The Electroosmotic Effects Arising from the Interaction of the Selectively Anion and Selectively Cation Permeable Parts of Mosaic Membranes

It has been previously shown, theoretically and in model system experiments, that mosaic membranes composed of anion-selective (electropositive) and cation-selective (electronegative) parts interposed between electrolytic solutions of different concentrations give rise to local electrical circuits. In this work with model systems it is shown that these currents produce electroosmosis. In systems with permselective electronegative membranes and KCl solutions, the electroosmotic water transport was 16 moles/faraday. With the permselective electronegative membrane replaced by more porous electronegative membranes, the electroosmotic effects were about twice as high. With Li salts, the water transport was considerably larger. A system with a permselective electropositive membrane of 50 cm² effective area and an electronegative membrane of 120 cm² gave internally generated currents up to 20 ma. In extrapolating from the results with macromodels to effects with true mosaics, i.e. microsystems, it is stressed that current depends on the linear distance over which membranes interact. In true mosaic membranes, the current pathways will be of the same order as the dimensions of individual membrane microelements; the sum of all local microcurrents will be correspondingly larger than the current in the macromodel, and the electroosmotic effects will be proportionately greater. Electroosmotic effects with true charge-mosaic membranes may be of the same order or larger than the liquid transport by normal and anomalous osmosis which might occur across the individual parts of the charge-mosaic.     

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: A comparison

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.     

Measurement of membrane potential and estimation of effective fixed-charge density in membranes.

Electrical potentials Em arising across cross-linked phenolsulfonate membrane separating NaCl solutions of molality M1 and M2 have been measured at 25 degrees C. These values of Em have been used in the Nernst equation to calculate values for the apparent transport number ti(app) for the counterion or the co-ion in the membrane. Values of ti(app) together with the limiting value for the cation transport number in the aqueous phase have been used in the equation developed by Kobatake and co-workers to evaluate the membrane permselectivity Ps as a function of external electrolyte concentration. With the help of the equation relating Ps to phiX, the effective fixed-charge density in the membrane (where phi is a constant, 0 less than phi less than 1, and X is the membrane stochiometric charge density and can be evaluated by chemical analysis of the membrane phase), values for phiX and phi have been determined. Values of phi were low in dilute solutions and increased with increase in the concentration of the external solution. Similar behavior was noted in the case of another membrane system, cross-linked polymethacrylic acid in contact with KOH solutions. On the other hand, the membrane system, "untreated" collodion in contact with KCl solutions, exhibited a behavior in which the values of phi, low in dilute solutions, increased and then decreased following a gradual increase in the external concentration. This slight divergence in its behavior was attributed to the heterogeneity of the collodion membrane structure. The reliability of this potentiometric method to estimate effective fixed-charge density in membranes has been discussed in relation to a similar but old method due to Teorell, Meyer and Sievers. Also the significance of the values derived for phi has been pointed out.     

The contributions of normal and anomalous osmosis to the osmotic effects arising across charged membranes with solutions of electrolytes

The osmotic effect arising across a porous membrane separating the solution of an electrolyte from water (or a more dilute solution) is ordinarily due to both normal osmosis, as it occurs also with non-electrolytes, and to "anomalous" osmosis. It is shown that the normal osmotic component cannot be measured quantitatively by the conventional comparison with a non-electrolytic reference solute. Anomalous osmosis does not occur with electroneutral membranes. Accordingly, with membranes which can be charged and discharged reversibly (without changes in geometrical structure), such as many proteinized membranes, the osmotic effects caused by an electrolyte can be measured both when only normal osmosis arises (with the membrane in the electroneutral state) and when normal as well as anomalous osmosis occurs (with the membrane in a charged state). The difference between these two effects is the true anomalous osmosis. Data are presented on the osmotic effects across an oxyhemoglobin membrane in the uncharged state at pH 6.75 and in two charged states, positive at pH 4.0 and negative at pH 10.0, with solutions of a variety of electrolytes using a concentration ratio of 2:1 over a wide range of concentrations. The rates of the movement of liquid across the membrane against an inconsequentially small hydrostatic head are recorded instead of, as conventional, the physiologically less significant pressure rises after a standard time.     

A METHOD FOR THE INVESTIGATION OF ELECTROSTENOLYSIS

A quantitative method for the investigation of electrostenolysis has been developed. Electrostenolysis is redefined in the light of the discovery that organic molecules are subject to it. The experimental requirements for a quantitative study are enumerated, and the apparatus and procedure described. It is found that with ferrous and ferric ions and a cellulose acetate membrane, the potential drop across the membrane must be above about 2150 v./cm. in order to effect any oxidation and reduction. With the present apparatus and conditions the ratio of equivalents oxidized or reduced to faradays passing the membrane is low,— of the order of one to several thousand.     

Application of a taste evaluation system to the monitoring of Kimchi fermentation

As an objective method, taste evaluation with an instrument is able to supplement the subjective sensory evaluation and to be applied to the optimization of food processing. Kimchi, a Korean traditional pickle fermented with lactic acid bacteria, is expanding its consumption worldwide. The fermentation control of it has been routinely done by measuring titratable acidity and pH. In this study, an eight-channel taste evaluation system was prepared, followed by an application to the monitoring of Kimchi fermentation. Eight polymer membranes which individually responded to cationic or anionic substances were prepared by mixing electroactive materials such as tri-n-octylmethylammonium chloride, bis(2-ethylhexyl)sebacate as the plasticizer and polyvinyl chloride in the ratio of 1:66:33. Each membrane prepared was separately installed onto the sensitive area of an ion-selective electrode to produce the respective taste sensor. The eight-channel sensor array and a double junction reference electrode were connected to a 16-channel high input impedance amplifier. The amplified sensor signals were stored to a personal computer via a multi-channel A/D converter. Two sensor groups composed of the cation-selective and anion-selective polymer membrane electrodes showed characteristic concentration-dependency to various artificial taste substances. As a whole, the response potentials of the sensor array increased during the fermentation period at 4, 10 and 25 degrees C. Even the response potentials of the anion-selective taste sensors slightly increased possibly due to the protonation of anions by liberated H+ ions, thereby leading to a decrease in the anion concentration. When the signal data were interpreted by principal component analysis (PCA), the first PC at 4 degrees C explained most of the total data variance. A close correlation was found between the values of titratable acidity and the first PC, which indicated a possible applicability of the multi-channel taste sensor of this study to the process monitoring of various pickle.     

An anion-selective analogue of the channel-forming peptide alamethicin.

The peptide alamethicin self-assembles to form helix bundle ion channels in membranes. Previous macroscopic measurements have shown that these channels are mildly cation-selective. Models indicate that a source of cation selectivity is a zone of partial negative charge toward the C-terminal end of the peptide. We synthesized an alamethicin derivative with a lysine in this zone (replacing the glutamine at position 18 in the sequence). Microscopic (single-channel) measurements demonstrate that dimeric alamethicin-lysine18 (alm-K18) forms mildly anion-selective channels under conditions where channels formed by the parent peptide are cation-selective. Long-range electrostatic interactions can explain the inversion of ion selectivity and the conductance properties of alamethicin channels.     

Membrane channel formation by antimicrobial protegrins.

Protegrins are small, arginine- and cysteine-rich, beta-sheet peptides with potent activity against bacteria, fungi, and certain enveloped viruses. We report that protegrins form weakly anion-selective channels in planar phospholipid bilayers, induce potassium leakage from liposomes and form moderately cation-selective channels in planar lipid membranes that contain bacterial lipopolysaccharide. The disruption of microbial membranes may be a central attribute related to the host defense properties of protegrins.     

Modification of ion transport in lipid bilayer membranes in the presence of 2,4-dichlorophenoxyacetic acid. II. Suppression of tetraphenylborate conductance and changes of interfacial potentials.

It has been shown that the blocking of negatively charged tetraphenylborate ion transport in phosphatidylcholine (PC)-cholesterol membranes by the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is dominated by suppression of TPhB- diffusion across the membrane interior, rather than by the decrease of adsorption of TPhB- ions at the membrane surface. The blocking effect can be associated with the decrease of electric potential inside the membrane with respect to that of the aqueous medium, this decreases being proportional to the concentration of 2,4-D in the aqueous solution. It has been estimated that 25 - 30% of the total 2,4-D-induced change of the potential difference is between the plane of absorption of TPhB- and the aqueous solution, and the remaining fraction is between the membrane interior and the absorption plane. The results of this study support the dipolar hypothesis of 2,4-D action in lipid membranes. These conclusions are further supported by measurements changes of electric potential difference across air/water and air/lipid monolayer/water interfaces. It has been found that the electric potential of the nonpolar side of the interface decreases in the presence of neutral molecules of 2,4-D and that this effect becomes more prominent in presence of electrolyte. We have confirmed that PC-cholesterol monolayer cannot be considered as a model for half of the bilayer membrane because of the disagreement between the changes of the interfacial potential difference of PC-cholesterol monolayers and those determined from studied of transport of positive and negative ions across bilayer membranes. In contract, we have found close agreement between the 2,4-D-induced changes of electric potential of the lipid hydrocarbon region in glycerolmonooleate (GMO) membranes and GMO monolayers. We suggest that the action of 2,4-D in lipid membranes is not associated with the changes of orientation of dipoles of lipids constituting the membranes, but rather with a layer of 2,4-D molecules absorbed at the nonpolar/polar membrane boundary.     

A study of the effects of externally applied sodium-ions and detection of spatial non-uniformity of the squid axon membrane under internal perfusion

Electrical properties of the axon membrane were examined under internal perfusion of squid giant axons with a dilute solution of NaF or CsF. The rate of propagation of the action potential was markedly enhanced when NaCl was added to the external CaCl₂ solution. The membrane conductance both at rest and during the action potential was increased with increasing Na-concentration in the external medium. In the perfusion zone of these axons, the action potentials in different parts of the membrane were found to terminate in a more-or-less spatially random and temporally irregular fashion. When the electric field outside the axon membrane was examined with hyperfine glass-pipette electrodes, small rectangular potential changes of uniform amplitude were observed. The small potential changes, which resemble those obtained by Bean in EIM-treated lipid bilayer, were interpreted as indicating spatial non-uniformity of the axon membrane during excitation. The importance of long-range electric interaction between different parts of the axon membrane is emphasized.     

Alzheimer''s disease amyloid beta-protein forms Zn(2+)-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons.

We have previously shown that the 40-residue peptide termed amyloid beta-protein (A beta P[1-40]) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether A beta P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A beta P[1-40] leads to the spontaneous formation of cation-selective channels. With Cs+ as the main cation in both the external as well as the internal saline, the amplitude of the A beta P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid beta-protein in solution, can block the flow of Cs+ through the A beta P[1-40] channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A beta P[1-40] channel pore. These properties of the A beta P[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid beta-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid beta-protein and neuronal membranes also occur in vivo, lending further support to the idea that A beta P[1-40] channel formation might be a mechanism of amyloid beta-protein neurotoxicity.     

Liquid junction potentials calculated from numerical solutions of the Nernst-Planck and Poisson equations

We present numerical solutions for the one-dimensional Nernst-Planck and Poisson system of equations for steady-state electrodiffusion. Commonly used approximate solutions to these equations invoke assumptions of local electroneutrality (Planck approximation) or constant electric field (Goldman approximation). Calculations were performed to test the ranges over which these approximate theories are valid. For a dilutional junction of a 1:1 electrolyte, separated from adjoining perfectly stirred solutions by sharp boundaries, the Planck approximation is valid for values of kappa dL greater than 10, where 1/kappa d is the Debye length of the more dilute solution. The Goldman approximation is valid for kappa cL less than 0.1 where 1/kappa c is the Debye length of the more concentrated solution. These results suggest that the modeling of electrodiffusive flows in and near membrane ion channels may require numerical solutions of this set of equations rather than the use of either limiting case.     

Protonation of lysine residues inverts cation/anion selectivity in a model channel

A dimeric alamethicin analog with lysine at position 18 in the sequence (alm-K18) was previously shown to form stable anion-selective channels in membranes at pH 7.0 [Starostin, A. V., R. Butan, V. Borisenko, D. A. James, H. Wenschuh, M. S. Sansom, and G. A. Woolley. 1999. Biochemistry. 38:6144-6150]. To probe the charge state of the conducting channel and how this might influence cation versus anion selectivity, we performed a series of single-channel selectivity measurements at different pH values. At pH 7.0 and below, only anion-selective channels were found with P(K(+))/P(Cl(-)) = 0. 25. From pH 8-10, a mixture of anion-selective, non-selective, and cation-selective channels was found. At pH > 11 only cation-selective channels were found with P(K(+))/P(Cl(-)) = 4. In contrast, native alamethicin-Q18 channels (with Gln in place of Lys at position 18) were cation-selective (P(K(+))/P(Cl(-)) = 4) at all pH values. Continuum electrostatics calculations were then carried out using an octameric model of the alm-K18 channel embedded in a low dielectric slab to simulate a membrane. Although the calculations can account for the apparent pK(a) of the channel, they fail to correctly predict the degree of selectivity. Although a switch from cation- to anion-selectivity as the channel becomes protonated is indicated, the degree of anion-selectivity is severely overestimated, suggesting that the continuum approach does not adequately represent some aspect of the electrostatics of permeation in these channels. Side-chain conformational changes upon protonation, conformational changes, and deprotonation caused by permeating cations and counterion binding by lysine residues upon protonation are considered as possible sources of the overestimation.     

A quantitative study of electroporation showing a plateau in net molecular transport.

Electroporation is believed to involve a temporary structural rearrangement of lipid bilayer membranes, which results in ion and molecular transport across the membrane. The results of a quantitative study of molecular transport due to electroporation caused by a single exponential pulse are presented; transport of four molecules of different physical characteristics across erythrocyte ghost membranes is examined as a function of applied field strength. Flow cytometry is used to quantitatively measure the number of molecules transported for 10(4) to 10(5) individual ghosts for each condition. This study has four major findings: 1) Net transport first increases with field strength, but reaches a plateau at higher field strengths. Significant transport is found at or below 1 kV/cm, and transport plateaus begin at field strengths between 2 and 5 kV/cm depending on the molecule transported. 2) A single population of ghosts generally exists, but exhibits a wide distribution in the amount of molecular transport. 3) Under the conditions used, the direction of transport across the ghost membrane does not appear to affect molecular transport significantly. 4) Large numbers of ghosts may be destroyed by the electroporation procedure.     

A theory of ion permeation through membranes with fixed neutral sites

Summary Some model membranes and biological membranes behave as if ion permeation were controlled by fixed neutral sites, i.e., by groups that are polar but lack net charge. By solving the boundary conditions and Nernst-Planck flux equations, this paper derives the expected properties of four types of membranes with fixed neutral sites: model 1, a membrane thick enough that microscopic electroneutrality is obeyed; model 2, same as model 1 but with a free-solution shunt in parallel; model 3, a membrane thin enough that microscopic electroneutrality is violated; and model 4, same as model 3 but with a free-solution shunt in parallel. The conductance-concentration relation and the current-voltage relation in symmetrical solutions are approximately linear for all four models. Partial ionic conductances are independent of each other for a thin membrane but not for a thick membrane. Sets of permeability ratios derived from conductances, dilution potentials, or biionic potentials agree with each other in a thin membrane but not in a thick membrane. The current-voltage relation in asymmetrical single-salt solutions is linear for a thick membrane but nonlinear for a thin membrane. Examples of potential and concentration profiles in a thin membrane are calculated to illustrate the meaning of space charge and the electroneutrality condition. The experimentally determined properties (by A. Cass, A. Finkelstein & V. Krespi) of thin lipid membranes containing “pores” of the anion-selective antibiotic nystatin are in reasonable agreement with model 3. Tests are suggested for deciding if a membrane of unknown structure has neutral sites, whether it is thick or thin, and whether the sites are fixed or mobile.     

Thermal membrane potential through charged membranes in electrolyte solutions.

Measurements of the thermal membrane potential across cation and anion exchange membranes were carried out by using the same solution of various 1-1 electrolytes on both sides of the membrane. In all cases a good linear relationship was observed between the thermal membrane potential increment psi and the temperature difference increment T. The slope of the linear plot varied with the concentration of the electrolyte. The value of increment psi/increment T versus logarithmic activity of the electrolyte plot was linear with a slope of +/- R/F if the transport number of counterion was unity. The magnitude of increment psi/increment T was independent of coion species but dependent on counterions. These experimental results are in agreement with a theory presented previously. The thermal membrane potential caused by the direct effect of temperature differences and that by the indirect effect arising from the changes in ionic and water chemical potentials due to the temperature difference are separately discussed.     

Pores formed in lipid bilayer membranes by nystatin, Differences in its one-sided and two-sided action

Nystatin and amphotericin B induce a cation-selective conductance when added to one side of a lipid bilayer membrane and an anion-selective conductance when added to both sides. The concentrations of antibiotic required for the one-sided action are comparable to those employed on plasma membranes and are considerably larger than those required for the two-sided action. We propose that the two-sided effect results from the formation of aqueous pores formed by the hydrogen bonding in the middle of the bilayer of two "half pores," whereas the one-sided effect results from the half pores alone. We discuss, in terms of the flexibility of bilayer structure and its thickness, how it is possible to have conducting half pores and "complete pores" in the same membrane. The role of sterol (cholesterol and ergosterol) in pore formation is also examined.     

A self-assembled pigmented BLM on a platinum support: the light-induced electrical effects

The light-induced voltage and current changes under continuous illumination have been investigated in pigmented self-assembled lipid bilayer membranes deposited on a platinum electrode. Such self-organized pigmented bilayer-platinum system containing Zn-Phthalocyanine (ZnPc) as a photosensitizer and glycerol-dioleate (GDO) as a bilayer forming solution has been found to shift its electrode potential to more positive value on light irradiation as well as to increase the cathodic current across the membrane. The results indicate a direct electron transfer from the platinum electrode to hydrogen ion in the electrolyte solution. Furthermore, it has also been demonstrated a dramatic increase of the photocurrent over the time course of BLM formation visualizing a role of the bulk quenching processes which are significantly diminished in thin bilayer membrane.     

Osmotic Transport across Cell Membranes in Nondilute Solutions: A New Nondilute Solute Transport Equation

The fundamental physical mechanisms of water and solute transport across cell membranes have long been studied in the field of cell membrane biophysics. Cryobiology is a discipline that requires an understanding of osmotic transport across cell membranes under nondilute solution conditions, yet many of the currently-used transport formalisms make limiting dilute solution assumptions. While dilute solution assumptions are often appropriate under physiological conditions, they are rarely appropriate in cryobiology. The first objective of this article is to review commonly-used transport equations, and the explicit and implicit assumptions made when using the two-parameter and the Kedem-Katchalsky formalisms. The second objective of this article is to describe a set of transport equations that do not make the previous dilute solution or near-equilibrium assumptions. Specifically, a new nondilute solute transport equation is presented. Such nondilute equations are applicable to many fields including cryobiology where dilute solution conditions are not often met. An illustrative example is provided. Utilizing suitable transport equations that fit for two permeability coefficients, fits were as good as with the previous three-parameter model (which includes the reflection coefficient, σ). There is less unexpected concentration dependence with the nondilute transport equations, suggesting that some of the unexpected concentration dependence of permeability is due to the use of inappropriate transport equations.