ultimately how the translocation is coupled to the redox reactions.