Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm⁻¹ and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm⁻¹) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.     

Resolution of Pump and Leak Components of Sodium and Potassium Ion Transport in Human Erythrocytes

Further support for the pump-leak concept was obtained. Net transport was resolved into pump and leak components with the cardiac glycoside, ouabain. The specificity of ouabain as a pump inhibitor was demonstrated by its ineffectiveness when the pump was already inhibited by lack of one of the three pump substrates, sodium ion, potassium ion, or adenosine triphosphate. In the presence of ouabain the rates of passive transport of sodium and potassium ions changed almost in proportion to changes in their extracellular concentrations when one ion was exchanged for the other. In the presence of ouabain and at the extracellular concentrations which produced zero net transport, the ratio of potassium ions to sodium ions was 1.2-fold higher inside the cells than outside. This finding was attributed to a residual pump activity of less than 2% of capacity. The permeability to potassium ions was 10% greater than the permeability to sodium ions. A test was made of the independence of pump and leak. Conditions were chosen to change the rate through each pathway separately or in combination. When both pathways were active, net transport was the sum of the rates observed when each acted separately. A ratio of three sodium ions pumped outward per two potassium ions pumped inward was confirmed.     

Hydrogen-bonding network at the cytoplasmic region of a light-driven sodium pump rhodopsin KR2

Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the “Ion-selectivity filter” for sodium ions, while retinal Schiff base acts as the light “Switch and Gate” for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies. According to the atomic structure of KR2, Q123 is involved in the hydrogen-bonding network at the cytoplasmic region, together with S64, protein-bound waters, and peptide carbonyl of K255 bound to the chromophore. To gain the detailed structural information around Q123, here we compared light-induced difference Fourier-transform infrared (FTIR) spectra at 77?K between the wild-type (WT) and mutant proteins of KR2, such as Q123A, Q123V, and S64A. The obtained spectra were very similar between WT and these mutants, whereas the observed mutation effects enabled us to identify vibrations of the hydrogen-bonding network at the Q123 and S64 region. This is unique for KR2, not for the corresponding mutations in a light-driven proton-pump bacteriorhodopsin (BR). Hydrogen-bonding alteration is absent for the mutants of KR2, suggesting that proper inter-helical connectivity of helices B, C, and G is important for protein structural changes for sodium-pump function, which is controlled by the region around Q123.     

Arginine residues in the D2 polypeptide may stabilize bicarbonate binding in photosystem II of Synechocystis sp. PCC

Bicarbonate (HCO3-) causes a significant and reversible stimulation of anion-inhibited electron flow in photosystem II of higher plants and cyanobacteria. To test if selected arginine (Arg) residues are involved in the binding of HCO3-, we utilized oligonucleotide-directed mutagenesis to construct Synechocystis sp. PCC 6803 mutants carrying mutations in Arg residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233----glutamine) and R251S (arginine-251----serine) were 10-fold more sensitive to formate than the wild type. The formate concentration giving half-maximal inhibition of the steady-state oxygen evolution rate was 48 mM, 4.5 mM and 4 mM for the wild type, R233Q and R251S, respectively. Measurements of oxygen evolution in single-turnover flashes confirm that the mutants are more sensitive to formate than the wild type. Measurements of chlorophyll a fluorescence decay kinetics after the second saturating actinic flash indicated that, after formate treatment, the halftime of QA- oxidation was decreased by approximately a factor of 2, 4 and 6 in the wild type, R251S and R233Q, respectively. The recombination rate between QA- and S2 was approx. 2-fold slower in R251S and R233Q than in the wild type. In the presence of 100 mM sodium formate, reactivation of the Hill reaction by bicarbonate showed that the wild type had an apparent Km for bicarbonate of 0.5 mM, while the Km values for R233Q and R251S were 1.4 and 1.5 mM, respectively. We suggest that Arg-233 and Arg-251 in the D2 polypeptide contribute to stabilization of HCO3- binding in Photosystem II.     

Potassium and sodium movements in the Ehrlich mouse ascites tumor cell

Studies have been conducted on the movements of sodium and potassium into and out of the Ehrlich ascites tumor cell. Under steady state conditions, at 22 degrees C., in the absence of an exogenous source of glucose, the cell flux for both potassium and sodium averaged 0.8 microM10(7) cells/hr, or 3.0 pM/cm.(2)/sec. The cell can accumulate potassium and extrude sodium against electrochemical gradients for both ions. It is possible under the experimental conditions reported to separate the transport systems for these two ions. Thus, it has been shown that under conditions of low temperature with a diminished metabolism, net fluxes for the two ions are different. Also, following periods of 24 hours at 2 degrees C., an exogenous source of glucose enhances the accumulation of potassium sevenfold while sodium extrusion is uninfluenced by the presence of glucose. Similarly potassium exchange rates are temperature-dependent, with Q(10) values as high as 5, while exchange rates for sodium are temperature-insensitive, with Q(10) values of 1.2 to 1.6. Glycolysis has been eliminated as an energy source for the transport processes since these processes go on in the absence of an exogenous source of glucose. It is estimated that a maximum of 0.3 per cent of the energy derived from the total oxidative metabolism of glucose would be required to support independent transport of potassium and sodium.     

Quantitative trait loci for component physiological traits determining salt tolerance in rice

Rice (Oryza sativa) is sensitive to salinity, which affects one-fifth of irrigated land worldwide. Reducing sodium and chloride uptake into rice while maintaining potassium uptake are characteristics that would aid growth under saline conditions. We describe genetic determinants of the net quantity of ions transported to the shoot, clearly distinguishing between quantitative trait loci (QTL) for the quantity of ions in a shoot and for those that affect the concentration of an ion in the shoot. The latter coincide with QTL for vegetative growth (vigor) and their interpretation is therefore ambiguous. We distinguished those QTL that are independent of vigor and thus directly indicate quantitative variation in the underlying mechanisms of ion uptake. These QTL independently govern sodium uptake, potassium uptake, and sodium:potassium selectivity. The QTL for sodium and potassium uptake are on different linkage groups (chromosomes). This is consistent with the independent inheritance of sodium and potassium uptake in the mapping population and with the mechanistically different uptake pathways for sodium and potassium in rice under saline conditions (apoplastic leakage and membrane transport, respectively). We report the chromosomal location of ion transport and selectivity traits that are compatible with agronomic needs and we indicate markers to assist selection in a breeding program. Based upon knowledge of the underlying mechanisms of ion uptake in rice, we argue that QTL for sodium transport are likely to act through the control of root development, whereas QTL for potassium uptake are likely to act through the structure or regulation of membrane-sited transport components.     

Potential-dependent membrane sodium pump current in snail giant neurons

The effect of the membrane potential on the pump current evoked by iontophoretic injection of sodium into the neuron and the effect of the intracellular sodium ion concentration on the potential dependence of the pump current were investigated by the voltage clamp method in isolated and semi-isolated neurons ofHelix pomatia andHelix italiana. The pump current was shown to change its direction in the presence of marked hyperpolarization of the membrane (by more than −80 to −120 mV). An increase in the intracellular sodium ion concentration following injection of excess ions into the neuron increases the potential dependence of the pump current. A possible connection between passive potassium permeability and the activity of the enzymic transport mechanism for the elimination of sodium from the cell is postulated.     

On the Concept of Resting Potential—Pumping Ratio of the Na+/K+ Pump and Concentration Ratios of Potassium Ions Outside and Inside the Cell to Sodium Ions Inside and Outside the Cell

In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na⁺/K⁺ pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na⁺/K⁺ pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na⁺/K⁺ pump to prove that the three Na⁺ to two K⁺ pumping ratio of the Na⁺/K⁺ pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na⁺/K⁺ pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is ?30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na⁺/K⁺ pump and the ion concentration ratios play a role in the evolution of animal cells.     

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from -30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.     

A kinetic model for determining the consequences of electrogenic active transport in cardiac muscle

When active transport is electrogenic in a tissue that is continuously active, such as cardiac muscle, the active transport current is as important in the generation of the action potential as are the passive currents. A thermodynamically constrained kinetic model of electrogenic active transport of sodium and potassium ions has been developed in which the influences of voltage and chemical composition are explicitly defined. This model is coupled to a system of passive permeabilities, of the minimum degree of complexity, to simulate the integrated activity of active and passive ion transport in the generation of the cardiac action potential. Results of preliminary simulations indicate that electrogenic active transport provides a mechanism for slowly changing currents both within the time scale of an action potential as well as of many action potentials. The presence of active transport also complicates the interpretation of isotopic flux measurements and the separation of currents.     

Potassium uniport and ATP synthesis in Halobacterium halobium.

Light-driven potassium ion uptake in Halobacterium halobium is mediated by bacteriorhodopsin. This uptake is charge-balanced by sodium ions and not by proton release. Light-induced shifts in concentrations of divalent cations were found to be negligible. The transient changes in extracellular pH (alkaline overshoot) can be understood by the concomitant processes of ATP synthesis, proton/sodium exchange and potassium uptake. The driving force of potassium ion uptake is the membrane potential, no ATP-dependent potassium transport process is found. Fluorescence measurements indicate a high permeability of the membrane to potassium ions compared to sodium ions. Therefore the potassium ion diffusion potential contributes to the membrane potential (about 30 mV/decade) and thereby influences the ATP level. Sudden enhancement of the diffusion potential by the potassium ionophore monactin leads to the expected transient increase in cellular ATP level. Due to the large size (up to 100-fold) of the potassium ion gradient and its high capacity (intracellular concentration up to 3 M) the potassium ion gradient can well serve the cell as a long term storage form of energy.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

The transmembranous passes of K+ in brain cortex under radiation and chemical influences and their modification with natural adaptogen

An investigation of the influence of chronic low-intensity irradiation with 0.25 Gr dose and of a mixture of heavy metal salts both apart and together, as well as when correcting with a natural adaptogen such as Spirulina platensis, on passive and active transport of potassium and the work efficiency of Na,K-pump in slices of brain cortex was the problem of this study. As a biological model for in vivo researches on molecular-cellular level the thin layers of a rat brain cortex were used. It was shown that both radiation and chemical factors cause reliable changes of passive membrane permeability and operation of Na,K-pump, whose function is the maintenance of the certain gradient of potassium ions on the plasmatic membrane, the normalization of ion homeostasis, the stabilization of membranous potential. The analysis of calculated indexes of passive and of active ion transport show that at modification of X-rays influence with a mixture of heavy metal salts exactly the last makes the main contribution to efficiency decrease of energy-dependent transport of potassium. The natural adaptogen spirulina renders a better effect in the case of toxic action of the mixture of heavy metal salts, enlarging not only active transport of potassium ions, but its efficiency as well.     

Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn(109), Asn(116), Asn(122), and Asn(125)) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A(1) extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.     

Active and Passive Components of Sulfate Uptake in Sunflower Plants

The aim of the investigation was to identify components of active and passive ion uptake and transport in roots of plants and to assess their quantitative relations under different external and internal conditions. The uptake of radiosulfate and water by young sunflower plants from complete nutrient solutions labelled with ³⁵S was studied. The metabolism-linked nature of the sulfate uptake in the root following the passive migration into the apparent free space (AFS) was demonstrated by the addition of sodium. selenate, 2,4-dinitrophenol, potassium cyanide, and sodium azide to the nutrient solutions. The magnitude of the AFS measured on a root volume basis varied between 14 and 57 per cent depending on the pretreatment of the plants and the sulfate concentration of the nutrient solution. The variations were supposed to be due to different capacity to bind sulfate by exchange-adsorption within the AFS. The amounts of sulfate in different fractions of the total AFS-uptake were computed under certain theoretical assumptions. A quantitative connection was proposed between the magnitude of the adsorbed sulfate fraction in the AFS and the rate of active uptake into the symplasm. The exchange-adsorption probably constitutes the initial stage of active ion uptake. The stimulating effect by water on ion uptake would be an increase of the speed of transporting ions to, from, or along the adsorption sites in the AFS. Experiments conducted at temperatures in the nutrient solution between 5 and 35 C elucidated the multistep nature of ion transport within a root.     

In?Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H⁺/Na⁺ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na⁺ and H⁺ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na⁺ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na⁺-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na⁺ transport and electrogenic H⁺ transport, respectively, in the presence and absence of Na⁺. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na⁺ transport by IAR requires Na⁺ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H⁺/Na⁺ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.     

D2O as a modifier of ionic specificity of Na, K-ATPase

Effect of heavy water D2O on the rate of hydrolysis of ATP and pNPP by Na,K-ATPase was studied. Heavy water of high concentration inhibits the rate of ATPase reaction in all the studied ratios of the ions Na/K at constant ionic strength 150 mM. Activation of the enzyme was observed in the solution with low concentration of heavy water (less than 5%). The value of isotope effects depended on the ratio between sodium and potassium ion concentrations in the medium. At low temperature no activation of the enzyme with heavy water in low concentration was observed. Substitution of usual water for the heavy one was accompanied by a decrease of apparent constants of enzyme activation with sodium and potassium ions. During pNPP hydrolysis with Na,K-ATPase an increase of reaction rate in the medium with heavy water was observed. Substitution of potassium ions by cesium resulted in an increase of isotope effects during ATP and pNPP hydrolysis. Analysis of isotope effects in terms of the molecular model of sodium pump proposed permits a conclusion that the isotope effects of heavy water are explained by its influence as a solvent, the binding centres of potassium and sodium ions are localized in different regions of the enzyme differing in physico-chemical properties. The structure of sodium centres is controlled by hydrogen bonds, and of potassium ones--by hydrophobic interactions; the transport of ions by the enzyme is accompanied by dehydration of ions.     

Extramembrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport

We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. (2002) Nature 419, 587-593). The extramembrane headpiece of the AcrB trimer contains a central pore composed of three alpha-helices. Each pore helix belongs to a different monomer. In this study, we constructed cysteine-scanning mutants as to the residues comprising the pore helix. Of the 21 mutants (D99C to P119C), 5 (D101C, V105C, N109C, Q112C, and P116C) showed significantly reduced drug resistance and drug-exporting activity. These residues are localized on one side of the pore helix, i.e. on the wall of the pore. These observations strongly indicate the important role of this pore in the drug transport process. A N-ethylmaleimide binding experiment revealed that the pore is in the closed state, and the thickness of the permeability barrier in the middle of the pore corresponds to 2.5 alpha-helical turns. Two mutants (V105C and Q112C), which showed the greatest loss of activity of all of the pore mutants, were detected in the form of disulfide cross-linking dimers under normal conditions, suggesting that a conformational change of the pore is indispensable during the transport process.     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.