Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

The effect of hydrostatic pressure on S-layer-supported lipid membranes

We report on the behavior of unsupported and surface layer (S-layer)-supported lipid membranes at the application of a uniform hydrostatic pressure. At a hydrostatic pressure gradient higher than 6 N/m(2), unsupported lipid membranes, independent from which side pressurized and S-layer-supported lipid membranes pressurized from the lipid-faced side revealed a pronounced increase in capacitance. A maximal hydrostatic pressure gradient of 11.0 N/m(2) resulted in an almost doubling of the capacitance of the (composite) membranes. S-layer-supported lipid membranes showed a hysteresis in the capacitance versus pressure plot, indicating that this composite structure required a certain time to reorient when the pressure gradient acting from the lipid-faced side was balanced. By contrast, the S-layer-supported lipid membrane pressurized from the protein-faced side revealed only a minute increase in capacitance (C/C(0,max)=1.17+/-0.05), reflecting only minor pressure-induced area expansion. In addition, no hysteresis could be observed, indicating that no rearrangement of the composite membrane occurred. The maximal induced tension was with 4.3+/-0.2 mN/m, significantly higher than that of unsupported (2.5+/-0.3 mN/m) and S-layer-supported lipid membranes pressurized from the lipid-faced side (2.6+/-0.1 mN/m).     

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.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Electroporation of heterogeneous lipid membranes

Electroporation is the basis for the transfection of genetic material and for drug delivery to cells, including electrochemotherapy for cancer. By means of molecular dynamics many aspects of membrane electroporation have been unveiled at the molecular detail in simple, homogeneous, lipid bilayers. However, the correspondence of these findings with the process happening in cell membranes requires, at least, the consideration of laterally structured membranes. Here, I present a systematic molecular dynamics study of bilayers composed of different liquid-ordered and liquid-disordered lipid phases subjected to a transversal electric field. The simulations reveal two significant results. First, the electric field mainly affects the properties of the disordered phases, so that electroporation takes place in these membrane regions. Second, the smaller the disordered domains are, the faster they become electroporated. These findings may have a relevant significance in the experimental application of cell electroporation in vivo since it implies that electro-induced and pore-mediated transport processes occur in particularly small disordered domains of the plasma membrane, thus locally affecting only specific regions of the cell.     

Polymerization in black lipid membranes

A variety of different lipids containing dienoyl groups in the side chains were tested for membrane formation using the planar lipid bilayer approach. One of these lipids formed stable bilayers which could be polymerized using UV-illumination. The influence of the polymerization was studied in monolayers, lipid vesicles and planar bilayers. The stability of the lipid bilayer membranes was increased by polymerization. Thus, the lifetime of the membranes increased from about 1 h to 4–5 h or longer. Furthermore, the specific conductance of unmodified membranes and of carrier-mediated transport is reduced. The transport of lipophilic ions was investigated as a function of polymerization using the charge-pulse method. The absorption of dipicrylamine (DPA^-) is not affected. The translocation of this compound and of tetraphenylborate (B(Ph) ₄ ^- ) showed a strong decrease with polymerization time. The influence of polymerization on the membrane structure may be explained on the basis of a strong viscosity increase in the lipid bilayer membrane.     

Surface areas of lipid membranes

Upon photolysis, alkyl pentacyanocobaltate complexes generate alkyl radicals which react rapidly and specifically with nitroxide radicals, and which do not penetrate phospholipid bilayers. By measuring the loss of paramagnetic resonance signal intensity when multilamellar liposomes containing a small amount of spin-labeled lipid are exposed to these radicals, we have measured the proportion of lipid on the external surface of liposomes. We have shown that liposomes prepared under specified conditions from dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and binary mixtures of dipalmitoylphosphatidylcholine and cholesterol all have the same proportion of external lipid.     

Branched bimolecular lipid membranes.

Branched bimolecular lipid membranes separating three and four aqueous phases have been formed. The procedure is based on the technique of Montal and Mueller generalized to three and four lipid surface films spanning an appropriate aperture. The technique to produce Teflon structures for the mechanical support of branched bilayers is presented. The existence of the branched bilayer was established by measuring the specific capacity, specific resistance, and the gramicidin-induced single channel conductance of each branch. These structures should facilitate the study of transport properties of ionophores and other molecules and may also serve as model systems for the study of cell fusion.     

Bilirubin diffusion through lipid membranes

The possibility that bilirubin can diffuse through lipid bilayers is investigated with liposomes prepared from dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (egg PC) with 22 mole percent cholesterol, and a lipid extract preparation from N115 neuroblastoma cells. Liposomes were prepared with internalized bilirubin and bovine or human serum albumin, and bilirubin efflux into an exogenous solution of human serum albumin was measured. Efflux from DPPC liposomes was significantly higher above the phase transition temperature than below it. This change was dependent on the lipid undergoing a phase transition and could not be accounted for by 6 K change in temperature. Maximum bilirubin efflux from egg PC-cholesterol liposomes was found to depend on the relative internal and external albumin pools, suggesting an equilibrium distribution of bilirubin between them. These observations demonstrate that bilirubin can diffuse freely through these lipid membranes.     

Interaction of smooth muscle plasma membranes with flat lipid membranes

The method of implantation of smooth muscle cells from plasma membranes (PM) of the rabbit intestine into flat lipid membranes (FLM) is described. The method is based on the pretreatment of PM vesicles with asolectin liposomes in the ratio that provides the activation of membrane ATPases. Thus modified FLM possesses channel conductivity.     

Spin-labelled vacuolar-ATPase inhibitors in lipid membranes

Two spin-labelled derivatives of the 5-(2-indolyl)-2,4-pentadienoyl class of inhibitors of the vacuolar ATPase have been synthesised and their EPR properties characterised in phospholipid membranes. One spin-labelled inhibitor is the amide derivative of pentadienic acid and 4-amino-TEMPO (INDOL6), and the other is the 3-hydroxymethyl-PROXYL ester (INDOL5). The response of the EPR spectra to the chain-melting transition of dimyristoyl phosphatidylcholine (DMPC) bilayers demonstrates that both derivatives incorporate in phospholipid membranes. The axially anisotropic EPR spectra of INDOL6 in fluid DMPC membranes indicate that the indolyl-pentadienoyl inhibitors intercalate between the lipid chains, in the membrane. INDOL5, designed to possess additional internal segmental mobility, exhibits more nearly isotropic motion of the spin-label moiety in fluid membranes than does INDOL6. The EPR characteristics of INDOL5 are therefore well suited to detecting specific ligand-protein interactions. Progressive saturation EPR experiments with polar and hydrophobic relaxation agents (aqueous Ni2+ and oxygen) show that the nitroxide group is buried in the membrane, with the indole moiety providing the anchor at the membrane polar-apolar interface. Rates of spin-label reduction by externally added ascorbate confirm this assignment. These two spin-labelled derivatives provide complementary EPR probes of the lipid environment (INDOL6), and of ligand-protein interactions (INDOL5), for this class of V-ATPase inhibitor.     

Fluorescence coupling across lipid bilayer membranes

Summary An energy transfer between donor and acceptor fluorophores across single lipid bilayer membranes is demonstrated. Anilino-naphthalene sulfonate is used as the donor chromophore: its fluorescence is enhanced by the presence of lipid and thus indicates association with the purely lipid membranes of our preparation of vesicles in suspension. Light emit ted by the donor molecules excites fluorescence of acriflavine, a suitable acceptor enclosed inside the vesicles. Absorption and fluorescence spectra of this system, in its components and as a whole, are presented in evidence for an energy transfer.Supported by a grant from the Medical Research Council of Canada. The results of this work were presented, in part, at the 17th Annual Meeting of the Biophysical Society, February 27–March 2, 1973, Columbus, Ohio.Scholar of the Medical Research Council of Canada.     

Cell membranes: the lipid perspective

Although cell membranes are packed with proteins mingling with lipids, remarkably little is known about how proteins interact with lipids to carry out their function. Novel analytical tools are revealing the astounding diversity of lipids in membranes. The issue is now to understand the cellular functions of this complexity. In this Perspective, we focus on the interface of integral transmembrane proteins and membrane lipids in eukaryotic cells. Clarifying how proteins and lipids interact with each other will be important for unraveling membrane protein structure and function. Progress toward this goal will be promoted by increasing overlap between different fields that have so far operated without much crosstalk.     

Elastic constants of polymer-grafted lipid membranes

The surface expansion that is induced by the lateral pressure in the brush region of lipid membranes containing grafted polymers is deduced from the scaling and mean-field theories for the polymer brush, together with the equation of state for a lipid monolayer at the equivalence pressure with fluid lipid bilayers. Depending on the length and mole fraction of the polymer lipid, the membrane expansion can be appreciable. Direct experimental evidence for this lateral expansion comes from recent spin-label measurements with lipid membranes containing poly(ethylene glycol)-grafted lipids. The expansion in lipid area modifies the elastic constants of the polymer-grafted membranes in a way that opposes the direct elastic response of the polymer itself. Calculations as a function of polymer lipid content indicate that the net change in isothermal area expansion modulus of the membrane is negative but small, in contrast to previous predictions. A similar situation applies to the curvature elastic moduli of membranes containing short polymer lipids. For longer polymer lipids, however, the direct contribution of the polymer brush to the bending elastic constants dominates, and the increase in bending moduli with increasing polymer lipid content rapidly exceeds the basal values of the bare lipid membrane. The spontaneous (or intrinsic) curvature of the component monolayer of polymer lipid-containing membranes is calculated for the first time. The polymer brush contribution to spontaneous curvature scales quadratically with the polymer length, and at least quadratically with the mole fraction of polymer lipid.     

Interaction of furosemide with lipid membranes

Summary The interaction of furosemide with different phospholipids was investigated. Its influence on the lipid structure was inferred from its effect on the phase transition properties of lipids and on the conductance of planar bilayer membranes. The thermotropic properties of dipalmitoyl phosphatidylcholine, phosphatidylethanolamine (natural), dipalmitoyl phosphatidylethanolamine, brain sphingomyelin, brain cerebrosides and phosphatidylserine in the presence and absence of furosemide were investigated by differential scanning calorimetry,. The modifying effect of furosemide seems to be strongest on phosphatidylethanolamine (natural) and sphingomyelin bilayers. The propensity of furosemide to decrease the electrical resistance of planar lipid membranes was also studied and it is shown that the drug facilitates the transport of ions. Partition coefficients of furosemide between lipid bilayers and water were measured.Abbreviations DSC differential scanning calorimetry - PLM planar lipid membranes - DPPC dipalmitoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - PE phosphatidyl ethanol