Permeability Of Bilayer Lipid Membranes Research Articles

Deformation of giant unilamellar vesicles under osmotic stress.

Biological membrane plays an important role in maintaining an osmotic equilibrium between the cytoplasm and the extracellular solution of cells. Here, the giant unilamellar vesicles (GUVs) as cell models were used to investigate the effect of osmotic stress on phospholipid membranes. The deformation of GUVs, including inward budding and outward budding, was systematically investigated by the osmotic press from glucose, sucrose, LiCl, and KCl solutions. The permeability (P) of DMPC, DMPC/10 mol% Chol GUVs, DMPC/25 mol% Chol GUVs, and DMPC/40 mol% Chol GUVs in glucose, sucrose, LiCl, and KCl solutions were all obtained. The P value decreases with the addition of more cholesterol in the bilayer. The monovalent cations caused higher permeability of lipid bilayer membranes due to their combination with phospholipids. The molar flux of water (J) value was found to be the key factor for determining the deformation state from mainly inward budding to mainly outward budding. The findings in this paper may help us to understand cell transformation triggered with osmotic stress.

Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells

Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this "acid burst" were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress.

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.

Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition

The enhanced permeability of lipid bilayer membranes at their gel-to-liquid phase transition has been explained using a “bilayer lipid heterogeneity” model, postulating leaky interfacial regions between still solid and melting liquid phases. The addition of lysolipid to dipalmitoylphosphatidylcholine bilayers dramatically enhances the amount of, and speed at which, encapsulated markers or drugs are released at this, already leaky, phase transition through these interfacial regions. To characterize and attempt to determine the mechanism behind lysolipid-generated permeability enhancement, dithionite permeability and doxorubicin release were measured for lysolipid and non-lysolipid, containing membranes. Rapid release of contents from lysolipid-containing membranes appears to occur through lysolipid-stabilized pores rather than a simple enhancement due to increased drug solubility in the bilayer. A dramatic enhancement in the permeability rate constant begins about two degrees below the calorimetric peak of the thermal transition, and extends several degrees past it. The maximum permeability rate constant coincides exactly with this calorimetric peak. Although some lysolipid desorption from liquid state membranes cannot be dismissed, dialyzation above T m and mass spectrometry analysis indicate lysolipid must, and can, remain in the membrane for the permeability enhancement, presumably as lysolipid stabilized pores in the grain boundary regions of the partially melted solid phase.

Permeability of the Nuclear Envelope at IsolatedXenopusOocyte Nuclei Studied by Scanning Electrochemical Microscopy

In interphase eukaryotic cells, molecular transport between the cytoplasm and the nucleus is mediated by the nuclear pore complex (NPC), which perforates the double-membraned nuclear envelope (NE). Local permeability of the NE at large intact nuclei (approximately 400 microm in diameter) isolated from Xenopus laevis oocytes was studied by scanning electrochemical microscopy (SECM). Steady-state tip current versus tip-nucleus distance curves (approach curves) were measured with 10- and 2-microm-diameter Pt disk microelectrodes at the nuclei in isotonic buffer solutions containing redox-active molecules. The approach curves in the normalized form are independent of the tip diameter, indicating diffusion-limited membrane transport of the redox molecules. SECM chronoamperometry demonstrated that a decrease in the steady-state tip current at short tip-nucleus distances is due to smaller diffusion coefficients and concentrations of the redox molecules in the nucleus than those in the buffer solution. The experimental approach curves fit very well with theoretical ones for freely permeable membranes, yielding the NE permeability to the molecules that is at least 2 orders of magnitude larger than permeability of bilayer lipid membranes and cell membranes. This result indicates that passive transport of the redox molecules across the NE is facilitated by open NPC pores. The flux of the redox molecules sustainable by a single NPC channel (>9.8 x 10(6) molecules per NPC per second) and the diameter of the channel pore (>15 nm) were estimated from the SECM data by assuming the NE as an array of nanometer-sized NPC pores. The effects of the redox molecules on the nucleus and the NPC function were examined by studying signal-mediated nuclear import of rhodamine-labeled bovine serum albumin with and without nuclear localization signals by fluorescence microscopy.

Theoretical analysis of localized heating in human skin subjected to high voltage pulses

Electroporation, the increase in the permeability of bilayer lipid membranes by the application of high voltage pulses, has the potential to serve as a mechanism for transdermal drug delivery. However, the associated current flow through the skin will increase the skin temperature and might affect nearby epidermal cells, lipid structure or even transported therapeutic molecules. Here, thermal conduction and thermal convection models are used to provide upper and lower bounds on the local temperature rise, as well as the thermal damage, during electroporation from exponential voltage pulses (70 V maximum) with a 1 ms and a 10 ms pulse time constant. The peak temperature rise predicted by the conduction model ranges from 19 °C for a 1 ms time constant pulse to 70 °C for the 10 ms time constant pulse. The convection (mass transport) model predicts a 18 °C peak rise for 1 ms time constant pulses and a 51 °C peak rise for a 10 ms time constant pulse. The convection model compares more favorably with previous experimental studies and companion observations of the local temperature rise during electroporation. Therefore, it is expected that skin electroporation can be employed at a level which is able to transport molecules transdermally without causing significant thermal damage to the tissue.

Changes of structural and dynamic properties of model lipid membranes induced by α-tocopherol: implication to the membrane stabilization under external electric field

The effects of α-tocopherol on electric properties of bilayer lipid membranes were investigated. Planar bilayer membranes formed by the Mueller-Rudin method were used. Voltammetric and chronopotentiometric measurements were performed using a four-electrode potentiostat-galvanostat. It was demonstrated that registration of membrane capacitance, resistance, and voltammetric characteristics provided information about the change in the structure and permeability of bilayer lipid membranes. The results suggested that incorporation of α-tocopherol into lipid membrane destabilized its structure and facilitated the electrogeneration of pores. The possible role of observed changes in physiological functions of α-tocopherol was discussed.

Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin

The effects of ethylene oxide and propylene oxide block copolymers (pluronics) on the permeability of several weak acids and bases through bilayer lipid membranes have been studied by the methods of monitoring (1) pH shifts near planar bilayers, (2) doxorubicin fluorescence quenching inside liposomes, and (3) current transients in the presence of hydrophobic anions. It has been shown that pluronics facilitate the permeation of comparatively large molecules (such as 2- n-undecylmalonic acid and doxorubicin) across lipid bilayers, while the permeation of small solutes (such as ammonium and acetic acid) remains unaffected. Pluronics also accelerate the translocation of large hydrophobic anions (tetraphenylborate). The effect of pluronics correlates with the content of propylene oxide units: it is enhanced when the portion of polypropylene oxide block in the copolymer is increased. The action of the pluronic on lipid membrane permeability differs from the effect of the conventional detergent Triton X-100, which does not affect doxorubicin transport if added at concentrations similar to those used for pluronics. It has been proposed that pluronics accelerate the processes of solute diffusion within lipid bilayers (in a structure-dependent manner) rather than influencing the rate of solute adsorption/desorption on the membrane surface. We suppose that the effect of pluronics on doxorubicin permeation across lipid bilayers along with the known effect on the multidrug resistance protein determines its influence on the therapeutic activity of anthracycline drugs.

Permeability of bilayer lipid membranes in phase transition. The significance of intermolecular phosphate-phosphate hydrogen bonding

The effect of pH on the phase transition temperature of 1,2-dipalmitoyl- sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl- sn-glycero-3-thionphosphate (thion-DPPA) has been investigated. The phase transition was detected using the jump like increase effect in the conductance of the planar bilayer membrane. It is shown that the steepness of pH-dependence of the phase transition temperature differs for the two kinds of lipids in the pH range of 3.5–8. This result is explained in terms of decreased intermolecular hydrogen bonding between the head groups of thion-DPPA. Calculations taking into account the ability of DPPA molecules to intermolecular phosphate-phosphate hydrogen bonding were made. The results of calculations are in good agreement with the experimental data obtained in this study.

Permeability of bilayer lipid membranes for superoxide (O 2−) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 Å in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O 2 −, were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O 2 −( P′ O 2 −  (7.6 ± 0.3) · 10 -8 cm/s), or HO 2 • ( P′ HO 2 −  4.9 · 10 -4 cm/s) were determined. The effect of the transmembrane electric potential (K + concentration gradient, valinomycin) on the permeability of liposomal membranes for O 2 − were investigated. It was found that O 2 − can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.

The effect of primary products of lipid peroxidation on the transmembrane transport of calcium ions

Permeability of lipid bilayer membranes to Ca 2− was increased when the membranes were formed from oxidized lipids or when initiators of lipid peroxidation, ascorbate and iron ions, were added to both sides of the membrane. Permeabilities for other divalent and monovalent cations also were increased, but there was a substantially higher selectivity for the divalent cations. Similarly, the Ca 2+ permeability of vesicles of sarcoplasmic reticulum increased when exposed to iron:ascorbate. However, the increase in Ca 2+ permeability was not as great as with addition of the ionophore A23187. Thus, it appears that the primary products of lipid peroxidation are the simplest inductors of transmembrane cation transport.

Ion transport mediated by the valinomycin analogue cyclo(L-Lac-L-Val-D-Pro-D-Val)3 in lipid bilayer membranes.

Cyclo(L-Lac-L-Val-D-Pro-D-Val)3 (PV-Lac) a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes by forming a 1:1 ion-carrier complex. The selectively sequence for PV-Lac is identical to that of valinomycin; i.e., Rb+ greater than K+ greater than Cs+ greater than or equal to NH+4 greater than Na+ greater than Li+. The steady-state zero-voltage conductance, G(0), is a saturating function of KCl concentration. A similar behavior was found for Rb+, Cs+, and NH+4. However, the ion concentration at which G(0) reaches a plateau strongly depends on membrane composition. The current-voltage curves present saturating characteristics, except at low ion concentrations of Rb+, K+, or Cs+. The ion concentration at which the saturating characteristics appear depends on membrane composition. These and other results presented in this paper agree with a model that assumes complexation between carrier and ion at the membrane-water interface. Current relaxation after voltage-jump studies were also performed for PV-Lac. Both the time constant and the amplitude of the current after a voltage jump strongly depend on ion concentration and membrane composition. These results, together with the stationary conductance data, were used to evaluate the rate constants of the PV-Lac-mediated K+ transport. In glycerolmonooleate they are: association rate constant, 2 x 10(6) M-1 s-1; dissociation rate constant, 4 x 10(5) s-1; translocation rate constant for complex, 5 x 10(4) s-1; and the rate of translocation of the free carrier (ks), 55 s-1. ks is much smaller for PV-Lac than for valinomycin and thus limits the efficiency with which the carrier is able to translocate cations across the membrane.

The permeability of bilayer lipid membranes on the incorporation of erythrocyte membrane extracts and the identification of the monosaccharide transport proteins

1. 1. Extracts of the human erythrocyte membrane have been prepared by five different procedures involving Triton X-100 solubilization and gel chromatography. 2. 2. The extracts have been analysed by gel electrophoresis and incorporated asymmetrically into phosphatidylcholine-cholesterol- n-decane planar bilayers. 3. 3. Removal of excess Triton X-100 from membrane extracts or prolonged storage facilities the proteolysis of membrane extracts with the partial or complete breakdown of band 3 polypeptides (notation of Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606–2616) and the appearance of an enhanced zone 4.5 and low molecular weight material. 4. 4. Incorporation of zone 4.5 polypeptides into bilayer lipid membranes increases their permeability to d-glucose at 27 and 5°C. 5. 5. It is suggested that the components of the monosaccharide system are present in band 3 polypeptides but that they can undergo proteolysis with some retention of transport activity.

Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts

1. 1. Extracts of the human erythrocyte membrane have been prepared by solubilization with Triton X-100 and analysed by electrophoresis and gel filtration techniques. 2. 2. The extracts have been incorporated asymmetrically into lecithincholesterol- n-decane planar bilayers. 3. 3. The electrical characteristics and glucose permeabilities of the bilayers have been measured. 4. 4. The extracts increased the electrical conductance of the bilayers and also markedly enhanced the d-glucose permeability but not the l-glucose permeability. 5. 5. The enhanced d-glucose permeability was inhibited by monosaccharide transport inhibitors. 6. 6. The results support the claim that a monosachharide facilitated diffusion system has been set-up in vitro which has many of the characteristics of the transport system in the human erythrocyte membrane. 7. 7. The data indicates that the trans membrane polypeptides of band 3 of the electrophoretogram of the erythrocyte membrane proteins (notation of Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606–2616) are implicated in d-glucose transport, although the possibility that a relatively minor component of the membrane could be responsible for glucose transport cannot be eliminated.

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide [formula omitted

The cyclic dodecapeptide PV, cyclo-( d-Val- l-Pro- l-Val- d-Pro ) 3 , a structural analogue of the ion-carier valinomycin, increase the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na + < NH 4 + < K + < Cs + < Rb +. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanims). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.

Permeability of lipid bilayer membranes to biogenic amines and cations: Changes induced by ionophores and correlations with biological activities

The permeation of various cations and biogenic amines across artificial lipid membranes (bilayer membranes) was investigated by means of electrical conductivity measurements and fluorescence spectroscopy. Their permeability properties were modified by doping them with five different carboxylic ionophores. The induced permeability changes were correlated with some biological activities of the ionophores. Four out of five ionophores increased the permeability of doped membranes for Li+, Na+, K+, Mg2+ and Ca2+. Two of them showed a preference for K+ whereas one (X-537A) increased the membrane permeability for K+ as well as for Ca2+. It was also found that the ionophores increased the permeability for serotonin, dopamine, norepinephrine and epinephrine. No direct coupling was found between the facilitated ion permeation and the permeation of biogenic amines induced by the ionophores. The measurements can be qualitatively explained by assuming that the permeation of biogenic amines is competitively inhibited by cations. It appears that one biogenic amine molecule forms a complex with one ionophore molecule, the complex acting as a carrier for biogenic amines. All ionophores investigated increased the bilayer permeability considerably for some biogenic amines. (A preference up to 420∶1 for serotonin over epinephrine was measured for one specific ionophore.) There was no correlation between thein vitro antibacterial activity (against bacillus E and bacillus TA) of the ionophores and their potency to change the ion permeability of doped membranes. The correlation found between the ionophore-induced permeation of biogenic amines through membranes and their antibacterial activity is probably without biological meaning. However, a rather good correlation was found between cardiac sympathetic effects of the ionophores and their ability to facilitate permeation of norepinephrine through artificial membranes.

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)(delta)) 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 10(4) times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K(+) greater, similar Rb(+) > Cs(+) > NH(4) (+) > TI(+) > Na(+) > Li(+); the ratio of GGm in K(+) to that in Na(+) is about 10 for PV compared to > 10(3) 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.

Vasopressin and water permeability of artificial lipid membranes

Vasopressin markedly stimulated the water permeability of bilayer lipid membranes: a two-fold increase was measured at 25° in presence of 1.7·10 −9 M (50 μunits/ml) vasopressin. Oxytocin and a mixture of the amino acids comprising the vasopressin molecule could not substitute for vasopressin at comparable concentration. The experimental activation energy of water transport was reduced in the presence of vasopressin from 14 to 4 kcal/mole, in agreement with the effect of the hormone on water permeability of toad bladder.

Kinetics of carrier-mediated ion transport across lipid bilayer membranes

A theoretical treatment of the electrical conductance and tracer permeability of lipid bilayer membranes in the presence of macrocyclic ion carriers is given. The analysis is based on the assumption that the complex between the ion and the carrier is formed in the membrane-solution interface. The translocation of the complex across the membrane is described as a transport over an activation energy barrier. It is shown that some information on the rate constants may be obtained from the existing conductance measurements. For instance, the current voltage characteristic of lecithin membranes in the presence of monactin indicates that the rate-determining step in the ion transport is the translocation of the complex across the interior of the membrane.

Properties of liquid bilayer membranes separating two aqueous phases: Temperature dependence of water permeability

The temperature dependence of the water permeability of lipid bilayer membranes has been determined over the interval 7 to 49 °C, by measuring the net volume flux produced by a NaCl gradient. The membranes were generated from solutions of lecithin and cholesterol in decane, either 0.5% (w/v) lecithin and 0.25% cholesterol (solution A) or 1% lecithin and 1% cholesterol (solution B). At 25 °C, P os, the osmotic permeability coefficient, is 31.7 ± 4.3 μ/sec and 24.4 ± 3.7 μ/sec for membranes formed from solutions A and B, respectively. Plots of In ( P os) versus 1 T are linear and yield values of 12.7 ± 0.3 and 13.1 ± 0.7 kcal./ mole for the experimental activation energies E a. These results, together with literature data for the solubility and diffusion of water in hydrocarbons, are consistent with a simple solubility-diffusion mechanism for the water permeation process. The similarity between the values of both P os and E a for cellular membranes and the lipid bilayer suggests that a similar mechanism may operate in the biological systems.