Effects of ethanol on lipid bilayers with and without cholesterol: the distearoylphosphatidylcholine system

Differential scanning calorimetry (DSC) and fluorescence spectroscopy are useful techniques for investigating the phase transitions of phospholipid bilayers. In this study, these methods have been extended to determine the effects of ethanol on DSPC and DSPC/2 mol.% cholesterol bilayers. The biphasic effect of the main transition was observed on the DSC heating scans above 0.60 M ethanol. In addition, the concentration at which the biphasic effect occurs is not significantly changed in the presence of 2 mol.% cholesterol. For the fluorescence studies, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into the bilayer to monitor the phase transitions through the displacement of DPH. This fluorescent probe is used to directly determine the onset of interdigitation in the bilayer systems as indicated by a large decrease in the DPH fluorescence intensity. The addition of cholesterol lowered and broadened the transition temperatures of the phosphatidylcholine (PC) system. However, 2 mol.% cholesterol did not have a significant effect on the induction of the interdigitated phase in DSPC as observed from the small difference in ethanol threshold concentration for the two systems. This suggests that DSPC forms a more stable interdigitated gel phase than other PCs with shorter acyl chains.     

Effects of ethanol and methanol on lipid metabolism in Bacillus subtilis

In Bacillus subtilis, the fatty acid moiety of the phospholipids was affected differently during growth in the presence of 1.1 M-methanol or 0.7 M-ethanol, though at these concentrations methanol and ethanol had the same effects on growth rate and completely inhibited sporulation. Synthesis of phosphatidylglycerol was also strongly inhibited and the amount of total cell phospholipids was reduced by 50% by both alcohols. The composition of fatty acids, especially the relative concentration of 12-methyltetradecanoic acid, was modified only by ethanol; in bacteria grown in the presence of methanol, changes in fatty acid composition were negligible. In non-sporulating mutants, synthesis of phosphatidylglycerol was much less affected than in the wild-type and synthesis of phosphatidylethanolamine was increased. In these strains, fatty acid composition was also modified by ethanol but unaffected by methanol.     

Cholesterol and lipid phases influence the interactions between serotonin receptor agonists and lipid bilayers

Solid state NMR techniques have been used to investigate the effect that two serotonin receptor 1a agonists (quipazine and LY-165,163) have on the phase behavior of, and interactions within, cholesterol/phosphocholine lipid bilayers. The presence of agonist, and particularly LY-165,163, appears to widen the phase transitions, an effect that is much more pronounced in the presence of cholesterol. It was found that both agonists locate close to the cholesterol, and their interactions with the lipids are modulated by the lipid phases. As the membrane condenses into mixed liquid-ordered/disordered phases, quipazine is pushed up toward the surface of the bilayer, whereas LY-165,163 moves deeper into the lipid chain region. In light of our results, we discuss the role of lipid/drug interactions on drug efficacy.     

The effect of temperature on lipid-n-alkane interactions in lipid bilayers

The relationship between age-related alterations in the lipid composition of cultured rat-heart fibroblasts and several biochemical and biophysical parameters was investigated. Aged (14-15-day-old) cultures displayed higher mole ratios of sphingomyelin to phosphatidylcholine, as well as elevated cholesterol levels. A concomitant increase was observed in the total protein content of the cells and in the Vmax values of both membranal and cytoplasmic marker enzymes. Fluorescence photobleaching recovery was employed to study the lateral mobility of the lipid probe NBD-phosphatidylethanolamine and of membrane glycoproteins that bind succinylated concanavalin A. The mobile fractions of both probes were higher in aged cultures, while the lateral diffusion coefficients were lower. To further demonstrate the dependence of the above parameters on the cellular lipid composition, we have manipulated the lipid composition of old cultures by treatments with liposomes (small unilamellar vesicles) of specific compositions. Treatments which reversed the lipid composition towards that of young (5-6-day-old) cultures caused a concomitant reversal of the measured biochemical and biophysical parameters to the values observed in young cultures. These findings suggest that alterations in the organization and mobility of cell membrane constituents are involved in mediating changes in cellular functions. In view of our previous findings on cultures of rat-heart myocytes (Yechiel, E., Barenholz, Y. and Henis, Y.I. (1985) J. Biol. Chem. 260, 9132-9136), it appears that the modulation of cellular properties through the membrane lipid composition may be a general phenomenon in many cell types.     

The condensing effect of cholesterol in lipid bilayers

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases nonlinearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol complexes. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.     

Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines the behavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the results in the context of the developed theories for DFS. We found that experiments performed at different temperatures and with different cantilever spring constants enabled a more effective comparison of experimental data with theory in comparison with previously published data.     

The influence of n-alkanols on the capacity per unit area of planar lipid bilayers

The 1000-1300 cm-1 region of the infrared spectrum of dipalmitoylphosphatidylcholine (DPPC) and other phosphate-containing molecules has been studied by the Fourier-transform technique. Three absorption bands have been assigned to various vibrational modes of the DPPC phosphate group, with maximum wavenumbers at 1060, 1086 and 1222 cm-1. These values are the same above and below Tc of the phospholipid. Dehydration produces band-shifts toward higher wavenumbers .     

Interaction of protegrin-1 with lipid bilayers: membrane thinning effect

Protegrins (PG) are important in defending host tissues, preventing infection via an attack on the membrane surface of invading microorganisms. Protegrins have powerful antibiotic abilities, but the molecular-level mechanisms underlying the interactions of their beta-sheet motifs with the membrane are not known. Protegrin-1 (PG-1) is composed of 18 amino acids with a high content of basic residues and two disulfide bonds. Here we focused on the stability of PG-1 at the amphipathic interface in lipid bilayers and on the details of the peptide-membrane interactions. We simulated all-atom models of the PG-1 monomer with explicit water and lipid bilayers composed of both homogeneous POPC (palmitoyl-oleyl-phosphatidylcholine) lipids and a mixture of POPC/POPG (palmitoyl-oleyl-phosphatidylglycerol) (4:1) lipids. We observed that local thinning of the lipid bilayers mediated by the peptide is enhanced in the lipid bilayer containing POPG, consistent with experimental results of selective membrane targeting. The beta-hairpin motif of PG-1 is conserved in both lipid settings, whereas it is highly bent in aqueous solution. The conformational dynamics of PG-1, especially the highly charged beta-hairpin turn region, are found to be mostly responsible for disturbing the membrane. Even though the eventual membrane disruption requires PG-1 oligomers, our simulations clearly show the first step of the monomeric effects. The thinning effects in the bilayer should relate to pore/channel formation in the lipid bilayer and thus be responsible for further defects in the membrane caused by oligomer.     

Effect of benzyl alcohol on lipid bilayers. A comparisons of bilayer systems.

The effect of the small anesthetic molecule, benzyl alcohol, on the structure of various bilayer system has been studied by optical, electrical, and x-ray diffraction techniques. We find that the modifications in bilayer thickness caused by benzyl alcohol differ dramatically for planar (or black lipid) bilayers containing solvent, planar bilayers containing little or no solvent, and vesicular bilayers. Benzyl alcohol increases the thickness of planar bilayers containing n-alkane solvents, yet decreases the thickness of "solvent-free" planar bilayers. The effect of benzyl alcohol on vesicular bilayers below the phase transition temperature also depends on whether solvent is present in the bilayers. Without solvent, gel-state bilayers are reduced in thickness by benzyl alcohol, whereas in the presence of solvent, the thickness is unchanged. Above the phase transition temperature, benzyl alcohol has no measurable effect on vesicular bilayer thickness, whether solvent is present or not. These results indicate that different model membrane systems respond quite differently to a particular anesthetic.     

The influence of ethanol on lipid metabolism in mouse tissues

• 1.1. The double isotope ratios and specific radioactivities of individual lipid fractions in major tissues of mice have been determined before and after ethanol ingestion.
• 2.2. Several significant alterations in these ratios were caused by this treatment, with marked increases in most liver lipids and diverse responses in other tissues.
• 3.3. These data establish that ethanol ingestion causes widespread perturbations in tissue lipid metabolism, with the main emphasis being directed towards an increase in the rate of synthesis of the liver lipid classes.
• 4.4. The data also provides indications of an increase in the peroxisomal oxidation of fatty acids following ethanol ingestion, with the different distribution of ethanol metabolizing systems giving rise to individual metabolic responses in the separate tissues.
• 5.5. These results have been discussed in relation to the eatablished involvements of lipid metabolism and tissue interactions in the whole animal.

     

The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

The influence of the trichorzianin C-terminal residues on the ion channel conductance in lipid bilayers

Four natural trichorzianin analogues, channel-forming peptaibols, differing in their C-terminal residues (Gln or Glu, Trpol or Pheol) were tested for their macroscopic and single-channel conductances in planar lipid bilayers. The results indicate that, as regards to the voltage threshold, the most efficient analogue is the charged Trpol-bearing one. In addition, Trpol brings about a drastic lengthening of the open channel life-times. This behaviour is attributed to the dipole moment of the end residues and to the bulkiness and hydrogen bonding ability of Trpol.     

The influence of 1-alkanols and external pressure on the lateral pressure profiles of lipid bilayers

The suggestion by Robert Cantor, that drug-induced pressure changes in lipid bilayers can change the conformational equilibrium between open and closed states of membrane proteins and thereby cause anesthesia, attracted much attention lately. Here, we studied the effect of both large external pressure and of 1-alkanols of different chain lengths—some of them anesthetics, others not—on the lateral pressure profiles across dimyristoylphosphatidylcholine (DMPC) bilayers by molecular dynamics simulations. For a pure DMPC bilayer, high pressure both reduced and broadened the tension at the interface hydrophobic/hydrophilic and diminished the repulsion between the phospholipid headgroups. Whereas the effect of ethanol on the lateral pressure profile was similar to the effect of a large external pressure on a DMPC bilayer, long-chain 1-alkanols significantly amplified local maxima and minima in the lateral pressure profile. For most 1-alkanols, external pressure had moderate effects and did not reverse the changes 1-alkanols exerted on the pressure profile. Nevertheless, assuming the bent helix model as a simple geometric model for the transmembrane region of a membrane protein, protein conformational equilibria were shifted in opposite directions by addition of 1-alkanols and additional application of external pressure.     

The effect of lanthanum on alamethicin channels in black lipid bilayers

The properties of alamethicin channels in dioleyl phosphatidylcholine bilayers were studied in 1 M LaCl3 and were compared with those in 1 M NaCl. Single-channel recordings demonstrated that the mean single-channel life-time is about 0.25 s in NaCl but only about 17 ms in LaCl3. Whereas in NaCl the conductance levels 2 and 3 are mostly populated, in LaCl3 the levels 0 and 1 are preferentially adopted. The single-level conductance are slightly smaller in LaCl3 if the higher bulk solution conductivity of LaCl3 is taken into account. Multipore experiments confirmed earlier results (Boheim, G., Irmscher, G. and Jung, G. (1978) Biochim. Biophys. Acta 507, 485--506) that the bilayer conductance is less strongly dependent on voltage in LaCl3 than in NaCl solution. Current-fluctuation analysis showed that this effect can be explained by a less strong dependence on voltage of the pore-formation rate as well as of the mean channel life-time in LaCl3. The data can be interpreted as an increased lateral diffusion mobility of the alamethicin monomers in the bilayer. This can be the result of the binding of La3+ to the polar headgroups which can induce cluster formation of the phospholipids.     

Actin and amphiphilic polymers influence on channel formation by Syringomycin E in lipid bilayers

The bacterial lipodepsipeptide syringomycin E (SRE) added to one (cis-) side of bilayer lipid membrane forms voltage dependent ion channels. It was found that G-actin increased the SRE-induced membrane conductance due to formation of additional SRE-channels only in the case when actin and SRE were applied to opposite sides of a lipid bilayer. The time course of conductance relaxation depended on the sequence of SRE and actin addition, suggesting that actin binds to the lipid bilayer and binding is a limiting step for SRE-channel formation. G-actin adsorption on the membrane was irreversible. The amphiphilic polymers, Konig’s polyanion (KP) and poly(Lys, Trp) (PLT) produced the actin-like effect. It was shown that the increase in the SRE membrane activity was due to hydrophobic interactions between the adsorbing molecules and membrane. Nevertheless, hydrophobic interactions were not sufficient for the increase of SRE channel-forming activity. The dependence of the number of SRE-channels on the concentration of adsorbing species gave an S-shaped curve indicating cooperative adsorption of the species. Kinetic analysis of SRE-channel number growth led to the conclusion that the actin, KP, and PLT molecules form aggregates (domains) on the trans-monolayer. It is suggested that an excess of SRE-channel formation occurs within the regions of the cis-monolayer adjacent to the domains of the adsorbed molecules, which increase the effective concentration of SRE-channel precursors.     

Surface changes induced by osmotic stress and its influence on the glycerol permeability in lipid bilayers

The penetration rate of glycerol across lipid bilayers can be assayed dispersing liposomes filled with a 0.1 M glucose solution in an isotonic or a hypertonic solution of glycerol. The kinetic of glycerol permeation is found to be different in each of those cases. Liposomes dispersed above the phase transition temperature in hypertonic solutions show an increase in the surface polarization as measured by means of merocyanine 540. Under this condition, the permeation of glycerol shows a two-step kinetic which is indicative of a non-fickean diffusion process. In contrast, liposomes dispersed in isotonic solutions of the permeant show a fickean behavior. The changes in polarization of the membrane interface are ascribed to variations in the surface potential due to the osmotic collapse and the glycerol concentration in contact with the outer surface. The permeability of polar molecules can, in consequence, be considered as a function of the surface potential of the liposome which is congruent with previous data in literature reporting that water permeability increases as a function of the zeta potential of liposomes shrunken in hypertonic solutions.     

State of water and its diffusion across lipid bilayers: effect of hydration degree

The state of adsorbed water (estimated from the dependence of the shape of the 1H NMR spectrum on the angle between the normal to the bilayers and the direction of the magnetic field) and the diffusion of water molecules in the direction of the normal to the bilayers (estimated by 1H NMR spectroscopy with the impulse gradient of magnetic field) in microscopically oriented dioleoylphosphatidylcholine bilayers have been studied depending on hydration. The dependences of the shape of the NMR spectrum on angle differ qualitatively only at concentrations of water greater and less than the concentration that is achieved upon hydration from saturated vapors chi(eq) (about 23 weight %). At concentrations below chi(eq), all water present in samples enters the hydrate shells of polar "heads" of lipids or is in the state of "rapid exchange" with the water of hydrate shells, with the result that the signal of spin echo for water is observed only in a narrow range of angles close to the "magic angle", 54 degrees C. At concentrations above xhi(eq), the signal of spin echo for water is retained at all orientations, indicating probably that part of water between the bilayers ("quasi-free water") is in the state of a "slow exchange" with water "bound" to polar "heads". It was found that the coefficient of self-diffusion of water across the system of bilayers inversely depends on the degree of hydration, which is described in the Tanner model with consideration of the self-diffusion of water molecules in the hydrophobic moiety of the bilayer. The permeability of the bilayer, the coefficient of distribution of molecules between the water and lipid phases, and the coefficient of self-diffusion of water in the hydrophobic moiety of the bilayer were estimated.     

Effect of the cosolutes trehalose and methanol on the equilibrium and phase-transition properties of glycerol-monopalmitate lipid bilayers investigated using molecular dynamics simulations

The influence of the cosolutes trehalose and methanol on the structural, dynamic and thermodynamic properties of a glycerol-1-monopalmitate (GMP) bilayer and on its main transition temperature \(T_m\) is investigated using atomistic molecular dynamics simulations (600 ns) of a GMP bilayer patch (2 × 8 × 8 lipids) at different temperatures in the range of 302 to 338 K and considering three different cosolute concentrations. Depending on the environment and temperature, these simulations present no or a single GL \(\rightarrow \) LC, LC \(\rightarrow \) GL or LC \(\rightarrow \) ID transition, where LC, GL and ID are the liquid crystal, gel and interdigitated phases, respectively. The trehalose molecules form a coating layer at the bilayer surface, promote the hydrogen-bonded bridging of the lipid headgroups, preserve the interaction of the headgroups with trapped water and induce a slight lateral expansion of the bilayer in the LC phase, observations that may have implications for the phenomenon of anhydrobiosis. However, this cosolute does not affect \(T_m\) and its dependence on hydration in the concentration range considered. On the other hand, methanol molecules intercalate between the lipid headgroups, promote a lateral expansion of the bilayer in the LC phase and induce a concentration dependent decrease of \(T_m\) , observations that may have implications for the phenomenon of anesthesia. The occurrence of an ID phase in the presence of this cosolute may be viewed as an extreme consequence of lateral expansion. The analysis of the simulations also suggests the existence of two basic conservation principles: (1) the hydrogen-bond saturation principle rests on the observation that for all species present in the different systems, the total numbers of hydrogen-bonds per molecule is essentially constant, the only factor of variability being their distribution among different partners; (2) the densest packing principle rests on the observation that the effective volume per methylene group in the interior of the bilayer is only weakly sensitive to the environment, with values comparable to those for liquid (LC) and solid (ID) alkanes, or intermediate (GL).