Alternating current studies of charge carrier transport in lipid bilayers. Pentachlorophenol in lecithin-cholesterol membranes.

Surface and interior electrical properties of lecithin-cholesterol bilayer membranes treated with the uncoupler pentachlorophenol have been determined on the basis of a.c. measurements over a wide range of frequencies (0.02 to 1000 kHZ). The method used depends on accurately determining the resistance of the aqueous solution in series with each individual membrane by extrapolating admittance data into infinite frequency. Loss tangent vs. frequency curves are corrected by subtracting out a loss contribution which is present in untreated membranes and is due, presumably, to dielectric relaxation. The results, which are useful below 100 kHZ, can be fitted to loss tangent curves computed for a three-element equivalent circuit consisting of frequency independent conductance-capacitance pairs, arranged in series to represent surface and interior properties of membranes. Interior conductances agree with net conductances obtained from d.c. measurements. The pH and concentration dependence of surface conductance is consistent with a scheme of transport in which a fixed number of surface binding sites are filled preferentially with neutral pentachlorophenol molecules, which in turn dissociate to supply protons to the aqueous phase. Surface capacitances range from 15 to 90 times that of interior capacitance and show a systematic increase with pentachlorophenol concentration at high pH, and a decrease with concentration at low pH.     

Uncoupler antagonism of valinomycin induced bilayer membrane conductance

We have found that an uncoupler of oxidative phosphorylation, tetrachloro-2-trifluoromethylbenzimidazole (TTFB), can block valinomycin induced potassium ion, (K⁺), conductance through bilayer membranes. Blocking is most pronounced at high K⁺ concentration, (≥ 0.1 M), and at a pH greater than the pK of TTFB. A blocking mechanism involving competition for membrane sites, together with association of oppositely charged species within the membrane, is proposed.     

Electrical conductivity in lipid bilayer membranes induced by pentachlorophenol.

Electrical conductivity induced in thin lipid bilayer membranes by pentachlorophenol has been studied. The membranes were formed from phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl glycerol and various amounts of cholesterol. The position and the magnitude of the maximum of the conductivity vs. pH curve depend on the type of lipids and cholesterol content. At low pentachlorophenol concentrations and low pH the concentration dependence of conductivity is quadratic and becomes linear at higher pH. Above 10(-5) M of pentachlorophenol the concentration dependence of the membrane conductivity tends to saturate. Presence of pentachlorophenol enhances membrane transport of nonactin-K+ complex. Increase of cholesterol content increases pentachlorophenol induced conductivity in all membranes and shifts the conductivity toward lower pH. For phosphatidyl choline the largest rate of change of membrane conductivity with cholesterol occurs at 1:1 phospholipid to cholesterol molar ratio. Pentachlorophenol is found to be a class II uncoupler and the experimental results are consistent with the hypothesis that the membrane permeable species are dimers formed by combination of neutral and dissociated pentachlorophenol molecules. Several schemes of membrane conduction, including dimer formation in the aqueous phase as well as at the membrane-water interface have been considered. Arguments are given in favor of the formation of dimers within the membrane surface.     

The effect of the polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation

Summary Bilayer membranes were formed from decane, cholesterol, and three lipids isolated fromStaphylococcus aureus: positively charged lysyl phosphatidylglycerol (LysPG), negatively charged phosphatidylglycerol (PG), and neutral diglucosyldiglyceride (DiGluDiGly). The uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and 3-t-butyl,5-chloro,2-chloro,4-nitrosalicylanilide (S 13), increased the electrical conductance of all three differently charged bilayers. S 13 was found to be the most effective reagent of the known uncouplers in increasing conductance of the bilayers. The conductance induced by uncouplers was investigated as a function of pH and uncoupler concentration. The pH of maximum conductance for each uncoupling agent was dependent on both the uncoupler and the lipid; it was lower for each uncoupler in LysPG and higher in PG compared to DiGluDiGly bilayers. At a pH below the optimum for LysPG, the conductance of the positively charged membrane was 500 times and of the neutral one 10 times higher than that of the negatively charged bilayer at equal uncoupler concentration and pH. Above the pH optimum for DiGluDiGly, the conductance was approximately equal for the positive and neutral membranes, but was lower in PG bilayers. Conductance depended linearly on uncoupler concentration. The bilayer conductance induced by S 13 was entirely due to increased proton permeability in all three lipids. The findings are consistent with the role of uncouplers as carriers for protons across the hydrocarbon interior of lipid membranes. The differences in conductance of differently charged lipid bilayers at equal uncoupler concentration, as well as the change of pH optimum of conductance with lipid charge, can be explained in terms of an electrostatic energy contribution of the fixed lipid charges to the distribution of the uncoupler anion between the aqueous and the membrane phases.     

The steady-state properties of an ion exchange membrane with mobile sites

A study of the properties of the steady states of a system composed of two solutions separated by an ion exchange membrane having mobile sites is presented. It is assumed that the membrane is impermeable to coions; the solutions contain no more than two species of counterions, both of the same valence; and no flow of bulk solution occurs. Assuming that all ions are completely dissociated, behave ideally, and have constant mobilities throughout the membrane, explicit expressions are derived for the steady states of the electric current, individual fluxes, and concentration profiles as functions of the compositions of the solutions and of the difference of electric potential between them. The derived expressions are compared with those for an ion exchange membrane having fixed sites; and it is found that the expressions of certain quantities, such as the difference of electric potential between the two solutions for zero current or the ratio of the fluxes of the counterions as functions of the external parameters of the system, are the same for both types of membranes. On the other hand, differences in the behavior of the two types of membranes are found from other expressions-for example, the current-voltage relationship. In the mobile site ion exchanger the current asymptotically approaches finite limiting values for high positive and negative voltages while in the fixed site ion exchanger it is the conductance which approaches finite limiting values.     

Current voltage curves of biomolecular lipid membranes.

The first part of this paper describes the current voltage curves of bimolecular membranes of oxidized cholesterol formed between two aqueous solutions of tetrabutylammonium chloride. These membranes are selectively permeable for cations and the membrane interfaces are electrically uncharged. The dependence of the membrane conductivity on the membrane potential can be described as the product of the conductivity at zero current ("zero conductivity") and a function called "overlinearity". The zero conductivity increases linearly with the concentration of tetrabutylammonium chloride. The overlinearity is independent of the concentration of tetrabutylammonium chloride. In the second part the Nernst-Planck and Poisson equations are integrated numerically for a three-phase system consisting of an aqueous electrolyte solution, a membrane and an aqueous electrolyte solution. Each phase is characterized by material constants. Appropriate boundary conditions cause the electric current to build up electrical double layers on both sides of the membrane. The opposing double layers with opposite electrical signs inject the soluble ions into the membrane. This ion injection accounts for the overlinearity of the current voltage curves, thus explaining the measured characteristics.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

Electrochemical and NMR spectroscopic investigations of the influence of the probe molecule Eu(fod)3 on the permeability of lipid membranes to ions

Investigating the action of the fluorinated europium complex Eu(fod)3 on lipid membranes we found that the complex facilitates the ion transfer through the membrane. Electric measurements on planar lipid membranes showed that the membrane conductivity increases considerably by insertion of the complex into the membrane. The increase in the conductivity was only obtained if both layers of the membrane were modified with the complex. 1H NMR spectroscopic studies using DOPC liposomes gave information about the location of the modifier complex in the lipid membrane. From chemical shift effects we concluded that the complex resides in the choline head group region of the membrane and also in the membrane interior near the -C =C- lipid double bond, but not in the center of the bilayer. For understanding of the mentioned conductivity effect we assume that the europium complex induces defects of yet unknown structure in the lipid matrix which provide paths for the ion transfer through the membrane. As appropriate measurements revealed, these paths seem to conduct cations predominantly. Investigating the current voltage behavior of the modified lipid membranes in dependence on the ion concentration we obtained different shaped current-voltage curves. Calculation showed that a model with only one energy barrier inside the membrane is unable to describe these curves kinetically. However, by assuming two energy barriers--one barrier in each membrane lipid layer--the observed curve can be described satisfactorily.     

An Ion Displacement Membrane Model

The usual assumption in treating the diffusion of ions in an electric field has been that the movement of each ion is independent of the movement of the others. The resulting equation for diffusion by a succession of spontaneous jumps has been well stated by Parlin and Eyring. This paper will consider one simple case in which a different assumption is reasonable. Diffusion of monovalent positive ions is considered as a series of jumps from one fixed negative site to another. The sites are assumed to be full (electrical neutrality). Interaction occurs by the displacement of one ion by another. An ion leaves a site if and only if another ion, not necessarily of the same species, attempts to occupy the same site. Flux ratios and net fluxes are given as functions of the electrical potential, concentration ratios, and number of sites encountered in crossing the membrane. Quantitative comparisons with observations of Hodgkin and Keynes are presented.     

Membrane bioenergetic parameters in uncoupler-resistant mutants of Bacillus megaterium.

Mutants of Bacillus megaterium displaying malate-driven ATP synthesis resistant to uncouplers of oxidative posphorylation are further characterized. Both the pH gradient and electrical potential generated across the membrane by malate respiration are equally sensitive to uncouplers in the wild type and uncoupler-resistant mutants. The mutants possess 0 to 10% of the wild type ATPase activity which is not activated by pretreatment with heat or trypsin. Despite this inability to measure ATPase activity, the mutants demonstrate acid-pulse-driven ATPase synthesis which is sensitive to uncouplers as well as malate-driven ATP synthesis which becomes uncoupler sensitive at pH 5.5. N,N' -Dicyclohexylcarbodiimide and valinomycin plus potassium inhibition of ATP synthesis is reversed by uncouplers in the mutants but not in the wild type. The data support the existence of a specific site on the ATPase complex for uncoupler binding which, if altered by mutation, affects uncoupler binding to the complex. The retention of malate-driven ATP synthesis in the absence of a significant pH gradient or electrical potential suggests that an alternative intermediate is involved in coupling oxidation to phosphorylation.     

Nonlinear Electrical Effects in Lipid Bilayer Membranes: III. The Dissociation Field Effect

In the course of an analysis of nonlinear electrical effects in lipid bilayer membranes, the influence of the dissociation field (or Wien) effect on the membrane conductivity is investigated. It is shown that the theory of Onsager for the Wien effect in a macroscopic phase can be applied to a thin membrane when the proper boundary conditions at the membrane-solution interface are introduced. It is assumed that an activation energy is associated with the passage of the ion across the interface. The mathematical treatment of the model is restricted to the case for which cations and anions have identical properties except for the charge sign. The resulting differential equations for the ion concentration within the membrane are integrated numerically. The analysis shows that the influence of the Wien effect on the membrane conductivity is appreciable only if the energy barrier at the interface is sufficiently high, i.e. if the rate limiting step for the ion transport is the passage of the ion across the interface.     

Dielectric properties of adsorption/ionization site of pentachlorophenol in lipid membranes

The results of three complementary studies focused on characterization of the local environment of the common pesticide pentachlorophenol (PCP) adsorbed to phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes are reported. The effect of cholesterol (Chol) was examined. These studies included: Measurements of solvatochromic shifts of the ultraviolet absorption spectra of PCP in membranes and in polar non-hydrogen-bonding (a red shift) and hydrogen-bonding (a blue shift) solvents. Pi-pi transition energies were analyzed in terms of the dielectric cavity models of Onsager, Block-Walker, which includes dielectric saturation, and a soft dipole model of Suppan, which accounts for PCP's polarizability. The estimates of dielectric constant of the PCP adsorption site yielded 8.1-8.7 for the PC and 16.8-20.1 for PG membranes. Solvatochromic effects indicate hydrogen bonding between the membrane-bound ionized PCP molecule and water, which is enhanced by the presence of cholesterol. Determinations of the pKa of PCP adsorbed to PC, PG, PC/Chol, PG/Chol membranes and dissolved in dioxane-water solutions of a known dielectric constant. The pKa value of PCP adsorbed to membranes was always greater than the standard pKa value and it increased with the membrane's negative charge. The pKa value sequence in 0.1 M KCl was 6.68 (PG), 6.32 (PG/Chol = 70:30 mole fractions), 5.97 (PC), and 5.75 (PC/Chol = 70:30). The intrinsic pKa values of PCP in membranes were 5.2-5.4 (PG) and 5.5-6.0 (PC). Estimates of the dielectric constant of PCP's ionization site in membranes yielded 10-22 (PC) and 27-37 (PG). Cholesterol facilitated the release of the hydrogen ion from membrane-bound PCP. Measurements of pH dependence of PCP-induced membrane electrical conductivity. pH values of conductivity maxima were always greater than the standard pKa of PCP, and their sequence corresponded to that of the pKa values of membrane-bound PCP. The anomalous properties of PCP as a Class 2 uncoupler are due to PCP's lipophilic character. In response to a low dielectric constant of the adsorption/ionization site, the physicochemical characteristics of PCP adsorbed to membranes are different from the standard values--a fact that needs to be taken into account in the development of models of PCP's toxicity.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Effect of high magnesium ion concentration on the electron transport rate and proton exchange in thylakoid membranes in higher plants]

The effects of magnesium ion concentration on the rate of electron transport in isolated pea thylakoids were investigated in the pH range from 4.0 up to 8.0. In the absence of magnesium ions in the medium and in the presence of 5 mM MgCl2 in the experiments not only without added artificial acceptors but also with ferricyanide or methylviologen as an acceptor, this rate had a well-expressed maximum at pH 5.0. It was shown that, after depression to minimal values at pH 5.5-6.5, it gradually rose with increasing pH. An increase in magnesium ion concentration up to 20 mM essentially affected the electron transfer rate: it decreased somewhat at pH 4.0-5.0 but increased at higher pH values. At this magnesium ion concentration, the maximum rate was at pH 6.0-6.5 and the minimum, at pH 7.0. Subsequent rise upon increasing pH to 8.0 was expressed more sharply. The influence of high magnesium ion concentration on the rate of electron transport was not observed in the presence of gramicidin D. It was found that without uncoupler, the changes in the electron transfer rate under the influence of magnesium ions correlated to the changes in the first-order rate constant of the proton efflux from thylakoids. It is supposed that the change in the ability of thylakoids to keep protons by the action of magnesium ions is the result of electrostatic interactions of these ions with the charges on the external surface of membranes. A possible role of regulation of the electron transport rate by magnesium ions in vivo is discussed.     

Generation of potential in lipid bilayer membranes as a result of proton-transfer reactions in the unstirred layers

The addition of an uncoupler in the presence of a concentration gradient of weak acids or bases (sodium acetate and ammonium chloride) leads to the generation of a potential on lipid bilayer membranes (LBM) which is positive in sign on the side of the membrane with a high concentration of sodium acetate and negative on the side with a high concentration of ammonium chloride. It is shown that the potential was caused by the pH gradient in the unstirred layers. These effects can be understood in terms of the previously described [Science,182, 1258 (1973)] model for the transfer of weak acids and bases through LBM. This system described may be useful for quantitation of permeabilities for weak acids and bases through bilayer membranes.     

Factor Xa Binding to Phosphatidylserine-Containing Membranes Produces an Inactive Membrane-Bound Dimer

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.     

Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pK_a of the uncoupler molecule.

The absorption and fluorescence excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca²⁺ strongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes.

With mitochondrial membranes an effect of Ca²⁺ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it.

From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholipid moiety of the membrane, with its anionic group pointing to the aqueous solution.     

Modulation of phospholipid transfer protein activity. Inhibition by local anesthetics

The transfer of phospholipid molecules between biological and synthetic membranes is facilitated by the presence of soluble catalytic proteins, such as those isolated from bovine brain which interacts with phosphatidylinositol and phosphatidylcholine and from bovine liver which is specific for phosphatidylcholine. A series of tertiary amine local anesthetics decreases the rates of protein-catalyzed phospholipid transfer. The potency of inhibition is dibucaine>tetracaine>lidocaine>procaine, an order which is compared with and identical to those for a wide variety of anesthetic-dependent membrane phenomena. Half-maximal inhibition of phosphatidylinositol transfer by dibucaine occurs at a concentration of 0.18 mM, significantly lower than the concentration of 1.9 mM required for half-maximal inhibition of phosphatidylcholine transfer activity of the brain protein. Comparable inhibition of liver protein phosphatidylcholine transfer activity is observed at 1.6 mM dibucaine. For activity measurements performed at different pH, dibucaine is more potent at the lower pH values which favor the equilibrium toward the charged molecular species. With membranes containing increasing molar proportions of phosphatidate, dibucaine is increasingly more potent. No effect of Ca²⁺ on the control transfer activity or the inhibitory action of dibucaine is noted. These results are discussed in terms of the formation of specific phosphatidylinositol or phosphatidylcholine complexes with the amphiphilic anesthetics in the membrane bilayer.     

Effects of the local anesthetic bupivacaine on oxidative phosphorylation in mitochondria. Change from decoupling to uncoupling by formation of a leakage type ion pathway specific for H+ in cooperation with hydrophobic anions

The effects of the local anesthetic bupivacaine on the oxidative phosphorylation in rat liver mitochondria were examined. Bupivacaine caused a maximum of about 7-fold stimulation of state 4 respiration at about 3 mM, released oligomycin-inhibited state 3 respiration, and activated ATPase to a similar extent to that by the weakly acidic uncoupler SF 6847. These effects were greatly enhanced by the addition of certain hydrophobic anions such as 1-anilino-8-naphthalenesulfonate, tetraphenyl borate, and picrate. In the absence of these anions, bupivacaine did not increase the proton conductance in either energized or nonenergized mitochondrial membranes or in artificial bilayer lipid membranes and did not have any effect on the proton motive force. However, it greatly enhanced the proton conductivity of these membrane systems and collapsed the proton motive force in the presence of hydrophobic anions. The results of noise analysis of artificial lipid bilayer membranes indicated that an ion pair complex of bupivacaine with hydrophobic anions formed a leakage-type ion pathway. Thus it is concluded that bupivacaine acts as a decoupler in the absence of added hydrophobic anions but in cooperation with certain anions as an uncoupler of oxidative phosphorylation due to formation of a H(+)-specific pathway in the membranes.