Bilayer-gel membranes. Formation and some properties

We developed a new procedure which induces multifunctional reagents to crosslink at one interface between a black bilayer and the adjacent water phase. This procedure yields ‘bilayer-gel’ membranes, i.e. membranes consisting of a bilayer and a polymer layer. The bilayer-gel membrane may tentatively be considered to be a new membrane system, because the formation of the polymer layer changes some bilayer properties. We studied bilayer-gel membranes composed of a bilayer of oxidized cholesterol and of a polymer layer of poly-l-lysine crosslinked by glutardialdehyde. Compared to unmodified bilayers, this membrane system has an electrical conductance of the same magnitude, the same electrical capacity and similar shapes of current-voltage dependences. However, this system is asymmetrical and differs in ion selectivity and increased stability from an unmodified bilayer.     

Comparison of double layer potentials in lipid monolayers and lipid bilayer membranes

Summary It is shown that the Gouy-Chapman double layer analysis adequately describes the variation of the surface potential of monolayers of acidic natural lipids over a wide range of surface charge density and salt concentration. It is also shown that the potential which initially appears when an electrolyte gradient is rapidly imposed across a bilayer membrane is due to a difference in the double layer potentials on the two sides of the membrane. This conclusion follows from the fact that the observed bilayer potentials arise much more rapidly than can be accounted for by charge migration across the membrane and from the observation that the bilayer membrane concentration potentials, when measured immediately after establishment of a gradient, are equal to the surface potential change observed when the subphase concentration of a monolayer of the same lipid is changed by an amount equal to the gradient across the bilayer. The bilayer potential and monolayer potential changes, so measured, agree in a number of different electrolyte solutions over a wide range of electrolyte concentrations and surface charge densities. Because of this agreement and the applicability of the Gouy theory to monolayers, initial bilayer potentials may be calculated if the composition of the mixture used to form the membrane is known, provided that the pK's and areas of such components are available. In the absence of this information, membrane potentials may be calculated from electrophoretic data on the membrane lipid mixture; the conditions under which the latter approach is possible have been determined. The experimental results indicate that the composition of monolyers and bilayers spread from the same lipid mixture in decane are very similar, that the composition of the two types of film closely resembles the composition of the solution used to generate them, and that bilayer membranes are close-packed. The evidence further indicates that if any hydrocarbon solvent remains in these bilayers, it must be so situated that it contributes little, if anything, to the surface area. The steady state potential in the bilayer membrane system is frequently not identical with the initial potential which supports the hypothesis that in many cases only a fraction of the electrical conductance of unmodified membranes is caused by the ions which constitute the bulk electrolyte. An expression for the relationship between diffusion and double layer potentials has been derived which shows that, in the absence of any intrinsic selectivity of the hydrocarbon region of the membrane for hydrogen, hydroxyl, or impurity, the two potentials should be identical.     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.     

Supported phospholipid/alkanethiol biomimetic membranes: insulating properties.

A novel model lipid bilayer membrane is prepared by the addition of phospholipid vesicles to alkanethiol monolayers on gold. This supported hybrid bilayer membrane is rugged, easily and reproducibly prepared in the absence of organic solvent, and is stable for very long periods of time. We have characterized the insulating characteristics of this membrane by examining the rate of electron transfer and by impedance spectroscopy. Supported hybrid bilayers formed from phospholipids and alkanethiols are pinhole-free and demonstrate measured values of conductivity and resistivity which are within an order of magnitude of that reported for black lipid membranes. Capacitance values suggest a dielectric constant of 2.7 for phospholipid membranes in the absence of organic solvent. The protein toxin, melittin, destroys the insulating capability of the phospholipid layer without significantly altering the bilayer structure. This model membrane will allow the assessment of the effect of lipid membrane perturbants on the insulating properties of natural lipid membranes.     

X-ray scattering from labeled membranes.

We present a new method for the determination of structural parameters in biological membranes. Recording the continuous scattering of heavy-atom labeled membranes and applying elementary Fourier methods we obtain the scattering of the heavy-atom distribution alone. The details of this distribution are explored by developing a simple model and testing for cases relevant to biological membranes. We find that the intensity distribution is highly sensitive to many key parameters. The increased signal from heavy-atom labeling and the use of an improved x-ray system make it possible to record patterns from dilute membrane suspensions. Thus determination of these parameters is possible in the same environment where many membrane biochemical studies are performed. Application of the method is made to a model lipid bilayer membrane, dipalmitoyl phosphatidylcholine by labeling with UO2++ ions. We determine the precise distance between UO2++ layers on either side of the membrane as well as the width of the label on each side. This determination permits estimation of phosphate separation across single labeled bilayers in an aqueous suspension.     

Functional tethered membranes

Phospholipid bilayer membranes at the interface between a substrate and an aqueous phase, supported by or tethered to the solid surface via a polymer cushion, a peptide-, protein-, or oligosaccharide-coupling layer have reached a stage at which they are important as a novel model membrane system but also offer potential for practical applications (e.g. for biosensing purposes with membrane-integral receptors). We briefly summarize some of the recent progress made in the structural characterization of the build-up of these rather complex interfacial architectures, in the functionalization of the pure lipid matrix by the reconstitution of proteins, and in the lateral patterning of the membranes as a prerequisite for the construction of membrane chips for massive parallel monitoring of binding events.     

Large divalent cations and electrostatic potentials adjacent to membranes. A theoretical calculation

We have extended the Gouy-Chapman theory of the electrostatic diffuse double layer by considering the finite size of divalent cations in the aqueous phase adjacent to a charged surface. The divalent cations are modeled as either two point charges connected by an infinitely thin, rigid "rod" or two noninteracting point charges connected by an infinitely thin, flexible "string." We use the extended theory to predict the effects of a cation of length 10 A (1 nm) on the zeta and surface potentials of phospholipid bilayer membranes. The predictions of the rod and string models are similar to one another but differ markedly from the predictions of the Gouy-Chapman theory. Specifically, the extended model predicts that a large divalent cation will have a smaller effect on the potential adjacent to a negatively charged bilayer membrane than a point divalent cation, that the magnitude of this discrepancy will decrease as the Debye length increases, and that a large divalent cation will produce a negative zeta potential on a membrane formed from zwitterionic lipids. These predictions agree qualitatively with the experimental results obtained with the large divalent cation hexamethonium. We discuss the biological relevance of our calculations in the context of the interaction of cationic drugs with receptor sites on cell membranes.     

Properties of lipid bilayer membranes separating two aqueous phases: The effects of Fe+3 on electrical properties

Summary Ferric ion has been found to alter the electrical properties of lecithincholesterol-decane bilayer membranes. Within minutes after the addition of microgram quantities of FeCl₃ to the ambient aqueous phase, the resistance of the membrane falls by a factor of 10⁵ to 10⁶. No change in capacitance is observed. The resistance change is obtained with membranes made from synthetic lecithin (fully saturated fatty acids) as well as by those formed from egg lecithin. The conductance of the modified membrane exhibits both time and voltage dependent behavior; the time dependence of the current is similar to that of an inductance, and the voltage dependence of the current is exponential. Concomitant with the resistance change, the modified membrane becomes permselective, passing chloride almost to the complete exclusion of sodium. Anion selectivity can be converted to cation selectivity by the subsequent addition of certain chelating agents. Area-conductance measurements show the resistance change occurs in the thin film. The addition of a reducing agent causes the effect of the ferric ion to be reversed, and the conductance returns to that characteristic of unmodified membranes. When ferric ion is added to only one side of the membrane, the system rectifies with current ratios of up to 201. It is concluded that the alteration of membrane properties owes its origin to the hydrolysis of membrane-bound ferric ion. The interaction of ferric ion with aqueous dispersions of lecithin has been investigated by several techniques, and evidence is presented that the dispersions bind charged species of iron and that this charge diminishes under conditions where iron hydrolysis occurs.     

Interactions between a membrane sialoglycoprotein and planar lipid bilayers

Summary Bilayer membranes formed from lipids dissolved in decane were exposed to glycophorin, a sialoglycoprotein which had been extracted from human red cell membranes. The interaction with the bilayer produced an increase in the steady state electrical conductance of the membrane proportional to the amount added. Fluctuations in membrane current when the electrical potential difference was constant were observed concommitantly with this increase in membrane conductance. The minimum size of the fluctuations corresponds to a conductance of 10^–10 mho. The increase in conductance as well as the current fluctuations persisted after extensive washout of the chamber containing the protein (cisside). Subsequent addition of lectins (wheat germ agglutinin and phytohemoagglutinin) to the cis-side produced rupture of the membranes, whilst these hemoagglutinins added to the trans-side failed to produce an effect. Measurements of changes in surface potential using K⁺ nonactin as a probe indicated that glycophorin induces a negative surface charge. At high protein concentrations, the magnitude of the induced surface potential became independent of glycophorin concentration. The maximum number of charges introduced onto the membrane under these conditions was 1.4×10⁵/m². Cis (but not trans)-side addition of neuraminidase abolished these charges, indicating that they can be ascribed to the sialic acid residues that the protein bears. These results suggest that glycophorin incorporates into bilayer membranes with its N-terminal end (where the sialic acid and carbohydrates are located) facing the cis-side. Spectrin reversibly lowered the glycophorin-induced membrane conductance when added to the trans-side. Cis-side additions failed to produce an effect. Trypsin present on the trans-side irreversibly lowered the membrane conductance. These results indicate that parts of the glycophorin molecule, probably the C-terminal end, are accessible to reagents in the solution bathing the trans-side of the membrane. Thus glycophorin spans the planar bilayer in much the same way as it spans the red cell membrane.     

A molecular toolkit for highly insulating tethered bilayer lipid membranes on various substrates

Tethered bilayer lipid membranes (tBLMs) are promising model architectures that mimic the structure and function of natural biomembranes. They provide a fluid, stable, and electrically sealing platform for the study of membrane related processes, specifically, the function of incorporated membrane proteins. This paper presents a generic approach toward the synthesis of functional tBLMs adapted for application to various surfaces. The central element of a tethered membrane consists of a lipid bilayer. Its proximal layer is covalently attached via a spacer unit to a solid support, either gold or silicon oxide. The membranes are characterized optically by using surface plasmon resonance spectroscopy (SPR) or ellipsometry and electrically by using electrochemical impedance spectroscopy (EIS). The bilayer membranes obtained show high electrical barrier properties and can be used to incorporate and study small membrane proteins in a functional form.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

Membrane on a chip: a functional tethered lipid bilayer membrane on silicon oxide surfaces

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin.     

Structure of planar membrane formed from liposomes

Lipid vesicles with incorporated ion channels from polyene antibiotic amphotericin B were used to investigate structures of planar membranes formed by Shindler's techniques. A planar membrane assembled on the aperture in a lavsan film from two layers generated at the air-aqueous liposome suspension interface is not a simple bilayer but a bimolecular membrane containing numerous partly fused liposomes. A complete fusion of liposomal membranes with the planar bilayer is an unlikely event during membrane formation. A planar bimolecular lipid membrane without incorporated liposomes can be made by a method consisting of three stages: formation of a lipid layer on the air-water interface of a suspension containing liposomes, transfer of this layer along the surface of the solution into a chamber containing a solution without liposomes where a lipid monomolecular layer forms gradually (within about 20 min) at the air-water interface, assembling of the planar bilayer membrane from this monolayer. The knowledge of the planar membrane structure may be useful in experiments on incorporation of membrane proteins into a planar lipid bilayer.     

Biological membranes as bilayer couples. III. Compensatory shape changes induced in membranes

We have previously proposed the hypothesis that asymmetric membranes behave like bilayer couples: the two layers of the bilayer membrane can respond differently to a particular perturbation. Such a perturbation, for example, can result in the expansion of one layer relative to the other, thereby producing a curvature of that membrane. In experiments with erythrocytes and lymphocytes, we now demonstrate that different membrane perturbations which have opposite effects on membrane curvature can compensate and neutralize one another, as expected from the bilayer couple hypothesis. This provides a rational basis, for example, for understanding the effects of amphipathic drugs on a variety of cellular phenomena which involve shape changes of membranes.     

Formation of a bilayer lipid membrane on rigid supports: an approach to BLM-based biosensors

Sensory transduction in living cells is thought to involve a change of electrical parameters at the receptor membrane following specific binding events at the membrane surface. Because of the complexity of the biomembrane structure and the environmental factors associated with it, experimental bilayer lipid membranes (BLMs) have been employed for elucidation of processes at the membrane level. This is because the BLM system can be easily probed by a host of powerful and sensitive electrochemical methods. Further, recent advances in microelectronics and biotechnology suggest that the development of a BLM-based electrochemical biosensor may be possible. This paper describes the use of bilayer lipid membranes on solid substrates for analysis of sensor development problems, with relevance to a possible novel type of biomolecular device. Some electrical parameters of the new structure were measured and compared to usual BLM results. The advantages of the self-assembled structure, together with the measuring system, are discussed in terms of stability and sensitivity.     

Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: rapid solution exchange with a new technique.

Adsorption of Na+/K+-ATPase containing membrane fragments from pig kidney to lipid membranes allows the detection of electrogenic events during the Na+/K+-ATPase reaction cycle with high sensitivity and time resolution. High stability preparations can be obtained using solid supported membranes (SSM) as carrier electrodes for the membrane fragments. The SSMs are prepared using an alkanethiol monolayer covalently linked to a gold surface on a glass substrate. The hydrophobic surface is covered with a lipid monolayer (SAM, self-assembled monolayer) to obtain a double layer system having electrical properties similar to those of unsupported bilayer membranes (BLM). As we have previously shown (, Biophys. J. 64:384-391), the Na+/K+-ATPase on a SSM can be activated by photolytic release of ATP from caged ATP. In this publication we show the first results of a new technique which allows rapid solution exchange at the membrane surface making use of the high mechanical stability of SSM preparations. Especially for substrates, which are not available as a caged substance-such as Na+ and K+-this technique is shown to be capable of yielding new results. The Na+/K+-ATPase was activated by rapid concentration jumps of ATP and Na+ (in the presence of ATP). A time resolution of up to 10 ms was obtained in these experiments. The aim of this paper is to present the new technique together with the first results obtained from the investigation of the Na+/K+-ATPase. A comparison with data taken from the literature shows considerable agreement with our experiments.     

Water and nonelectrolyte permeability of lipid bilayer membranes

Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n- butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility- diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).     

Influence of polyelectrolyte chemical structure on their interaction with lipid membrane of zwitterionic liposomes

In this paper we extend our previous experimental work on interaction between polyelectrolytes and liposomes. First, the adsorption of chitosan and alkylated chitosan (cationic polyelectrolytes) with different alkyl chain lengths on lipid membranes of liposomes is examined. The amount of both chitosans adsorbed remains the same even if more alkylated polysaccharide has to be added to get saturation if compared with unmodified chitosan. It is demonstrated that alkyl chains do not specifically interact with the lipid bilayer and that electrostatic interaction mechanism governs the chitosan adsorption. The difference observed between unmodified and alkylated chitosans behavior to reach the plateau can be interpreted in terms of a competition between electrostatic polyelectrolyte adsorption on lipid bilayer and hydrophobic autoassociation in solution (which depends on the alkyl chain length). Second, interaction of liposomes with hyaluronan (HA) and alkylated hyaluronan (anionic polyelectrolytes) is analyzed. The same types of results as discussed for chitosan are obtained, but in this case, autoassociation of alkylated HA only occurs in the presence of salt excess. Finally, a first positive layer of chitosan is adsorbed on the lipid membrane, followed by a second negative layer of HA at three different pHs. This kind of multilayer decoration allows the control of the net charge of the composite vesicles. A general conclusion is that whatever the pH and, consequently, the initial charge of the liposomes, chitosan adsorption gives positively charged composite systems, which upon addition of hyaluronan, give rise to negatively charged composite vesicles.     

Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study

Summary Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50–200 sec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10⁸ to 0.1 cm²), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential differenceV _c. For 1m alkali ion chloridesV _c was about 1 V. The temperature dependence of the electrical breakdown voltageV _c is comparable to that being observed with cell membranes.V _c decreases between 2 and 48°C from 1.5 to 0.6 V in the presence of 1m KCl.Breakdown experiments were also performed with lipid bilayer membranes composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability.The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).     

A theory for the effects of neutral carriers such as the macrotetralide actin antibiotics on the electric properties of bilayer membranes

Summary To develop a quantitiative theoretical treatment for the effects of neutral macrocyclic antibiotics on the electrical properties of phospholipid bilayer membranes, this paper proceeds from the known ability of such molecules to form stoichiometric, lipid-soluble complexes with cations and deduces the electrical properties that a simple organic solvent phase would have if it were made into a membrane of the thinness of the phospholipid bilayer. In effect, we postulate that the essential barrier to ion movement across a bilayer membrane is its liquid-like hydrocarbon interior and that the neutral macrocyclic antibiotics bind monovalent cations and solubilize them in the membrane as mobile positively charged complexes. Using the Poisson-Boltzmann equation to describe the equilibrium profile of the electrical potential, it is shown that an excess of the positive complexes over all the other ions is expected in the membrane as a net space charge for appropriate conditions of membrane thickness and values of the partition coefficients of the various ionic species and without requiring the presence of fixed charges. Describing the fluxes of these complexes by the Nernst-Planck equation and neglecting the contribution to the electric current of uncomplexed ions, theoretical expressions are derived for the membrane potential in ionic mixtures, as well as for the limiting value of the membrane conductance at zero current when the membrane is interposed between identical solutions. The expressions are given in terms of the ionic activities and antibiotic concentrations in the aqueous solutions so as to be accessible to direct experimental test. Under suitable experimental conditions, the membrane potential is described by an equation recognizible as the Goldman-Hodgkin-Katz equation, in which the permeability ratios are combinations of parameters predicted from the present theory to be independently determinable from the ratio of membrane conductances in single salt solutions. Since this identity between permeability and conductance ratios is expected also for systems obeying the Independence Principle of Hodgkin and Huxley, the applicability of this principle to membranes exposed to antibiotics is discussed, and it is shown that this principle is compatible with the permeation mechanism proposed here.