Study of ion translocation by respiratory complex I. A new insight using (23)Na NMR spectroscopy

The research on complex I has gained recently a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism(s). The proton has been identified as the coupling ion, although in the case of some bacterial complexes I Na(+) has been proposed to have that role. We have addressed the relation of some complexes I with Na(+) and developed an innovative methodology using (23)Na NMR spectroscopy. This allowed the investigation of Na(+) transport taking the advantage of directly monitoring changes in Na(+) concentration. Methodological aspects concerning the use of (23)Na NMR spectroscopy to measure accurately sodium transport in bacterial membrane vesicles are discussed here. External-vesicle Na(+) concentrations were determined by two different methods: 1) by integration of the resonance frequency peak and 2) using calibration curves of resonance frequency shift dependence on Na(+) concentration. Although the calibration curves are a suitable way to determine Na(+) concentration changes under conditions of fast exchange, it was shown not to be applicable to the bacterial membrane vesicle systems. In this case, the integration of the resonance frequency peak is the most appropriate analysis for the quantification of external-vesicle Na(+) concentration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Use of Na-Nuclear Magnetic Resonance To Follow Sodium Uptake and Efflux in NaCl-Adapted and Nonadapted Millet (Panicum miliaceum) Suspensions

Cellular Na⁺ transport was followed in vivo by ²³Na nuclear magnetic resonance (NMR) using anionic dysprosium-based shift reagents to resolve internal and external ²³Na⁺ resonances. Proso millet (Panicum miliaceum) cell suspensions adapted for rapid growth on 130 mm NaCl had biphasic ²³Na efflux kinetics when shifted to low Na⁺ medium, while nonadapted cells had little measurable Na⁺ efflux after preloading with ²³NaCl. Uptake of ²³Na was also observed using ²³Na NMR. The resonance frequency of the external Na⁺-dysprosium (III) triphosphate, relative to that of the ²³Na in the cells, was sensitive to pH, permitting the pH of the external medium to be followed during the course of in vivo experiments.     

Direct measurement of increased myocardial cellular23Na NMR signals in perfused guinea-pig heart induced by dihydroouabain and grayanotoxin-I

The effects of the cardiac glycoside dihydroouabain (DHO), and the ericaceous toxin grayanotoxin-I (GTX-I) on myocardial cellular sodium (Na_i) concentrations were investigated using sodium-23 nuclear magnetic resonance (²³Na NMR) spectroscopy at 30°C in isolated perfused guinea-pig hearts. The Na_i NMR signals from perfused Langendorff heart preparations were obtained by the modified inversion recovery (IR) method based on the previous observation that the spin-lattice relaxation time (T₁) of the Na_i (25 or 34 msec at 8.46 Tesla (T)) is much faster than that of extracellular sodium (64 msec at 9.4 T). Na_i was estimated from the calibration curve of the frequency area of the²³Na NMR FT spectra plotted against the standard Na concentration. The Na_i concentration of the heart increased concomitantly with the positive inotropic effects (PIE) of DHO, GTX-I and monensin (MON). The cumulative sequential addition of DHO (5×10^–6 M), GTX-I (7×10^–8 M) and MON (5×10^–6 M), each of which alone induced no appreciable PIE, produced a 22% elevation in Na_i concentration relative to that of the control (100%) accompanying a PIE of 44%. The mechanism of this Na_i elevation induced by combinational addition of DHO, GTX-I and MON may be mediated as follows: GTX-I increases the net Na-influxvia Na⁺ channels; DHO inhibits the pumping out of Na⁺ from the cell; and MON transports external Na⁺ into the cell, acting as a sodium ionophore. Consequently, these drugs act synergistically to increase the Na_i, thereby increasing the intracellular Ca²⁺ concentrationvia Na⁺–Ca²⁺ exchange.     

The Na transport in gram-positive bacteria defect in the Mrp antiporter complex measured with Na nuclear magnetic resonance

²³Na nuclear magnetic resonance (NMR) has previously been used to monitor Na⁺ translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na⁺. In this work, the ²³Na NMR method was adapted for measurements of internal Na⁺ concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na⁺ translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na⁺ concentration than an external one.     

Pulsed NMR studies on Na + binding to simple carbohydrates

Pulsed NMR spectroscopy has been used to study Na⁺ binding to several simple carbohydrates in aqueous solution. Changes in the ²³Na spin-lattice relaxation time (T₁) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na⁺ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, $\text{c i s}$-inositol forms the strongest complexes with the stability constant about 1.2 M^?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na⁺ and Ca²⁺ was done, indicating that both cations have the same binding sites.     

Identification of a 3rd Na+ Binding Site of the Glycine Transporter,GlyT2

The Na⁺/Cl^- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na⁺ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na⁺ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na⁺-dependent leucine transporter, LeuT_Aa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na⁺ binding sites in GlyT2, but the physical location of the third Na⁺ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuT_Aa. Although betaine transport by BetP is coupled to the co-transport of two Na⁺ ions, the first Na⁺ site is not conserved between BetP and LeuT_Aa, the so called Na1'' site. We hypothesized that the third Na⁺ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1'' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na⁺ placed at the location consistent with the Na1'' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na⁺ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.     

Direct High-resolution Nuclear Magnetic Resonance Studies of Cation Transport in Vivo: Na+ Transport in Yeast Cells

A new nuclear magnetic resonance (NMR) method for monitoring transmembrane metal cation transport is reported. It is illustrated with a study of Na⁺ efflux from Na⁺-rich yeast cells. The technique involves the use of an anionic paramagnetic shift reagent, present only outside the cells, to induce a splitting of the sodium-23 NMR peak, in this case, into components representing intra- and extracellular Na⁺. The time course of the efflux is in good agreement with the literature and can be well fitted with a double exponential decay expression. Splitting of the lithium-7 NMR signal from a suspension of Li⁺-rich respiratory-deficient, petite yeasts is also reported.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

Changes of intracellular Na+ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na⁺ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na⁺concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na⁺ concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na⁺ , K⁺ -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell perme-abilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell mem-branes are discussed.     

Matrix-dependent modulation of anisotropic effects on NMR spectra from 7Li+ and 23Na+ encapsulated in cryptands

⁷Li and ²³Na NMR spectra of the respective cations in gelatin and ι-carrageenan gels containing cryptand-[2.1.1] (for Li⁺) or cryptand-[2.2.2] (for Na⁺) displayed two transitions: the one at higher frequency corresponded to the cation surrounded by gel, the other to cation inside its appropriately sized cryptand. While binding to cryptands yielded much broader lines and shorter T ₁ relaxation times, anisotropic splitting in first order ⁷Li or ²³Na NMR spectra was not detected. Stretching the gels resulted in increasing the anisotropic electric field gradient tensor; thus, the NMR transitions of the cation in the gel were split (removal of degeneracy) to display its characteristic 3:4:3 triplet for spin = 3/2 nuclei. The transitions of the cryptand-bound cations (Li⁺-cryptand-[2.1.1] and Na⁺-cryptand-[2.2.2]) showed different extents of interaction with the electric field gradient tensor depending on the composition of the gel matrix. The NMR signal for ⁷Li⁺-cryptand-[2.1.1] in stretched gelatin gel showed a five-fold increased splitting as compared to the ⁷Li⁺ signal in the reference gel. In stretched ι-carrageenan gels, no anisotropic splitting from the cryptand-bound Li⁺ was recorded. Steady-state irradiation envelopes or z-spectra showed evidence of Li⁺ exchange between isotropic (cryptand) and anisotropic (gel) sites only at higher temperatures (55 °C). For Na⁺ bound to the cryptand-[2.2.2], anisotropic splitting (three-fold smaller compared with the ²³Na signal in the reference gel) was only recorded in stretched ι-carrageenan gels, whereas gelatin gels showed only anisotropic splitting for the ²³Na signal in the reference gel.     

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li+ competition

Double quantum and triple quantum filtered ²³Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na⁺ in different cellular systems, in the absence and presence of Li⁺. The kinetics of Li⁺ influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li⁺ than red blood cells (RBCs), while a higher intracellular Li⁺ concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na⁺ in all cellular systems studied. Isotropic intracellular Na⁺ motions were observed in all types of cells, while anisotropic Na⁺ motions in the intracellular compartment were only detected in RBCs. ²³Na triple quantum signal efficiency for intracellular Na⁺ was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. ²³Na double quantum signal efficiency for intracellular Na⁺ was non-zero only in RBCs, and for extracellular Na⁺ the order RBCs > chromaffin > SH-SY5Y cells was observed. Li⁺ loading generally decreased intracellular Na⁺ isotropic movements in the cells, except for astrocytes incubated with a low Li⁺ concentration and increased anisotropic intracellular Na⁺ movements in RBCs. Li⁺ effects on the extracellular signals were more complex, reflecting Li⁺/Na⁺ competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li⁺ action.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.     

Specificity of Na+ binding to phosphatidylserine vesicles from A 23Na NMR relaxation rate study

²³Na NMR relaxation rate measurements show that Na⁺ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K⁺ and Ca²⁺ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na⁺ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na⁺-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na⁺) fall in the range 0.4–1.2 M^?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na⁺ at 0.1 M Na⁺ concentration.     

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na⁺/K⁺-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na⁺ form of Tel23 G4, which is the same hybrid form as the K⁺ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na⁺ form to K⁺ form during Na⁺/K⁺ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na⁺/K⁺ spectral conversion. In addition, the K⁺ titration monitored by NMR reveals that the Na⁺/K⁺ exchange in Tel23 G4 is a two-step process. The addition of K⁺ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na⁺/K⁺ exchange and insight into the mechanism of Na⁺/K⁺ structural conversion in human telomeric G4s.     

Nuclear Magnetic Resonance of Tissue 23Na: I. 23Na Signal and Na+ Activity in Homogenate

The ability to depress the resonance intensity of ²³Na in rat liver tissue was not found in the supernatant fraction. It was exclusively localized in particulate fractions. The intensity and saturation behavior of the ²³Na signal was examined in suspensions containing various amounts of the particulate fraction of rat liver homogenate. The results strongly suggest that the ²³Na signal of tissue reflects quadrupole interactions and does not result from a slow exchange between the free and bound fractions of Na⁺. The activity coefficient of Na⁺ in rat liver homogenate (no medium was added) was 0.59, about 20% less than that in the isotonic saline. Available evidences and discussion indicate that the bound Na⁺ in the homogenate is much less than the so-called “NMR-invisible” fraction of Na⁺.     

NMR Evidence for Complexing of Na+ in Muscle, Kidney, and Brain, and by Actomyosin. The Relation of Cellular Complexing of Na+ to Water Structure and to Transport Kinetics

The nuclear magnetic resonance (NMR) spectrum of Na⁺ is suitable for qualitative and quantitative analysis of Na⁺ in tissues. The width of the NMR spectrum is dependent upon the environment surrounding the individual Na⁺ ion. NMR spectra of fresh muscle compared with spectra of the same samples after ashing show that approximately 70% of total muscle Na⁺ gives no detectable NMR spectrum. This is probably due to complexation of Na⁺ with macromolecules, which causes the NMR spectrum to be broadened beyond detection. A similar effect has been observed when Na⁺ interacts with ion exchange resin. NMR also indicates that about 60% of Na⁺ of kidney and brain is complexed. Destruction of cell structure of muscle by homogenization little alters the per cent complexing of Na⁺. NMR studies show that Na⁺ is complexed by actomyosin, which may be the molecular site of complexation of some Na⁺ in muscle. The same studies indicate that the solubility of Na⁺ in the interstitial water of actomyosin gel is markedly reduced compared with its solubility in liquid water, which suggests that the water in the gel is organized into an icelike state by the nearby actomyosin molecules. If a major fraction of intracellular Na⁺ exists in a complexed state, then major revisions in most theoretical treatments of equilibria, diffusion, and transport of cellular Na⁺ become appropriate.     

Sodium Transport in Capillaries Isolated from Rat Brain

Abstract: Brain capillary endothelial cells form a bloodbrain barrier (BBB) that appears to play a role in fluid and ion homeostasis in brain. One important transport system that may be involved in this regulatory function is the Na⁺,K⁺-ATPase that was previously demonstrated to be present in isolated brain capillaries. The goal of the present study was to identify additional Na⁺ transport systems in brain capillaries that might contribute to BBB function. Microvessels were isolated from rat brains and ²²Na ⁺ uptake by and efflux from the cells were studied. Total ²²Na ⁺ uptake was increased and the rate of ²²Na ⁺ efflux was decreased by ouabain, confirming the presence of Na⁺,K⁺-ATPase in capillary cells. After inhibition of Na⁺,K⁺-ATPase activity, another saturable Na ⁺ transport mechanism became apparent. Capillary uptake of ²²Na ⁺ was stimulated by an elevated concentration of Na ⁺or H⁺ inside the cells and inhibited by extracellular Na⁺, H⁺, Li⁺, and NH₄⁺. Amiloride inhibited ²²Na ⁺ uptake with a K_i between 10^?5 and 10^?6M but there was no effect of 1 mM furosemide on ²²Na⁺ uptake by the isolated microvessels. These results indicate the presence in brain capillaries of a transport system capable of mediating Na ⁺/ Na ⁺ and Na ⁺/H ⁺ exchange. As a similar transport system does not appear to be present on the luminal membrane of the brain capillary endothelial cell, it is proposed that Na ⁺/H ⁺ exchange occurs primarily across the antiluminal membrane.     

Thallium inhibition of ouabain-sensitive sodium transport and of the (Na+K)-ATPase in human erythrocytes

The influence of Tl⁺ on Na⁺ transport and on the ATPase activity in human erythrocytes was studied. 0.1–1.0 mM Tl⁺ added to a K⁺-free medium inhibited the ouabain-sensitive self-exchange of Na⁺ and activated both the ouabain-sensitive ²²Na outward transport and the transport related ATPase. 5–10 mM external Tl⁺ caused inhibition of the ouabain-sensitive ²²Na efflux as well as the (Na⁺ + Tl⁺)-ATPase. Competition between the internal Na⁺ and rapidly penetrating thallous ions at the inner Na⁺-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl⁺. An increase of the internal Na⁺ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl⁺. A protective effect of Na⁺ was also demonstrated on the (Na⁺ + Tl⁺)-ATPase of fragmented erythrocyte membranes studied at various Na⁺ and Tl⁺ concentrations.     

Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an Occluded State of LeuT

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.     

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.