Conductance channels in neutral lipid bilayers

Summary A model channel for the conduction of ions (positive or negative but not both) through a lipid bilayer is presented. The transition-state theory is used to relate the current with voltage and ionic concentrations. Sites within the channel are considered to act cooperatively so that the ion is subjected to a ligand field in which it has complete freedom along the channel axis. The ions in the channel are treated as an ionic gas. Effects due to space-charges within the channel arising from the conducting ions are considered whereas surface-charge effects are neglected.The ionic specificity of the channel is indicated and the theory compared to that in which equilibrium free energy changes are the dominant influence.     

Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine

The tricyclic antidepressant desipramine causes a decrease in cellular acid sphingomyelinase (A-SMase, EC 3.1.4.12) activity when added to culture medium of human fibroblasts. This effect can be prevented by incubation of the cells with the protease inhibitor leupeptin, which suggests that desipramine induces proteolytic degradation of the lysosomal enzyme. By using surface plasmon resonance (SPR, Biacore) we were able to monitor the interactions of A-SMase and substrate-containing lipid bilayers immobilized on the surface of a Pioneer trade mark L1 sensor chip. SPR binding curves show that the enzyme hardly dissociates from the lipid surface at acidic pH values. On the other hand, a drop in binding signals (resonance units, RU) of approximately 50% occurred after injection of 20 mM desipramine. Our findings indicate that desipramine interferes with the binding of A-SMase to the lipid bilayers and thereby displaces the enzyme from its membrane-bound substrate. The application of control substances suggests a key role for the cationic moiety of desipramine. We hypothesize that the displacement of the glycoprotein A-SMase from the inner membranes of late endosomes and lysosomes by desipramine renders it susceptible to proteolytic cleavage by lysosomal proteases.     

Molecular dynamics simulations of charged and neutral lipid bilayers: treatment of electrostatic interactions

Molecular dynamics (MD) simulations complement experimental methods in studies of the structure and dynamics of lipid bilayers. The choice of algorithms employed in this computational method represents a trade-off between the accuracy and real calculation time. The largest portion of the simulation time is devoted to calculation of long-range electrostatic interactions. To speed-up evaluation of these interactions, various approximations have been used. The most common ones are the truncation of long-range interactions with the use of cut-offs, and the particle-mesh Ewald (PME) method. In this study, several multi-nanosecond cut-off and PME simulations were performed to establish the influence of the simulation protocol on the bilayer properties. Two bilayers were used. One consisted of neutral phosphatidylcholine molecules. The other was a mixed lipid bilayer consisting of neutral phosphatidylethanolamine and negatively charged phosphatidylglycerol molecules. The study shows that the cut-off simulation of a bilayer containing charge molecules generates artefacts; in particular the mobility and order of the charged molecules are vastly different from those determined experimentally. In the PME simulation, the bilayer properties are in general agreement with experimental data. The cut-off simulation of bilayers containing only uncharged molecules does not generate artefacts, nevertheless, the PME simulation gives generally better agreement with experimental data.     

Effect of lactose permease presence on the structure and nanomechanics of two-component supported lipid bilayers

In this paper we present a comparative study of supported lipid bilayers (SLBs) and proteolipid sheets (PLSs) obtained from deposition of lactose permease (LacY) of Escherichia coli proteoliposomes in plane. Lipid matrices of two components, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), at a 3:1, mol/mol ratio, were selected to mimic the inner membrane of the bacteria. The aim was to investigate how species of different compactness and stiffness affect the integration, distribution and nanomechanical properties of LacY in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). Both compositions displayed phase separation and were investigated by atomic force microscopy (AFM) imaging and force-spectroscopy (FS) mode. PLSs displayed two separated, segregated domains with different features that were characterised by FS and force-volume mode. We correlated the nanomechanical characteristics of solid-like gel phase (L_β) and fluid liquid-crystalline phase (L_α) with phases emerging in presence of LacY. We observed that for both compositions, the extended PLSs showed a L_β apparently formed only by lipids, whilst the second domain was enriched in LacY. The influence of the lipid environment on LacY organisation was studied by performing protein unfolding experiments using the AFM tip. Although the pulling experiments were unspecific, positive events were obtained, indicating the influence of the lipid environment when pulling the protein. A possible influence of the lateral surface pressure on this behaviour is suggested by the higher force required to pull LacY from DPPE:POPG than from POPE:POPG matrices. This is related to higher forces governing protein–lipid interaction in presence of DPPE.     

Increased adhesion between neutral lipid bilayers: interbilayer bridges formed by tannic acid.

Tannic acid (TA) is a naturally occurring polyphenolic compound that aggregates membranes and neutral phosolipid vesicles and precipitates many proteins. This study analyzes TA binding to lipid membranes and the ensuing aggregation. The optical density of dispersions of phosphatidylcholine (PC) vesicles increased upon the addition of TA and electron micrographs showed that TA caused the vesicles to aggregate and form stacks of tightly packed disks. Solution calorimetry showed that TA bound to PC bilayers with a molar binding enthalpy of -8.3 kcal/mol and zeta potential measurements revealed that TA imparted a small negative charge to PC vesicles. Monolayer studies showed that TA bound to PC with a dissociation constant of 1.5 microM and reduced the dipole potential by up to 250 mV. Both the increase in optical density and decrease in dipole potential produced by TA could be reversed by the addition of polyvinylpyrrolidone, a compound that chelates TA by providing H-bond acceptor groups. NMR, micropipette aspiration, and x-ray diffraction experiments showed that TA incorporated into liquid crystalline PC membranes, increasing the area per lipid molecule and decreasing the bilayer thickness by 2 to 4%. 2H-NMR quadrupole splitting measurements also showed that TA associated with a PC molecule for times much less than 10(-4) s. In gel phase bilayers, TA caused the hydrocarbon chains from apposing monolayers to fully interdigitate. X-ray diffraction measurements of both gel and liquid crystalline dispersions showed that TA, at a critical concentration of about 1 mM, reduced the fluid spacing between adjacent bilayers by 8-10 A. These data place severe constraints on how TA can pack between adjacent bilayers and cause vesicles to adhere. We conclude that TA promotes vesicle aggregation by reducing the fluid spacing between bilayers by the formation of transient interbilayer bridges by inserting its digallic acid residues into the interfacial regions of adjacent bilayers and spanning the interbilayer space.     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Structure of lipid bilayers

The quantitative experimental uncertainty in the structure of fully hydrated, biologically relevant, fluid (L(alpha)) phase lipid bilayers has been too large to provide a firm base for applications or for comparison with simulations. Many structural methods are reviewed including modern liquid crystallography of lipid bilayers that deals with the fully developed undulation fluctuations that occur in the L(alpha) phase. These fluctuations degrade the higher order diffraction data in a way that, if unrecognized, leads to erroneous conclusions regarding bilayer structure. Diffraction measurements at high instrumental resolution provide a measure of these fluctuations. In addition to providing better structural determination, this opens a new window on interactions between bilayers, so the experimental determination of interbilayer interaction parameters is reviewed briefly. We introduce a new structural correction based on fluctuations that has not been included in any previous studies. Updated measurements, such as for the area compressibility modulus, are used to provide adjustments to many of the literature values of structural quantities. Since the gel (L(beta)') phase is valuable as a stepping stone for obtaining fluid phase results, a brief review is given of the lower temperature phases. The uncertainty in structural results for lipid bilayers is being reduced and best current values are provided for bilayers of five lipids.     

Cholestryl-phosphoryl-choline in lipid bilayers.

Cholesteryl-phosphoryl-choline (CPC), a hybrid between cholesterol and lecithin, is incorporated into sonicated liposomes and erythrocyte membranes similarly to cholesterol. The effect of CPC on lipid microviscosity and degree of order is smaller, but not significantly than that of cholesterol. It is proposed that CPC may be employed as an efficient modulator of lipid dynamics.     

Functional tethered lipid bilayers

Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.     

Properties of bilayer lipid membranes with different surface charges in the presence of tetraalkylammonium salts]

It is shown that with the growth of the radius of tetraalkylammonium ions the conductivity of the membrane increases. With an increase of the dimensions of penetrating ions the maximum cation selectivity reached in the beginning decreases with a further growth of cation radius. A negative charge on the membrane surface results in a higher cation selectivity and conductance mechanism is observed at the transition to the cations with a large radius at their high enough concentration in the aqueous solution. It can be explaind by the formation of triplet in the membrane phase which aid in the penetration of C1-.     

Structure determination of lipid bilayers

A method of determining the phases of X-ray reflections from oriented model membrane systems at low resolution is described. The method involves deconvolution and requires that d less than or equal to 2v where v is the width of the head group region within the bilayer and d is the thickness of the bilayer. The method can be used with a single set of X-ray data and applies to lipid bilayers which have a relatively constant density in the hydrocarbon region. Phases for the first five or six orders of phosphatidylethanolamine and lecithin are derived. A refined analysis based upon deconvolution but using information inherent in the Fourier profile is also described.     

Proton permeation of lipid bilayers

Proton permeation of the lipid bilayer barrier has two unique features. First, permeability coefficients measured at neutral pH ranges are six to seven orders of magnitude greater than expected from knowledge of other monovalent cations. Second, proton conductance across planar lipid bilayers varies at most by a factor of 10 when pH is varied from near 1 to near 11. Two mechanisms have been proposed to account for this anomalous behavior: proton conductance related to contaminants of lipid bilayers, and proton translocation along transient hydrogen-bonded chains (tHBC) of associated water molecules in the membrane. The weight of evidence suggests that trace contaminants may contribute to proton conductance across planar lipid membranes at certain pH ranges, but cannot account for the anomalous proton flux in liposome systems.Two new results will be reported here which were designed to test the tHBC model. These include measurements of relative proton/potassium permeability in the gramicidin channel, and plots of proton flux against the magnitude of pH gradients. (1) The relative permeabilities of protons and potassium through the gramicidin channel, which contains a single strand of hydrogenbonded water molecules, were found to differ by at least four orders of magnitude when measured at neutral pH ranges. This result demonstrates that a hydrogen-bonded chain of water molecules can provide substantial discrimination between protons and other cations. It was also possible to calculate that if approximately 7% of bilayer water was present in a transient configuration similar to that of the gramicidin channel, it could account for the measured proton flux. (2) The plot of proton conductance against pH gradient across liposome membranes was superlinear, a result that is consistent with one of three alternative tHBC models for proton conductance described by Nagle elsewhere in this volume.     

Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.     

Hydrocarbon chain dynamics in lipid bilayers

A model is proposed for hydrocarbon chain dynamics in lipid bilayers. In the upper and middle parts of the chain all motion occurs by concerted rotations around at least two carbon carbon bonds at a time, preserving a structural with kinks (that is gauche^±trans gauche^? conformations) as the only deviations from the all-trans chain. At the end, independent rotations around carboncarbon bonds play a larger and larger part. This gives a reasonable interpretation of deuterium NMR data.     

Diffusion in lipid bilayers containing barriers

In epithelial cells, a barrier or tight junction restricts the diffusion of lipid probes from the apical to the basolateral side of the outer membrane bilayer. This phenomenon is studied theoretically with the diffusion equation on planar and spherical surfaces. Two models for the tight junction are considered: a penetrable barrier embedded in a monolayer and an impenetrable obstacle in the outer membrane of a bilayer than must be bypassed by flip-flopping between inner and outer membranes. The rate of passing from one side of the cell to the other is calculated for each of these models under steady state conditions. The results are compared with recent fluorescent photobleaching recovery experiments. The theoretical interpretation indicates that it would be difficult to distinguish experimentally between the flip-flop case and the barrier crossing case. Assuming a flip-flop model, large differences in the magnitude of the flip-flop rates of probes are necessary to explain the experimental results as suggested by Dragsten et al. (Dragsten, P. R., R. Blumenthal, and J. S. Handler, 1981, Nature [Lond.], 294:718--722).     

Two-dimensional microelectrophoresis in supported lipid bilayers

We report the application of supported bilayers for two-dimensional microelectrophoresis. This method allows the lateral separation and accumulation of charged amphiphilic molecular probes in bilayers by application of an electric field parallel to the bilayer surface. Diffusion coefficient and mobility of the fluorescent probes are determined by observation of the fluorescence recovery after photobleaching (pattern bleaching). The diffusion coefficients and the mobilities of oppositely charged fluorescent probes in one bilayer can be determined independently from a single measurement. By analysis of the motion of charged and uncharged probes in one membrane one can distinguish between the motion caused by the electric field acting on the charge of individual probes and that caused by frictional forces due to electroosmosis.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.