Insulin-induced changes in mechanical characteristics of lipid bilayers modified by liver plasma membrane fragments

Insulin interaction with BLM with incorporated fragments of rat liver plasma membranes, containing hormone receptors, was studied by determining Young modulus of elasticity of bilayer lipid membranes in direction perpendicular to the surface, E. The presence of membrane proteins in a concentration of 60 micrograms.ml-1 induced a significant decrease in parameter E (to approx. 50%) as compared with values obtained in non-modified membranes during insulin action (concentration interval 10(-11)-10(-9) mol.l-1). The extent of the effect was dependent on the initial phase state of the membrane, on cholesterol content in BLM as well as on membrane proteins concentration in lipid bilayer.     

Effects of local anesthetics on mechanical characteristics of lipid bilayers and on the ion transport dynamics.

Bilayer lipid membranes (BLM) of various composition were used to study the effects of local anesthetics (LA) carbisocaine and lidocaine on mechanical membrane characteristics and on the transport dynamics of ions across gramicidin D ionic channels. Carbisocaine concentrations of 20 mumols/l-0.1 mmol/l caused a considerable decrease (by 15-40%) in modulus of elasticity E1 in direction perpendicular to membrane surface. The effect of lidocaine was approx. one order of magnitude weaker. LA-induced changes in E1 were shown to depend on both the lipid composition of the membrane and the electrolyte pH. Neutral forms of LA induce marked changes in E1. An analysis of current-voltage (I-V) characteristics of BLM modified by the channel forming agent gramicidin D revealed that carbisocaine significantly affects the superlinear segment of the I-V relationship; this suggests a strong effect on the transport dynamics of ions through the internal channel region. The results of the study suggest that the action of both carbisocaine and lidocaine may be non-specific. The effectivity of the non-specific action of LA is determined by the hydrophobic moiety of the local anesthetic molecule.     

Optical and electrical properties of thin monoolein lipid bilayers

Summary Monoolein lipid bilayers were formed using a monolayer transfer technique and from dispersions of monoolein in squalene, triolein, 1-chlorodecane and 1-bromodecane. Measurements of optical reflectance and electrical capacitance were used to determine the thickness and dielectric constant of the bilayers. The thickness of the hydrocarbon region of the five bilayer systems ranged from 2.5 to 3.0 nm. Two of the bilayer systems (made from 1-chlorodecane and 1-bromodecane solvents) had a high dielectric constant (2.8 to 2.9) whereas the other bilayer systems had dielectric constants close to that of pure hydrocarbons (2.2). The charge-pulse technique was used to study the transport kinetics of three lipophilic ions and two ion carrier complexes in the bilayers. For the low dielectric constant bilayers, the transport of the lipophilic ions tetraphenylborate, tetraphenylarsonium and dipicrylamine was governed mainly by the thickness of the hydrocarbon region of the bilayer whereas the transport of the ion-carrier complexes proline valinomycin-K⁺ and valinomycin-Rb⁺ was nearly independent of thickness. This is consistent with previous studies on thicker monoolein bilayers. The transport of lipophilic anions across bilayers with a high dielectric constant was 20 to 50 times greater than expected on the basis of thickness alone. This agrees qualitatively with predictions based on Born charging energy calculations. High dielectric constant bilayers were three times more permeable to the proline valinomycin-K⁺ complex than were low dielectric constant bilayers but were just as permeable as low dielectric constant bilayers to the valinomycin-Rb⁺ complex.     

Voltage-dependent conductance induced by alamethicin-phospholipid conjugates in lipid bilayers.

Alamethicin, a linear 20-amino acid antibiotic, forms voltage-dependent channels in lipid bilayer membranes. We show here that alamethicin-phospholipid conjugates can be prepared by photolysis of unilamellar vesicles containing alamethicin and a phosphatidylcholine analogue with a carbene precursor at the end of the C-2 fatty acyl chain. This result indicates that at least a portion of the alamethicin molecule is in contact with the hydrocarbon moiety of the membrane in the absence of an applied voltage. Furthermore, the alamethicin-phospholipid photoproduct is able to induce a voltage-gated conductance similar to that of natural alamethicin. The importance of these results in terms of mechanisms for channel gating is discussed.     

Comparison between orientational and conformational orders in fluid lipid bilayers.

The orientational order as determined by 2H NMR and the infrared frequencies of the C--H stretching modes of the methylene groups have been measured for several systems (POPC, POPC/cholesterol and POPE), all in the fluid phase, and then were compared; this work reveals an unexpected linear correlation between them. This experimental result shows that both measurements are essentially sensitive to a common motion, most likely trans/gauche isomerisation. This new correlation with those already found in the literature suggest that several measurements related to the hydrophobic core of the fluid bilayer describe different aspects of a universal behavior. The correlation presented here does not extend to the lipid in gel phase where slower motions affect the NMR lineshape.     

Water permeability and mechanical strength of polyunsaturated lipid bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21 degrees C ranged from approximately 9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to approximately 5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower approximately 3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21 degrees C varied modestly in a range from approximately 30 to 40 microm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to approximately 50 microm/s for C18:0/2, then strikingly to approximately 90 microm/s for diC18:2 and approximately 150 microm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.     

Morphological alterations in erythrocyte membranes induced by 9-amino-1,2,3,4-tetrahydroacridine and 9-aminoacridine

The effects of 9-amino-1,2,3,4-tetrahydroacridine (THA) and its fully aromatic analogue 9-aminoacridine (9-AA) on erythrocyte membrane morphology were investigated via scanning electron microscopy. The ghost population was categorized into four distinct classes and alterations in the relative amounts of these populations with drug addition were noted. The samples incubated in 9-AA had a significantly higher (p less than 0.001) flat, two-dimensional cell population. This shift in morphology may be attributable to the unwinding of spectrin and the subsequent collapse of the membrane.     

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.     

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.     

Helix-helix interactions in lipid bilayers.

Using a continuum model, we calculated the electrostatic interaction free energy between two alpha-helices in three environments: the aqueous phase, a low dielectric alkane phase, and a simple representation of a lipid bilayer. As was found in previous work, helix-helix interactions in the aqueous phase are quite weak, because of solvent screening, and slightly repulsive, because of desolvation effects that accompany helix assembly. In contrast, the interactions can be quite strong in a hypothetical alkane phase because desolvation effects are essentially nonexistent and because helix-helix interactions are not well screened. In this type of environment, the antiparallel helix orientation is strongly favored over the parallel orientation. In previous work we found that the free energy penalty associated with burying helix termini in a bilayer is quite high, which is why the termini tend to protrude into the solvent. Under these conditions the electrostatic interaction is strongly screened by solvent; indeed, it is sufficient for the termini to protrude a few angstroms from the two surfaces of the bilayer for their interaction to diminish almost completely. The effect is consistent with the classical model of the helix dipole in which the dipole moment is represented by point charges located at either terminus. Our results suggest, in agreement with previous models, that there is no significant nonspecific driving force for helix aggregation and, hence, that membrane protein folding must be driven by specific interactions such as close packing and salt-bridge and hydrogen bond formation.     

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.     

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.     

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of the electrical double layer in the quenching of 9-aminoacridine fluorescence by negatively charged surfaces.

The addition of 9-aminoacridine monohydrochloride to carboxymethyl-cellulose particles or azolectin liposomes suspended in a low cation medium results in a quenching of its fluorescence. This quenching can be released on the addition of cations. The effectiveness of cations is related only to their valency in the series of salts tested, being monovalent less than divalent less than trivalent, and is independent of the associated anions. These results indicate an electrical rather than a chemical effect, and the relative effectiveness of the various cations can be predicted by the application of classical electrical double layer theory. Fluorescence quenching can also be released on protonation of the fixed negatively charged ionisable groups, and the quenching release curve follows the ionisation curve of these groups. We postulate that when 9-aminoacridine molecules are in the electrical diffuse layer adjacent to the charged surface their fluorescence is quenched, probably due to aggregate formation. As cations are added the 9-aminoacridine concentration at the surface falls as it is displaced into the bulk solution, where it shows a high fluorescence yield with a fluorescence lifetime of 16.3 ns. The fluorescence quenching is associated with an absorbance decrease, which is pronounced with carboxymethyl-cellulose particles and can probably be attributed to self-shielding. The negative charges carried by lipoprotein membranes are primarily due to carboxyl and phosphate groups. Therefore these results with carboxymethyl-cellulose (carboxyl) and azolectin (phosphate) support our earlier suggestion that 9-aminoacridine may be used to probe the electrical double layer associated with negatively charged biological membranes.     

Conductance transition induced by an electric field in lipid bilayers

A cooperative phenomenon showing a structural change in the organization of bilayer lipid membranes at a critical value of the applied electric field is presented. The transition is characterized by a sharp increase in conductance. The phenomenon can be observed under current-clamp conditions (rather than the usual voltage-clamp conditions) to avoid rupturing the membrane. At a critical potential value the conductance increases and therefore the potential decreases to keep the current constant. Results refer to membranes made of egg phosphatidylcholine (PC), diphytanoylphosphatidylcholine and cholesterol/egg PC. It is found that the critical potential at which the transition occurs depends dramatically on pH and ionic concentration, indicating that the electrical properties of the external surface determine the major characteristics of such a transition.     

Direct measurement of the mechanical properties of lipid phases in supported bilayers

Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ~200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (k(A)) as 106 pN/nm and 199 pN/nm and the bending stiffness (k(c)) as 18 k(B)T and 57 k(B)T for the liquid and gel phases, respectively.     

Genetic analysis of clear-plaque mutations induced in bacteriophage lambda by 9-aminoacridine

Clear-plaque mutations were induced in the cI and cII genes of λ by treating lysogenic cells with 9-aminoacridine (9AA). Mapping of the mutations revealed that there were two hot spots for 9AA mutagenesis in cI, and one strong hot spot in cII. The hot spots in cI mapped close to 1 of the 3 runs of 4 G/C base-pairs and near the only run of 5 G/Cs, respectively, in this gene. Of 36 cI mutations tested, at most one mapped near a run of 6 A/T base-pairs. By analogy, the sequence responsible for the strong hot spot in cII may be the run of 6 G/Cs in this gene.     

Effect of retinol and retinoic acid on permeability,electrical resistance and phase transition of lipid bilayers

Retinol and retinoic acid have been incorporated into the artificial membrane systems, planar bimolecular lipid membranes and liposomes, and their effects on several membrane parameters have been measured. 1. Retinol and retinoic acid increased the permeability of egg lecithin liposomes to K⁺, I^? and glucose when incorporated into the membranes at levels as low as 0.5 membrane mol%. Retinoic acid influenced permeability more than did retinol for each of the solutes tested. 2. Retinol and retinoic acid both decreased the electrical resistance of egg lecithin-planar bimolecular lipid membranes from 0.5 to 8 membrane mol%. Retinoic acid effected a larger change than did retinol. 3. Retinol and retinoic acid increased the permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine liposomes to water at 1.0 and 3.0 membrane mol%. A larger effect on water permeability was measured for retinoic acid than for retinol. 4. Retinol and retinoic acid at 1.0 and 3.0 membrane mol% were shown to lower the phase-transition temperature of liposomes composed of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. Phase-transition temperatures were monitored by abrupt changes in water permeability and liposome size associated with the transition. Retinoic acid lowered the phase-transition temperature of dimyristoylphosphatidylcholine liposomes more than did retinol, while both retinoids had almost the same effect on dipalmitoylphosphatidylcholine liposomes.     

delta pH-induced fluorescence quenching of 9-aminoacridine in lipid vesicles is due to excimer formation at the membrane.

The fluorescence of 9-aminoacridine (9-AA) is quenched in vesicular suspensions containing negatively charged lipid headgroups (e.g., phosphatidylserine) upon imposition of a transmembrane (inside acidic) pH-gradient. It is shown that this fluorescence loss is accompanied by the formation of 9-AA dimers that undergo a transition in the dimer excited state to a dimer-excimer state. This result has been obtained on the basis of the specific dimer fluorescence excitation and hypochromic absorbance spectra that are redshifted by maximally 275 cm-1 (4.4 nm) with respect to the corresponding monomer spectra, as well as by the detection of the characteristic broad excimer emission band, centered at 560 nm. The existence of the spectrally distinct dimer-excimer is further corroborated by fluorescence life-time measurements that indicate an increased lifetime of up to 24 ns for this complex as compared with the normal monomer fluorescence lifetime of 16 ns. The formation of this dimer-excimer complex from the monomers can be reversed completely and the original monomeric spectral properties restored after the abolishment of the electrochemical proton gradient. In addition to the delta pH-induced dimer redshift in absorbance and fluorescence excitation, a further small redshift in monomer absorbance, fluorescence excitation, and emission spectra is observed due solely to the presence of the negatively charged phospholipid headgroups.