Water permeability of bilayer lipid membranes: Sterol-lipid interaction

Summary Bilayer lipid membranes were generated in an aqueous medium from synthetic, egg or plant phosphatidyl choline (PC) or from plant monogalactosyl diglyceride (MG). The water permeability of the black membranes was determined by measuring the net volume flux produced by a NaCl gradient. The osmotic permeability coefficient,P _os, was markedly affected by the number of double bonds in the fatty acid conjugates of the lipids: the greater the degree of unsaturation, the higher the value ofP _os. The temperature dependence ofP _os of the lipid membranes was studied over a range of 29 to 40°C. The experimental activation energy,E _a , estimated from the linear plots of log (P _os)versus 1/T, was significantly higher for MG membranes (17 kcal/mole) than for the various PC membranes (11 to 13 kcal/mole), probably owing to hydrogen bonding between MG and water molecules. In comparison with PC membranes, the membranes generated from PC and cholesterol (11 molar ratio) had lowerP _os but similarE _a values. Likewise, either stigmasterol or -sitosterol decreasedP _os of MG membranes, whileE _a was not affected by the sterols. MG-cholesterol membranes were specifically characterized by a unique value ofE _a (–36 kcal/mole) thus indicating temperature dependent structural changes.     

A barrier model for current flow in lipid bilayer membranes

Summary Theshape of the energy barrier inside thin, insulating membranes can be an important factor in determining the detailed behavior of transmembrane ionic flows. In particular, a model is developed in which the shape of the barrier is expected to have direct influence on such experimentally important membrane properties as: (a) the shape of the current-voltage relation; (b) the dependence of zero current conductivity on asymmetric concentrations; (c) the dependence of the rectification ratio on the concentration ratio.Current-voltage curves were measured for a wide range of symmetrical and asymmetrical concentrations in black lipid (phosphatidyl ethanolamine) films in the presence of nonactin and potassium. A single barrier shape was found to describe accurately the experimental results in terms of the model.     

Lipid content and lipid type as determinants of the epidermal permeability barrier

During terminal differentiation, mammalian epidermal lipids undergo striking changes in both composition and distribution. Phospholipids and neutral lipids are replaced by a mixture of ceramides and neutral lipids organized in intercellular lamellar bilayers. Whether all of these lipids and/or whether specific lipid classes regulate permeability barrier function is not known. When hairless mice were treated with acetone, the degree of barrier perturbation (measured as transepidermal water loss, TEWL) increased linearly with the amount of lipid removed. Moreover, virtually all lipid species appeared to be removed by acetone treatment. In contrast, the nonpolar organic solvent, petroleum ether, while removing greater amounts of lipids, provoked lesser barrier abnormalities. As determined by both quantitative thin-layer chromatography and histochemistry, petroleum ether selectively extracted nonpolar lipids leaving sphingolipids and free sterols in place. In petroleum ether-treated animals, subsequent acetone treatment removed additional sphingolipids and produced a dramatic increase in TEWL. A linear relationship existed for the quantities of sphingolipid removed and degree of barrier disruption in acetone-treated, but not petroleum ether-treated animals. These results support a relationship between the total lipid content of the stratum corneum and barrier function. Secondly, although the results demonstrate the participation of the total lipid mixture in the barrier, removal of nonpolar species alone appears to cause only a modest level of barrier disruption, while removal of sphingolipids and free sterols leads to a more profound level of barrier perturbation.     

Differential effects induced by α- and β-endosulfan in lipid bilayer organization are reflected in proton permeability

The effects of two insecticides isomers, α- and β-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 °C, the global kinetic constant (K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of α-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 °C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for α-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, α-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to α-endosulfan, β-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all β-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 °C, are similar to those described for DPPC. The β-isomer induced a very small effect, and α-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of α- and β-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of α-endosulfan in vivo, contributing to its higher degree of toxicity as compared with β-endosulfan.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.     

Electroporation of a lipid bilayer as a chemical reaction

When a cell's transmembrane potential is increased from a physiological one to more than 370 mV, the transmembrane current increases more than hundredfold within a millisecond. This is due to the formation of conductive pores in the membrane. We construct a model in which we conceive of pore formation as a voltage sensitive chemical reaction. The model predicts the logarithm of the pore formation rate to increase proportionally to the square of the voltage. We measure currents through frog muscle cell membranes under 8 ms pulses of up to 440 mV. The experimental data appear consistent with the model.     

Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. We now show that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. We find that the integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs we show that wun function in the trachea is distinct from its role in germ cell migration.     

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-. microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform     

Structural rearrangements induced by glycerol increase the permeability of bilayer lipid membranes for amphotericin

It has been shown that structural rearrangements induced by glycerol in bilayer lipid membranes (BLM) containing cholesterol facilitate the transmembrane transport of amphotericin B molecules in the direction of glycerol gradient. The addition of amphotericin B to the same side with glycerol results in a change in bilayer selectivity from the cation to the anion one. Besides, the final conductivity is blocked by tetraethylammonium from the solution with no amphotericin B added. It testifies to the transport of amphotericin molecules to the opposite side of the membrane. The transport effect depends on the cholesterol content in bilayer, ionic strength of the medium and slightly depends on temperature. It is concluded that transport of amphotericin B in such conditions differs from the diffusive one and is due to the formation of intermediate lipid phases in the course of structural rearrangements of bilayers.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 degrees C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Effect of peptide PV on the ionic permeability of lipid bilayer membranes

This paper reports the effects of peptide PV (primary structure: cyclo-(D-val-L-pro-L-val-D-pro)_δ) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (G_m) induced by PV in either Na⁺ or K⁺ medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in G_m in K⁺ medium is about 10⁴ times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K⁺ ≳ Rb⁺ > Cs⁺ > NH₄ ⁺ > TI⁺ > Na⁺ > Li⁺; the ratio of GGm in K⁺ to that in Na⁺ is about 10 for PV compared to > 10³ for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na⁺ or K⁺ salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10^–5 M. From a model which assumes that V_m equals the equilibrium potential for the PV-cation complexes (MS ⁺) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10^–5 cm s^–1, which is about 10^–7 times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of V_m are in agreement with the calculated equilibrium potential for MS⁺ over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS⁺ concentrations at the membrane surfaces.     

Thermodynamic factors determining the permeability barrier properties of lipid bilayers

The permeability barrier properties of lipid bilayers are usually determined by the rate of swelling of multilamellar liposomes or by the exchange of radioactively labeled molecules in sonicated vesicles. The values reported in the literature for the permeability of water and non electrolytes differ according to which method is applied in their determination. In addition, drastic assumptions (i.e. homogeneity of the membrane) are commonly introduced for the interpretation of the phenomenological permeability coefficients. This paper discusses the permeability coefficient considering the departures from the ideality of the membrane system. The non ideal terms can be put in function of measurable quantities such as the excluded volume of the membrane and the hydration degree of the lipid molecules. By means of this formalism it is possible to explain quantitatively the experimental values found for the permeability coefficient of water in sonicated vesicles below and above the phase transition temperature. In addition, different magnitudes of the energies of activation for the permeation of non electrolytes have been found depending on if the liposomes are dispersed in isotonic or hipertonic solutions of a permeant. The formalism described allows to explain such differences in terms of the influence of the solute concentration on the density of the lipid membrane. The reasons for which the simple formalism for homogeneous membranes can not be applied to lipid membranes are discussed in detail.     

Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism

Many of the ichthyoses are associated with inherited disorders of lipid metabolism. These disorders have provided unique models to dissect physiologic processes in normal epidermis and the pathophysiology of more common scaling conditions. In most of these disorders, a permeability barrier abnormality "drives" pathophysiology through stimulation of epidermal hyperplasia. Among primary abnormalities of nonpolar lipid metabolism, triglyceride accumulation in neutral lipid storage disease as a result of a lipase mutation provokes a barrier abnormality via lamellar/nonlamellar phase separation within the extracellular matrix of the stratum corneum (SC). Similar mechanisms account for the barrier abnormalities (and subsequent ichthyosis) in inherited disorders of polar lipid metabolism. For example, in recessive X-linked ichthyosis (RXLI), cholesterol sulfate (CSO(4)) accumulation also produces a permeability barrier defect through lamellar/nonlamellar phase separation. However, in RXLI, the desquamation abnormality is in part attributable to the plurifunctional roles of CSO(4) as a regulator of both epidermal differentiation and corneodesmosome degradation. Phase separation also occurs in type II Gaucher disease (GD; from accumulation of glucosylceramides as a result of to beta-glucocerebrosidase deficiency). Finally, failure to assemble both lipids and desquamatory enzymes into nascent epidermal lamellar bodies (LBs) accounts for both the permeability barrier and desquamation abnormalities in Harlequin ichthyosis (HI). The barrier abnormality provokes the clinical phenotype in these disorders not only by stimulating epidermal proliferation, but also by inducing inflammation.