Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol.

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action. At antibiotic levels above 1:1 antibiotic: cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentraion, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Nonequilibrium behavior in supported lipid membranes containing cholesterol

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Sch?fer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action.At antibiotic levels above 1 : 1 antibiotic : cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentration, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Properties of lipid bilayer membranes made from lipids containing phytanic acid

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phophatidylcholine was systhesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis.Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm^?2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Ionic transport in lipid bilayer membranes.

The current-voltage relationships of model bilayer membranes have been measured in various phospholipid systems, under the influence of both a gradient of potential and an ionic concentration, in order to describe the ion translocation through hydrated transient defects (water channels) across the bilayer formed because of lipid structure fluctuations and induced by temperature. The results have been analyzed in the light of a statistical rate theory for the transport process across a lipid bilayer, recently proposed by Skinner et al. (1993). In order to take into account the observed I-V curves and in particular the deviation from an ohmic behavior observed at high potential values, the original model has been modified, and a new version has been proposed by introducing an additional kinetic process. In this way, a very good agreement with the experimental values has been obtained for all of the systems we have investigated (dimyristoylphosphatidyl ethanolamine bilayers and mixed systems composed by dimyristoylphosphatidyl ethanolamine/dimyristoylphosphatidylcholine mixtures and dimyristoylphosphatidyl ethanolamine/phosphatidic acid dipalmitoyl mixtures). The rate constants governing the reactions at the bilayer interfaces have been evaluated for K+ and Cl- ions, as a function of temperature, from 5 to 35 degrees C and bulk ionic concentrations from 0.02 to 0.2 M. Finally, a comparison between the original model of Skinner and the modified version is presented, and the advantages of this new formulation are briefly discussed.     

Calculation of deformation energies and conformations in lipid membranes containing gramicidin channels.

In this paper we calculate surface conformation and deformation free energy associated with the incorporation of gramicidin channels into phospholipid bilayer membranes. Two types of membranes are considered. One is a relatively thin solvent-free membrane. The other is a thicker solvent-containing membrane. We follow the approach used for the thin membrane case by Huang (1986) in that we use smectic liquid crystal theory to evaluate the free energy associated with distorting the membrane to other than a flat configuration. Our approach is different from Huang, however, in two ways. One is that we include a term for surface tension, which Huang did not. The second is that one of our four boundary conditions for solving the fourth-order differential equation describing the free energy of the surface is different from Huang's. The details of the difference are described in the text. Our results confirm that for thin membranes Huang's neglect of surface tension is appropriate. However, the precise geometrical form that we calculate for the surface of the thin membrane in the region of the gramicidin channel is somewhat different from his. For thicker membranes that have to deform to a greater extent to accommodate the channel, we find that the contribution of surface tension to the total energy in the deformed surface is significant. Computed results for the shape of the deformed surface, the total energy in the deformed surface, and the contributions of different components to the total energy, are presented for the two types of membranes considered. These results may be significant for understanding the mechanisms of dimer formation and breakup, and the access resistance for ions entering gramicidin channels.     

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.     

Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

Phosphatidylcholine exchange between the boundary lipid and bilayer domains in cytochrome oxidase containing membranes.

A phospholipid spin label, 16-doxylphosphatidylcholine, is employed in a study of lipid--protein interactions in cytochrome oxidase containing membranes. Two methods are used to label the membranous cytochrome oxidase: dispersion in cholate with subsequent detergent removal, and fusion with vesicles of the pure phospholipid label in the absence of detergent. A fraction of the label is immobilized, which is calculated to fall in the range of 0.17--0.21 mg of phospholipid/mg of protein (0.15--0.19 after correction for lipids not extracted by chloroform--methanol). This narrow range of values is independent of methods of labeling, protein isolation, and lipid depletion within experimental error. When labeling by fusion is utilized, the patches of pure phosphatidylcholine spin label diffuse in the plane of the bilayer, become diluted, and demonstrate exchange with bound phospholipid. These observations are evidence that boundary lipid, as reflected by the partitioning of the phosphatidylcholine label, is in equilibrium with adjacent bilayer regions and that it consists of a relatively constant amount of phospholipid associated with the hydrophobic portion of the protein.     

Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage

We have employed four lipids in the present study, of which two are cationic and two bear phosphatidylcholine (PC) headgroups. Unlike dipalmitoylphosphatidylcholine, the other lipids employed herein do not have any ester linkage between the hydrocarbon chains and the respective lipid backbones. Small unilamellar vesicles formed from each of the PC and cationic lipids with or without varying amounts of cholesterol have been examined using the steady-state fluorescence anisotropy method as a function of temperature. The anisotropy data clearly indicate that the order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect is similar irrespective of the electrostatic character of the lipid employed. The influence of cholesterol inclusion on multi-lamellar lipid dispersions has also been examined by 1H-nuclear magnetic resonance spectroscopy above the phase transition temperatures. With all the lipids, the line widths of (CH2)n protons of hydrocarbon chains in the NMR spectra respond to the addition of cholesterol to membranes. The influence on the bilayer widths of various lipids upon inclusion of cholesterol was determined from X-ray diffraction studies of the cast films of the lipid-cholesterol coaggregates in water. The effect of cholesterol on the efflux rates of entrapped carboxyfluorescein (CF) from the phospholipid vesicles was determined. Upon incremental incorporation of cholesterol into the phospholipid vesicles, the CF leakage rates were progressively reduced. Independent experiments measuring transmembrane OH- ion permeation rates from cholesterol-doped cationic lipid vesicles using entrapped dye riboflavin also demonstrated that the addition of cholesterol into the cationic lipid vesicles reduced the leakage rates irrespective of lipid molecular structure. It was found that the cholesterol induced changes on the membrane properties such as lipid order, linewidth broadening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3beta-OH of cholesterol. These findings emphasize the importance of hydrophobic interaction between lipid and cholesterol and demonstrate that it is not necessary to explain the observed cholesterol induced effects on the basis of the presence of hydrogen bonding between the 3beta-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region.     

Spontaneous vesicle formation at lipid bilayer membranes.

Unilamellar vesicles are observed to form spontaneously at planar lipid bilayers agitated by exothermic chemical reactions. The membrane-binding reaction between biotin and streptavidin, two strong transmembrane neutralization reactions, and a weak neutralization reaction involving an "antacid" buffer, all lead to spontaneous vesicle formation. This formation is most dramatic when a viscosity differential exists between the two phases bounding the membrane, in which case vesicles appear exclusively in the more viscous phase. A hydrodynamic analysis explains the phenomenon in terms of a membrane flow driven by liberated reaction energy, leading to vesicle formation. These results suggest that energy liberated by intra- and extracellular chemical reactions near or at cell and internal organelle membranes can play an important role in vesicle formation, membrane agitation, or enhanced transmembrane mass transfer.     

Opioid peptide interactions with lipid bilayer membranes.

The interaction of the delta-opioid receptor selective peptides, cyclic [D-Pen2, D-Pen5]-enkephalin [DPDPE] and its acyclic analog, DPDPE(SH)2, with neutral phospholipid bilayer membranes was examined by permeability and calorimetry measurements. The permeabilities were accomplished by entrapping either peptide inside of unilamellar liposomes (composed of a mixture of a molar ratio 65:25:10 phosphatidylcholine/phosphatidylethanolamine/cholesterol) then monitoring the peptide efflux through the bilayer. The initial permeability of DPDPE (first 12 h) averaged over four experiments was (0.91 +/- 0.47).10(-12) cm s-1. In contrast the average permeability of the acylic DPDPE(SH)2 was (4.26 +/- 0.23).10(-12) cm s-1. The effect of these peptides on the phase transition, Tm, of 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by high sensitivity differential scanning calorimetry. The Tm, the calorimetric enthalpy, and the van 't Hoff enthalpy of DPPC were not significantly altered by the presence of DPDPE, whereas the calorimetric data for DPPC with DPDPE(SH)2 showed a small, yet significant, increase (0.2 degrees C) in the Tm with a 30% decrease in the cooperative unit. Both the permeability and calorimetry data reveal a stronger peptide-membrane interaction in the case of the more flexible acyclic peptide.     

Kinetic characteristics of the excitability-inducing material channel in oxidized cholesterol and brain lipid bilayer membranes

The kinetic characteristics of the opening and closing of the excitability-inducing material (EIM) channel in oxidized cholesterol and in brain lipid bilayers are compared. The kinetics of the opening and closing of individual ion-conducting channels in bilayers doped with small amounts of EIM are determined from discrete fluctuations in ionic current. The kinetics for approach to steady-state conductance are determined for lipid bilayers containing many channels. Steady-state and kinetic characteristics for the EIM channel incorporated in brain lipid bilayers can be accounted for by the model developed for the EIM channel incorporated in oxidized cholesterol membranes. Relaxation time, calculated from rate constants of single-channel membranes or directly measured in many-channel membranes is strongly temperature dependent, and is always shorter in brain lipid membranes. Changes in temperature do not affect the interaction of the electric field and the open channel, but the open configuration of the EIM channel in brain lipid bilayers is stablized with increasing temperature. The configurational energy difference between the open and closed channel, calculated from temperature studies, is larger in brain lipid bilayers. The energy barrier which separates the two configurations of the channel is larger in oxidized cholesterol bilayers.