Facilitated transport of di- and trinitrophenolate ions across lipid membranes by valinomycin and nonactin.

The conductance of black lipid membranes in the presence of 2,4,6-trinitrophenol (or 2,4-dinitrophenol) is considerably enhanced, if the cation carriers valinomycin, enniatin B or nonactin are added. The effect is, however, largely independent of the cation concentration and is identical for the cations Li+, Na+ and Ba2+. This finding, as well as the sign and magnitude of the diffusion potential in the presence of a gradient of picrate are consistent with the assumption that the transport of picrate anions is facilitated by the above-mentioned macrocyclic compounds, but that cations are not directly involved. A model is suggested which, based on the generation of mobile defect structures by the incorporation of large molecules, allows one to explain facilitated transport without the assumption of stable chemical bonds between a carrier and its transported substrate. If K+ is present in the aqueous phase, the conductance is largely determined by the permeation of the cation complexes of valinomycin and nonactin. The conductance is, however, increases by adsorption of picrate anions to the membrane surface. The negative surface potential generated by the adsorption layer seems to be responsible for the saturation of the conductance at high picrate concentrations in the absence of valinomycin and nonactin.     

Cation binding by valinomycin and trinactin at the air-water interface: Cooperativity in cation binding by valinomycin

A previous communication reported the uptake of monovalent cations by a valinomycin monolayer at the air-water interface (Colacicco, G., Gordon, E. E. and Berchenko, G. (1968) Biophys. J. 8,22a). A similar study has been done with trinactin. As in the case of valinomycin, an elevated surface potential is obtained when the cation-ionophore complex is formed. A surface potential of 0.82 V was obtained for the trinactin-cation complex, as compared with 0.54 V for uncomplexed trinactin. The observed cation selectivity NH₄⁺ > K⁺ > Rb⁺ > Cs⁺, Na⁺ and Li⁺ is in agreement with partition and bilayer conductance experiments.A minimum packing area of 130 Ų obtained for the trinactin-cation complex was in excellent agreement with the 125 Ų predicted from space filling models, reinforcing the suggestion that area-per-molecule calculations obtained at the air-wate interface can provide useful information on the molecular dimensions of these hydrophobic, relatively low molecular weight transport antibiotics.Comparison of the data obtained previously with valinomycin and with trinactin revealed two striking differences: (1) a large inflection in the force-area curve concurrent with cation binding and indicative of a conformational change was obtained with valinomycin,, but no evidence was found with trinactin; (2) the uptake of cations by trinactin could be predicted by simple equilibrium expressions, but the uptake of cations by valinomycin was strongly cooperative. Possible mechanisms for this cooperative association fo cations are discussed.     

Two-phase partition studies of alkali cation complexation by ionophores

Summary The partition of alkali cations and anions between an aqueous and an immiscible organic phase has been studied in the absence and presence of neutral and carboxylic ionophores of the valinomycin and nigericin types, respectively. Cation extraction into the organic phase was augmented considerably by the ionophores, and a cation specificity of K⁺Rb⁺>Cs⁺Na⁺ was found for all the neutral ionophores tested. Evidence is given that the actual values of ion specificity are a function of the solvent polarity, especially for valinomycin where an inversion of the K⁺/Rb⁺ specificity was observed. The ionophores examined have the following rank order of effectiveness for K⁺ extraction into a standard organic phase consisting of 70% toluene-30%n-butanol: valinomycin>18-crown-6trinactin>enniatin Bdinactin>monactin>nonactin. The ion affinity and selectivity data thus obtained have been compared with data previously reported.In a toluene-butanol solvent, extraction of cations in the absence of ionophores occurs as ion pairs. On the other hand, the neutral ionophores extract the cations by the mechanism of complexation, with the lipophilic anions coextracted as free gegenionic species at lower ionophore complex concentrations. When the concentration of extracted cations exceeds 1×10^–4 m, ion pairing between the ionophore complex and the anion occurs, and this tendency increases with increasing concentration and decreasing polarity of the organic phase. Anion pairing with the complexed cations is much less than for the free cations and this effect appears to be due to the larger distance of closest approach of the anion for the complexed cation.     

Crystal structure of peptide cyclo-(D-Val-L-Pro-L-Val-D-Pro)

The crystal and molecular structure of the rubidium/picrate complex of the peptide cyclo-(D-val-L-pro-L-val-D-pro)₃, called prolinomycin, has been determined by X-ray crystallography and found to be similar to the well known ion-carrier valinomycin. Prolinomycin crystallizes in the triclinic system with two prolinomycin molecules and two each rubidium cations and picrate anions in the unit cell. There are also ordered toluene and chloroform molecules, which are the solvents of crystallization, in the unit cell. The conformation of the two crystallographically independent prolinomycin molecules in the unit cell are very similar. Potential energy calculations show that the cation is bound more strongly in prolinomycin compared to valinomycin. This was also observed in solution (7).     

Gated ion fluxes involved in photophobic responses of the blue-green alga,Phormidium uncinatum

The sensory transduction chain of photophobic responses in the blue-green alga, Phormidium uncinatum seems to involve a gating cation transport through membrane bound ion channels which provides an effective amplification.The calcium conducting ionophore A23187 inhibits the photophobic response totally and induces frequent reversals which resemble phobic responses but occur without any light stimulation. This indicates that the electrogenic ion conductance may depend on a gradient of divalent cations, esp. calcium. The calcium conductance during a photophobic response is further confirmed by the inhibitory effect of ruthenium red and lanthanum, blockers of the electrogenic calcium transport. In the case of lanthanum this inhibition is found at a concentration at which neither the number of motile filaments nor the average speed of movement is impaired.Incorporation of ionophores for monovalent cations (gramicidin and valinomycin) only partially impairs the response. Similarly, inhibition of the Na⁺/K⁺ pump by ouabain is less effective. Thus, the existence of a countercurrent of monovalent cations during the response, which has been described for e.g. ciliates, is yet obscure in blue-green algae.     

Mechanism of active shrinkage in mitochondria II. Coupling between strong electrolyte fluxes

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCl causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner.2. A passive penetration of K⁺ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude.3. Passive swelling and active shrinkage occurs also when K⁺ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration.4. Active shrinkage, either with K⁺ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%.5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.     

Current-voltage studies on the thylakoid membrane in the presence of ionophores

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1 %) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K ≈ 10⁴) and the turnover numbers (≈ 3 · 10⁴/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.     

Transport of alkali cations through thin lipid membranes by (222)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of K⁺ and Na⁺ transport across the membrane of large unilamellar vesicles (L.U.V.) were compared at two pH's, with two carriers: (222)C ₁₀-cryptand (diaza-1, 10-decyl-5-hexaoxa-4,7,13,16,21,24-bicyclo[8.8.8.]hexacosane) and valinomcyin, i.e. an ionizable macrobicyclic amino polyether and a neutral macrocyclic antibiotic. The rate of cation transport by (222)C₁₀ saturated as cation and carrier concentrations rose. The apparent affinity of (222)C₁₀ for K⁺ was higher and less pH dependent than that for Na⁺ but resembled the affinity of valinomycin for K⁺. The efficiency of (222)C₁₀ transport of K⁺ decreased as the pH fell and the carrier concentration rose, and was about ten times lower than that of valinomycin. Noncompetitive K⁺/Na⁺ transport selectivity of (222)C₁₀ decreased as pH, and cation and carrier concentrations rose, and was lower than that of valinomycin. Transport of alkali cations by (222)C₁₀ and valinomycin was noncooperative. Reaction orders in cationn(S) and carrierm(M) varied with the type of cation and carrier and were almost independent of pH;n(S) andm(M) were not respectively dependent on carrier or cation concentrations. The apparent estimated constants for cation translocation by (222)C₁₀ were higher in the presence of Na⁺ than of K⁺ due to higher carrier saturation by K⁺, and decreased as pH and carrier concentration increased. Equilibrium potential was independent of the nature of carrier and transported cation. Results are discussed in terms of the structural, physicochemical and electrical characteristics of carriers and complexes.     

The respiration of brain mitochondria and its regulation by monovalent cation transport

The Na⁺ and K⁺ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose.Respiration was uncoupled by Na⁺ and less so by K⁺. Uncoupling was maximal in the presence of EDTA plus P_i and was decreased by Mg²⁺. Maximal uncoupler-stimulated respiration rates were inhibited by Na⁺ but largely unaffected by K⁺. The inhibition by Na⁺ was relatively insensitive to Mg²⁺. Membrane Na⁺ and K⁺ conductances as well as neutral exchanges (Na⁺/H⁺ and K⁺/H⁺ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations.Cation conductance, i.e. electrophoretic Na⁺ or K⁺ permeation, was increased by EDTA (Na⁺ > K⁺) and decreased by Mg²⁺. Magnesium preferentially suppressed Na⁺ conductance so as to reverse the cation selectivity (K⁺ > Na⁺). Neutral cation/H⁺ exchange rates (Na⁺ > K⁺) were not influenced by chelator or Mg²⁺.The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K⁺ conductor valinomycin. The metabolic influences of Na⁺ and K⁺ can be explained in terms of coupled flow of these ions with protons and their effect upon the H⁺ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Cation transport and electrogenesis byStreptococcus faecalis

Summary Uptake of the lipid-soluble cations dibenzyldimethylammonium (DDA⁺) and triphenylmethylphosphonium (TPMP⁺) byStreptococcus faecalis is biphasic. The initial phase is a rapid binding of the ions which does not require a source of metabolic energy and apparently consists of cation exchange at the cell surface. Upon addition of glucose further uptake of the cations occurs, by exchange for Na⁺ and H⁺. Evidence is presented suggesting that this metabolic uptake of DDA⁺ and TPMP⁺ is not due to active transport. It rather appears that uptake results from the generation of an electrical potential, interior negative, by the extrusion of H⁺ and, indirectly, of Na⁺. Accumulated DDA⁺ and TPMP⁺ are discharged by proton-conducting uncouplers. The cationconducting antibiotics valinomycin, monactin, nigericin and monensin do not inhibit uptake. Potassium and, under certain conditions, H⁺ displace DDA⁺ and TPMP⁺. Generation of an electrical difference across the membrane was verified by the accumulation of K⁺ in the presence of valinomycin. The concentration ratios achieved correspond to potentials of the order of –150 to –200 mV.     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

The K+-ionophores nonactin and valinomycin interact differently with the protein of reconstituted cytochromec oxidase

The K⁺-ionophores valinomycin and nonactin induce a qualitatively identical change of the visible spectrum of isolated oxidized cytochromec oxidase (red shift), but the amplitude is half with nonactin. Valinomycin, in the presence or absence of a protonophore, stimulates the respiration of the reconstituted enzyme to a higher extent than nonactin and results in a higherK _m for cytochromec. In contrast, nonactin causes a fivefold rate of proton conductivity across a liposomal membrane, after induction of a K⁺-diffusion potential. The data indicate that respiratory control by these antibiotics is not only due to degradation of a membrane potential, but rather to specific interaction with and modification of cytochromec oxidase.     

Sodium and potassium binding by rat liver cell microsomes

The effects of ion concentration, pH, and presence of competing ions on the sodium and potassium binding properties of rat liver cell microsomes were studied. Typical adsorption isotherms were obtained in the concentration dependence studies, with saturation being reached when 1.2 to 1.4 m.eq. cations were retained per gm. of microsome Kjeldahl nitrogen. The retention was shown to be due to a binding to specific sites rather than to a trapping of the cations. The binding showed a sharp pH dependence in the range 6.0 to 7.5. The presence of one cation depressed the binding of the other, indicating that Na⁺ and K⁺ as well as H⁺ ions compete for the same sites. Potassium was bound slightly more strongly than sodium, while hydrogen was bound about 10⁵ times more strongly than either. Calculations show that the binding follows the simple mass law. Similarities between adsorption by microsomes and adsorption by synthetic cation exchange resins are discussed and compared to some of the characteristics of electrolyte behavior in living systems. A possible ion exchange elution, active cation transport mechanism is suggested, involving the preferential elution of Na⁺ out of the cell by H⁺ ions produced by metabolism.     

A laser-T-jump study of the adsorption of dipolar molecules to planar lipid membranes

The adsorption of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as well as of other dipolar molecules to the interface of artificial lipid membranes gives rise to a change of the dipole potential between the membrane interior and water. As a consequence of the adsorption of the neutral species, the conductance of planar membranes, observed in the presence of the macrocyclic ion carriers nonactin or valinomycin, may change by many orders of magnitude. Using this effect in combination with a laser-T-jump technique, the kinetics of the adsorption process were measured and were interpreted on the basis of a Langmuir-isotherm. A partition coefficient (at small concentrations) of _HA =4.7·10^–4 cm, a rate constant of desorption k _HA100 s^-1 and a maximum surface density N _D=7.7·10¹³/cm² were found. The concentration at half saturation is K _HA=2.7·10^-4 M.Using these values the membrane conductance induced by the ion carrier nonactin and the shape of the current-voltage relationship as a function of the ligand concentration in water was analyzed. A maxiumum dipole potential of V _D ^max =-239 m V and a contribution of b=3.1·10^-15V cm² per single adsorbed 2,4-D molecule was found. 74% of the dipole potential acts on the inner membrane barrier separating the two interfacial adsorption planes of nonactin. The remainder (26%) favours interfacial complex formation between nonactin and K⁺ from the aqueous phase. The data hold for membranes formed from dioleoyllecithin in n-decane.     

Pressure effects on mechanisms of charge transport across bilayer membranes

Ion transport across diphytanoylphosphatidylcholine/decane bilayer membranes was measured as a function of hydrostatic pressure over the range 0.1-100 MPa (1-1000 atm). Carrier-mediated K+ conductance decreased with increasing pressure, yielding positive activation volumes of 45 A3 per complex for valinomycin mediated transport, and 74 A3 per complex in the case of nonactin. Comparison with the known pressure dependence of the viscosity of bulk alkane liquids supports the view that the rate limiting step for carrier-mediated transport is the translocation of the carrier-cation complex across an essentially fluid hydrocarbon membrane core. The parameters characterizing transient conductance by the hydrophobic anions, dipicrylaminate and tetraphenylborate, by contrast, were found to be insensitive to pressure over the range available. This was also the case for the steady-state conductance observed at elevated concentrations of both tetraphenylborate and the hydrophobic cation, tetraphenylarsonium. The quasi-stationary conductance observed at elevated concentrations of dipicrylaminate did, however, decrease significantly with increasing pressure, indicating a positive activation volume of 20 A3 per ion. Alternative explanations of this more complex response of hydrophobic ions to pressure are considered. Ancillary measurements of specific membrane capacitance revealed an increase of about 10% with an increase of pressure to 100 MPa, yielding an estimated membrane compressibility on the order of 10(-9) m2 X N-1, comparable to that of bulk liquid hydrocarbons.     

Stability of sodium and potassium complexes of valinomycin

Stability constants of sodium and potassium complexes of valinomycin in some alcohols and water—organic solvent mixtures have been determined by titration, using circular dichroism to monitor complex formation. Constants range from 10¹ to 10⁶ M⁻¹. Stability of the potassium and sodium complexes increases with decreasing dielectric constant, but the ratio of the constants remains about 10³–10⁴. As others have shown, a similar selectivity for K⁺ is observed in a number of other types of measurements involving valinomycin. These include the permeability and conductance ratios which characterize the selectivity of cation transport through membranes and the ratio of salt extraction equilibrium constants. On the basis of data presented here, and elsewhere, it is suggested that conformational constraints within the depsipeptide part of the complexes aid ion selectivity and that differences in cation solvation and carbonyl ligand binding energies make an important, roughly equal, contribution.     

On the mechanism of anion uptake by plant roots

Summary The effect of the valence of the associated cation on Cl^–-uptake by excised barley roots grown in CaSO₄ has been studied at 26°, 6° and 2°C. The uptake of Cl^– relative to that of the associated cation was found to increase in the order: trivalent > divalent > monovalent. This was explained on the expected effect of the cation on the negative charge and potential of root surfaces. A lyotropic order was observed in case of monovalent cations, whereas divalent cations showed no such order. The order observed in Cl^–-uptake from chloride solutions of monovalent cations is associated with the ability of the absorbed cation to remove Ca and Mg from the roots. Li⁺ behaved similar to divalent cations in affecting the relative Cl^–-uptake from LiCl.As to the effect of temperature on the uptake of Cl^– and associated cation, it appears that Cl^– is not taken up to any great extent at 2°C whereas cations are still adsorbed at this low temperature. This has been explained on the assumption of the presence of negative adsorption spots on the root surface which can hold cations but not anions. It appears that Cl^–-uptake by roots requires the expenditure of energy to overcome repulsion arising from the negative surface.This work is supported by AEC contract AT (11-1) — 34 project 55.     

Efficiency,Na+/K+ selectivity and temperature dependence of ion transport through lipid membranes by (221)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of Na⁺ and K⁺ transport across the membrane of large unilamellar vesicles (LUV) were determined at two pH's when transport was induced by (221)C₁₀-cryptand (diaza-1,10-decyl-5-pentaoxa-4,7,13,16,21-bicyclo [8.8.5.] tricosane) at various temperatures, and by nonactin at 25°C and (222)C₁₀-cryptand at 20 and 25°C. The rate of Na⁺ and K⁺ transport by (221)C₁₀ saturated with the cation and carrier concentrations. Transport was noncooperative and exhibited selectivity for Na⁺ with respect to K⁺. The apparent affinity of (221)C₁₀ for Na⁺ was higher and less pH-dependent than that for K⁺, and seven times higher than that of (222)C₁₀ for K⁺ ions (20.5vs. 1.7 kcal·mole^–). The efficiency of (221)C₁₀ transport of Na⁺ was pH-and carrier concentration-dependent, and was similar to that of nonactin; its activation energy was similar to that for (222)C₁₀ transport of K⁺ (35.5 and 29.7 kcal · mole^–1, respectively). The reaction orders in cationn(S) and in carrierm(M), respectively, increased and decreased as the temperature rose, and were both independent of carrier or cation concentrations; in most cases they varied slightly with the pH.n(S) varied with the cation at pH 8.7 and with the carrier for Na⁺ transport only, whilem(M) always depended on the type of cation and carrier. Results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes.     

Discrimination between Alkali Metal Cations by Yeast : II. Cation interactions in transport

K⁺ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb⁺ is a competitive inhibitor of K⁺ uptake, but Li⁺, Na⁺, and Cs⁺ act like H⁺. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K⁺ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K⁺ transport competitively. Ca⁺⁺ and Mg⁺⁺ in relatively low concentrations partially inhibit K⁺ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K⁺ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb⁺ > Cs⁺ > Na⁺ > K⁺ > Li⁺. The conductance of the membrane was increased up to a value of about 10⁻² ohm⁻¹/cm² with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn²⁺ > Cd²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺.