Non-isothermal potential of phospholipid bilayer films

The thermal electrical potential generated in isochemical conditions by a temperature gradient at the two sides of lipid bilayer leaflets is measured. The experimental results agree rather well with the theoretical predictions. The cephaline (from sheep brain) bilayer behaves like a film with zero charge while the phosphatidylcholine (from egg yolk) film performs like a charged membrane. The results presented suggest that the measurement of electrical thermal potential is an interesting method to investigate the electrical behaviour of bilayer membranes.     

Biomimetic lipid/polymer colorimetric membranes: molecular and cooperative properties

Characterization of membranes and of biological processes occurring within membranes is essential for understanding fundamental cellular behavior. Here we present a detailed biophysical study of a recently developed colorimetric biomimetic membrane assembly constructed from physiological lipid molecules and conjugated polydiacetylene. Various analytical techniques have been applied to characterize the organization of the lipid components in the chromatic vesicles and their contributions to the observed blue-to-red color transitions. Experiments reveal that both the polymerized units as well as the lipids exhibit microscopic phases and form domains whose properties and bilayer organization are interdependent. These domains are interspersed within mixed lipid/polymer vesicles that have a size distribution different from those of aggregates of the individual molecular constituents. The finding that fluidity changes induced within the lipid domains are correlated with the chromatic transitions demonstrates that the colorimetric platform can be used to evaluate the effects of individual molecular components, such as negatively charged lipids and cholesterol, upon membrane fluidity and thermal stability.     

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.     

Penetration of Lysozyme and Cytochrome C in Lipid Bilayer: Fluorescent Study

Lysozyme and cytochrome c (CytC) are well-investigated proteins. Their specific interactions with lipid membranes, however, keep surprising secrets. Lysozyme destroys bacterial membrane; CytC binds hydrophobically to alkyl chains of the membrane lipid tails, indicating that both proteins are able to interact directly with the inner membrane components, especially with the fatty acyl chains of membrane lipids. The degrees of integration, depth of localization in the hydrophobic interior of different types of model membranes, and the type of interaction of lysozyme and CytC with surrounding lipids were investigated by fluorescent spectroscopy. Three different fluorescent markers, located at approximately 6.5, 9, and 18 Å into the lipid bilayer, were used. In addition, liposomes were designed as electrically neutral or positively or negatively charged to unravel the importance of the net electrical charge for lipid/protein interaction. CytC penetrates deeper into the lipid bilayer in comparison with lysozyme, and data are discussed in the terms of Stern–Volmer quenching of fluorescence.     

Electrical oscillation and fluctuation in phospholipid membranes. Phospholipids can form a channel without protein

Fluctuations and/or step-wise changes in membrane potential and electrical current were observed in bilayer membranes of dioleoylphosphatidylcholine (DOPC) in the absence of any channel protein. The DOPC membranes consisted of three types: black lipid membranes, pipette-clamp membranes and lipid membranes transferred to porous filter paper by conventional Langmuir-Blodgett techniques. This finding is significant since phospholipids are the main constituents of biomembranes. Lipid molecules with a cis double bond in their carbon skeleton are suggested to be important in the gating or excitation of biomembranes.     

Electrical Properties of Structural Components of the Crystalline Lens

The electrical properties of the crystalline lens of the frog eye are measured with stochastic currents applied with a microelectrode near the center of the preparation and potential recorded just under the surface. The stochastic signals are decomposed by Fourier analysis into sinusoidal components, and the impedance is determined from the ratio of mean cross power to input power. The data are fit by an electrical model that includes two paths for current flow: one through the cytoplasm, gap junctions, and outer membrane; the other through inner membranes and the extracellular space between lens fibers. The electrical properties of the structures of the lens which appear as circuit components in the model are determined by the fit to the data. The resistivity of the extracellular space within the lens is comparable to the resistivity of Ringer. The outer membrane has a normal resistance of 5 kohm · cm² but large capacitance of 10 μF/cm², probably because it represents the properties of several layers of fibers. The inner membranes have properties reminiscent of artificial lipid bilayers: they have high membrane resistance, 2.2 megohm · cm², and low specific capacitance, 0.8 μF/cm². There is so much membrane within the lens, however, that the sum of the current flow across all the inner membranes is comparable to that across the outer surface.     

The electrical interplay between proteins and lipids in membranes

All molecular interactions that are relevant to cellular and molecular structures are electrical in nature but manifest in a rich variety of forms that each has its own range and influences on the net effect of how molecular species interact. This article outlines how electrical interactions between the protein and lipid membrane components underlie many of the activities of membrane function. Particular emphasis is placed on spatially localised behaviour in membranes involving modulation of protein activity and microdomain structure.The interactions between membrane lipids and membrane proteins together with their role within cell biology represent an enormous body of work. Broad conclusions are not easy given the complexities of the various systems and even consensus with model membrane systems containing two or three lipid types is difficult. By defining two types of broad lipid–protein interaction, respectively Type I as specific and Type II as more non-specific and focussing on the electrical interactions mostly in the extra-membrane regions it is possible to assemble broad rules or a consensus of the dominant features of the interplay between these two fundamentally important classes of membrane component. This article is part of a special issue entitled: Lipid–protein interactions.     

Painted supported lipid membranes

We report herein measurements on a novel type of supported lipid films, which we call painted supported membranes (PSM). These membranes are formed in a self-assembly process on alkylated gold films from an organic solution. The formation process was investigated with surface plasmon resonance microscopy. The optical and electrical properties of the films were determined for various types of lipids and as a function of temperature by means of cyclic voltammetry and potential relaxation after charge injection. We could show that these films exhibit an extraordinarily high specific resistivity which, depending on the lipid, may be as high as 10⁹ ohm/cm². We could also show that due to this low conductivity, an electrical polarization across the PSM relaxes with characteristic time constants of up to 20 min. The electrical properties together with their high mechanical stability and accessibility to surface sensitive techniques make these films well suitable model membranes for optical and electrical investigations. Examples for such applications are given in the subsequent article by Seifert et al.     

Effects of double-layer polarization on ion transport.

It has been proposed that changes in ionic strength will alter the shape of current-voltage relations for ion transport across a lipid membrane. To investigate this effect, we measured currents across glyceryl monooleate membranes at applied potentials between 10 and 300 mV using either gramicidin and 1 mM NaCl or valinomycin and 1 mM KCl. A bridge circuit with an integrator as null detector was used to separate the capacitative and ionic components of the current. The changes in the current-voltage relations when ionic strength is varied between 1 and 100 mM are compared with predictions of Gouy-Chapman theory for the effects of these variations on polarization of the electrical diffuse double-layer. Double-layer polarization accounts adequately for the changes observed using membranes made permeable by either gramicidin or valinomycin.     

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.     

Hydrofluoric and nitric acid transport through lipid bilayer membranes

Hydrofluoric and nitric acid transport through lipid bilayer membranes were studied by a combination of electrical conductance and pH electrode techniques. Transport occurs primarily by nonionic diffusion of molecular HF and HNO3. Membrane permeabilities to HF and HNO3 ranged from 10(-4) to 10(-3) cm . s-1, five to seven orders of magnitude higher than the permeabilities to NO-3, F- and H+. Our results are consistent with the hypothesis that F- transport through biological membranes occurs mainly by nonionic diffusion of HF. Our results also suggest that of the two principal components of 'acid rain', HNO3 may be more toxic than H2SO4.     

Lipid components of sarcotubular membranes

The lipid composition of a highly purified preparation of rat skeletal muscle sarcoplasmic reticulum was studied. These membranes contain per mg of protein 0.50 ± 0.02 μmoles of phosphatidylcholine, 0.13 ± 0.02 μmoles of phosphatidylethanolamine and 0.07 ± 0.02 μmoles ofphosphatidylinositol. These three components account for 97.3% of total lipid phosphorus. Unlike other mammalian membranes so far studied, this preparation contains neither sphingomyelin nor phosphatidylserine. Neural lipids were also measured and it is concluded that neither cholesterol nor other neutral lipids are components of the membranes studied. The results of this study indicate therefore that the lipid profile of sarcotubular membranes is relatively simple compared with most other membranes.     

Role of membrane lipids in the interaction of daunomycin with plasma membranes from tumor cells: implications in drug-resistance phenomena

Equilibrium binding studies on the interaction between the anthracycline daunomycin and plasma membrane fractions from daunomycin-sensitive and -resistant murine leukemia P-388 cells are presented. Drug binding constants (KS) are 15,000 and 9800 M-1 for plasma membranes from drug-sensitive and drug-resistant cells, respectively. Drug binding to the membranes is not affected by either (i) thermal denaturation of membrane proteins or (ii) proteolytic treatment with trypsin, thus suggesting that the protein components of the membranes do not have a major role in determining the observed drug binding. Also, fluorescence resonance energy transfer between tryptophan and daunomycin in the membranes indicates that interaction of protein components with the drug should not be responsible for the observed differences in drug binding exhibited by plasma membranes from drug-sensitive and -resistant cells. Plasma membranes from drug-sensitive cells contain more phosphatidylserine and slightly less cholesterol than membranes from drug-resistant cells. Differences in the content of the acidic phospholipid between the two plasma membranes seem to produce a different ionic environment at membrane surface domains, as indicated by titration of a membrane-incorporated, pH-sensitive fluorescence probe. The possible role of membrane lipids in modulating drug binding to the membranes was tested in equilibrium binding studies using model lipid vesicles made from phosphatidylcholine, phosphatidylserine, and cholesterol in different proportions. The presence of phosphatidylserine greatly increases both the affinity and the stoichiometry of daunomycin binding to model lipid vesicles. The similarity between the effects of phosphatidylserine and other negatively charged compounds such as dicetyl phosphate, cardiolipin, or phosphatidic acid suggests that electrostatic interactions are important in the observed binding of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin-label ESR studies of lipid-protein interactions in thylakoid membranes

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.     

Electrodynamics of Lipid Membrane Interactions in the Presence of Zwitterionic Buffers

Due to thermal motion and molecular polarizability, electrical interactions in biological systems have a dynamic character. Zwitterions are dipolar molecules that typically are highly polarizable and exhibit both a positive and a negative charge depending on the pH of the solution. We use multilamellar structures of common lipids to identify and quantify the effects of zwitterionic buffers that go beyond the control of pH. We use the fact that the repeat spacing of multilamellar lipid bilayers is a sensitive and accurate indicator of the force balance between membranes. We show that common buffers can in fact charge up neutral membranes. However, this electrostatic effect is not immediately recognized because of the concomitant modification of dispersion (van der Waals) forces. We show that although surface charging can be weak, electrostatic forces are significant even at large distances because of reduced ionic screening and reduced van der Waals attraction. The zwitterionic interactions that we identify are expected to be relevant for interfacial biological processes involving lipid bilayers, and for a wide range of biomaterials, including amino acids, detergents, and pharmaceutical drugs. An appreciation of zwitterionic electrodynamic character can lead to a better understanding of molecular interactions in biological systems and in soft materials in general.     

pH modulation of transport properties of alamethicin oligomers inserted in zwitterionic-based artificial lipid membranes

Electric features of biological membranes are major determinants of the function and physiological manifestation of membrane-penetrating peptides, and such features are prone to be modulated by the properties of the surrounding aqueous medium. In this work, we demonstrate that pH plays crucial roles in modulating electric characteristics of zwitterionic-based artificial lipid membranes. The effect of pH on electrical properties of such membranes was probed by evaluating the transport properties of embedded alamethicin oligomers over a wide range of pH values (i.e., 0.65, 2.08, 2.94, 7 and 10.1). Our data strongly support the paradigm of a pH-dependent variation of the surface and membrane dipole potential which, in conjunction with possible lateral pressure effects within the lipid membrane, lead to a non-monotonic modulation of the electrical conductance of alamethicin oligomers. As expected, pH modulation of transport properties through the alamethicin oligomer is more visible for narrower pores (that is, the 1st conductive state) with slightly better cation selectivity as compared to larger oligomers.     

Chronopotentiometric Technique as a Method for Electrical Characterization of Bilayer Lipid Membranes

The basic electrical parameters of bilayer lipid membranes are capacitance and resistance. This article describes the application of chronopotentiometry to the research of lipid bilayers. Membranes were made from egg yolk phosphatidylcholine. The chronopotentiometric characteristic of the membranes depends on the current value. For low current values, no electroporation takes place and the voltage rises exponentially to a constant value. Based on these kinds of chronopotentiometric curves, a method of the membrane capacitance and the membrane resistance calculations are presented.     

Structure and hydration of purple membranes in different conditions

The unit cell dimension of the bacteriorhodopsin lattice in purple membranes decreases by the same amount (2%) upon drying the membranes at room temperature as when they are cooled to liquid nitrogen temperatures. Neutron diffraction experiments with H2O:2H2O exchange, however, show that whereas in the dry membranes the lipid headgroups are dehydrated and the decrease in dimension is due to a smaller area occupied by the lipid molecules, the water of hydration remains in place in the cooled membranes, and the decrease in dimension is due to thermal contraction only. These data suggest a hypothesis that functional bacteriorhodopsin, in the wet state at room temperature, has a relatively soft environment that would allow large amplitude motions of the protein; in the dry membranes at room temperature (which are inactive), the amplitudes of protein motions would be inhibited by a more close-packed environment as they are reduced, due to thermal contraction, in the cold membranes.     

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.     

Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.