Properties Of Planar Lipid Bilayers Research Articles

The role of lipid oxidation on electrical properties of planar lipid bilayers and its importance for understanding electroporation

Electroporation is a useful tool for the manipulation with the cell membrane permeability. Underlying physicochemical processes taking place at the molecular level during electroporation are relatively well studied. However, various processes remain unknown, one of them is lipid oxidation, a chain reaction that causes degradation of lipids, and might explain the long-lasting membrane permeability after the electric field has ceased. The aim of our study was to observe the differences in the electrical properties of planar lipid bilayers, as in vitro cell membrane models, due to lipid oxidation. Phospholipids were chemically oxidized and oxidation products were analysed using mass spectrometry. Electrical properties, resistance R (Ω) and capacitance C (F) were measured using an LCR meter. Using a previously developed measuring device, a linear increasing signal was applied to a stable bilayer in order to measure its breakdown voltage Ubr (V) and lifetime tbr (µs). We observed an increase in conductance and capacitance of the oxidized planar lipid bilayers when compared to their non-oxidized counterparts. With increasing lipid oxidation, the core of the bilayer becomes more polar, and consequently more permeable. Our findings can explain the long-lasting permeability of the cell membrane after electroporation.

Probing actin coated planar lipid bilayers with light and electricity in a microelectrode cavity array.

Planar lipid bilayers play a central role in nanopore sensing and ion-channel electrophysiology. However, bilayer fragility, especially in the presence of osmotic stress, can limit their application. We have been developing methods to enhance the mechanical properties of planar lipid bilayers with a layered, molecularly thin, minimal actin cortex (MAC). Approaches involve both single- and multiple-layered MAC structures (multi-MACs). In this work, we describe the MAC formation process in the context of different microelectrode cavity array chips (MECA and MECA-opto), as well as on glass-supported bilayers. We explore the response of these bilayers to osmotic stress and report single-molecule level imaging results that reveal the kinetic motion of actin filaments on the bilayer. We also discuss our latest permeability tests at both the single-molecule and macroscopic levels that show that multi-MACs do not significantly block access to the bilayer.

Lipid peroxidation induced changes in electrical properties of planar lipid bilayers

Cell membrane can be damaged by the oxidative breakdown of phospholipid molecules, e.g. lipid peroxidation, a chain reaction that causes degradation of phospholipids and formation of unstable intermediate product termed lipid peroxide. Lipid peroxidation can cause selective alterations in cell signaling, protein and DNA damage. Furthermore, oxidation is involved in the pathogenesis of various diseases such as atherosclerosis, Alzheimer’s disease and other immunological disorders. Oxidized phospholipids are known to destabilize biological membranes, therefore the study of lipid oxidation effects on cell membrane is of more general interest.

Effects of valinomycin doping on the electrical and structural properties of planar lipid bilayers supported on polyelectrolyte multilayers

Supported Lipid Bilayers (SLBs) on Polyelectrolyte Multilayers (PEMs) have large potential as models for developing sensor devices. SLBs can be designed with receptors and channels, which benefit from the biological environment of the lipid layers, to create a sensing interface for ions and biomarkers. PEMs assembled by the Layer-by-Layer (LBL) technique and used as supports for a lipid bilayer enable an easy integration of the bilayer on almost any surface and device. For electrochemical sensors, LBL assembly enables nanoscale tunable separation of the lipid bilayer from the electrode surface, avoiding undesired effects of the electrode surface on the lipid bilayers. We study the fabrication of valinomycin-doped SLBs on PEMs as a model system for biophysical studies and for selective ion sensing. SLBs are fabricated from dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) 50:50 vesicles doped with valinomycin, as a K+-selective carrier. SLBs were deposited on electrodes coated with poly(allyl amine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS) multilayers. Lipid bilayer formation was monitored by using Quartz Crystal Microbalance with Dissipation (QCMD) technique and Atomic Force Microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and potentiometric measurements were performed to assess K+ selectivity over other ions and the potential of valinomycin-doped SLBs for K+-sensing.

Ruggedized Chip-Based Lipid Bilayer Arrays with Permeable multi-MAC Support

Planar lipid bilayers play a central role in nanopore sensing and ion-channel electrophysiology. However, bilayer fragility often limits their application. Previously, we enhanced the mechanical properties of planar lipid bilayers with a single-layered, molecularly thin, minimal actin cortex (MAC). To further increase bilayer robustness, we are developing multiple layered MAC structures (multi-MACs) that can be chemically cross-linked to arbitrary thicknesses, without blocking diffusive access to the outer leaflet of the bilayer. In this work, we describe the multi-MAC formation process in the context of a bilayer array on a fluidic chip, as well as on glass supported bilayers. We report on permeability tests at both the single-molecule and macroscopic levels that show multi-MACs do not significantly block access to the bilayer. We discuss how permeability is achieved by controlling the openness of the F-actin network, which can be tailored using the density of linkage sites, the electrostatic interactions between linkers and filaments, and specific F-actin growth and deposition techniques. We also present results from single-molecule electrical and optical measurements that characterize the multi-MAC structure, permeability, and the approximate pore size of the multi-layered network.

Annexins V and XII alter the properties of planar lipid bilayers seen by conductance probes.

Annexins are proteins that bind lipids in the presence of calcium. Though multiple functions have been proposed for annexins, there is no general agreement on what annexins do or how they do it. We have used the well-studied conductance probes nonactin, alamethicin, and tetraphenylborate to investigate how annexins alter the functional properties of planar lipid bilayers. We found that annexin XII reduces the nonactin-induced conductance to approximately 30% of its original value. Both negative lipid and approximately 30 microM Ca(2+) are required for the conductance reduction. The mutant annexin XIIs, E105K and E105K/K68A, do not reduce the nonactin conductance even though both bind to the membrane just as wild-type does. Thus, subtle changes in the interaction of annexins with the membrane seem to be important. Annexin V also reduces nonactin conductance in nearly the same manner as annexin XII. Pronase in the absence of annexin had no effect on the nonactin conductance. But when added to the side of the bilayer opposite that to which annexin was added, pronase increased the nonactin-induced conductance toward its pre-annexin value. Annexins also dramatically alter the conductance induced by a radically different probe, alamethicin. When added to the same side of the bilayer as alamethicin, annexin has virtually no effect, but when added trans to the alamethicin, annexin dramatically reduces the asymmetry of the I-V curve and greatly slows the kinetics of one branch of the curve without altering those of the other. Annexin also reduces the rate at which the hydrophobic anion, tetraphenylborate, crosses the bilayer. These results suggest that annexin greatly reduces the ability of small molecules to cross the membrane without altering the surface potential and that at least some fraction of the active annexin is accessible to pronase digestion from the opposite side of the membrane.

Formation of ion-conducting channels by the membrane attack complex proteins of complement

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.

Wave-guide spectroscopy on planar lipid bilayers doped with hydrophobic ions

Wave-guide spectroscopy exploits the light pipe properties of planar lipid bilayers by propagating a light wave along the plane of the bilayer. Applying this technique to the optical absorption of chromophore in the membranes, results in an enhanced sensitivity when compared to normal incidence spectroscopy. This gain factor is of the order of 100 per mm optical path along the bilayer, thus transforming the weak absorbances in lipid membranes into easily measurable quantities. Wave-guide spectroscopy has been used to measure the adsorption isotherm of hydrophobic dipicrylamine ions in a phosphatidylcholine membrane. The adsorption isotherm is linear for low aqueous concentrations, in the micromolar range however, it changes into a sublinear dependence. The addition of an inert alkali salt to the electrolyte favours the adsorption of hydrophobic ions. Current saturation is observed with the transition to the sublinear isotherm. When using the time constant for current relaxation as an indicator of changes in the magnitude of the surface potential, it does not seem to vary with the additional dipicrylamine which adsorbs in the presence of high concentrations of alkali salt in the electrolyte. A compensation of hydrophobic charge by the alkali ions from the inert electrolyte is proposed.

Electrogenic and selective transport of biogenic amines across lipid bilayer membranes mediated by a neutral ionophore (AS701)

The neutral noncyclic imide and ether containing ionophore (AS701), a selective carrier for Li + among alkali cations, was found to be capable of mediating the transport of NH 4 + and of biogenic amines (catechols and indoles) across lipid bilayer membranes also. Ionophore-mediated electrical properties of planar lipid bilayers were studied under experimental conditions where the positively-charged amine species was dominant. The ionophore was found to act as a selective carrier of the biogenic amines, mediating their electrogenic transport across the membrane, forming 2:1 carrier-amine permeant complexes, carrying a net-charge of +1. Selectively among the amines corresponding to the following sequence: tryptamine (35) > Li + (1) > serotonin (0.60) > dopamine (0.19) > norepinephrine (0.13) > epinephrine (0.05) > NH 4 + (0.05). The molecular factors involved in determining these selectivities are assessed.

Planar Lipid Bilayers as Light Guides

Abstract The light guide properties of planar lipid bilayers are discussed and experimentally verified. The interaction between the light and the constituents of the bilayer is substantially increased since the light path is as large as the diameter of the film, e.g. of the order of several millimeters. The influence of electric fields on the bilayer and on the bilayer-torus transition region has been investigated. Field induced generation of scattering centers is detected in solvent containing bilayers.

The Light Guide Effect of Planar Lipid Bilayers and Its Application to Bilayer Spectroscopy

Abstract The light guide properties of planar lipid bilayers offer a novel approach to bilayer spectroscopy. Potential applications are outlined.

Electrically silent anion transport through lipid bilayer membranes containing a long-chain secondary amine.

The permeability properties of planar lipid bilayers made from egg lecithin, n-decane and a long-chain secondary amine (n-lauryl [trialkylmethyl]amine) are described. Membranes containing the secondary amine show halide selectivity and high conductance at pH less than 6, as estimated by measurements of zero-current potentials generated by NaBr activity gradients. In the absence of halide ions, the membranes show H+ selectivity, although the total membrane conductance is relatively low. In 0.1 M NaBr both the membrane conductance (Gm) and the Br- self-exchange flux (JBr) are proportional to H+ concentration over the pH range of 7 to 4, and both JBr and Gm saturate at pH less than 4. However, JBr is always more than 100 times the flux predicted from Gm and the transference number for Br-. Thus, greater than 99% of the observed (tracer) flux is electrically silent and is not a Br2 or HBrO flux because the reducing agent, S2O3=, has no effect on JBr. At pH 7, JBr is proportional to Br- concentration over the range of 1-340 mM, with no sign of saturation kinetics. Both urea and sulfate tracer permeabilities are low and are unaffected by pH. The results can be explained by a model in which the secondary amine behaves as a monovalent, titratable carrier which exists in three chemical forms (C, CH+, and CHBr). Br- crosses the membrane primarily as the neurtal complex (CHBr). The positively charged carrier (CH+) crosses the membrane slowly compared to CHBr, but CH+ is the principal charge carrier in the membrane. At neurtal pH greater than 99% of the amine is in the nonfunctional form (C), which can be converted to CH+ or CHBr by increasing the H+ or Br- concentrations. The permeability properties of these lipid bilayers resemble in many respects the permeability properties of red cell membranes.