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.     

The effect of rimantadine on the structure of model and biological membranes

The action of the antiviral drug rimantadine on the structure of bilayer lipid membranes (BLM) and RBC membranes was investigated. Structural changes in BLM were recorded by ionophore conductivity changes and by changes in the third harmonic of capacity current signal due to lateral compression of BLM in an electric field. It was shown that the adsorption of rimantadine on BLM results in an increase in ionophore mobility in bilayer membranes of dioleolyllecithin (DOL) and common lipids of bovine brain (CL) and in a decrease in those of azolectin (A). Relative changes in the third harmonic signal also depend on the membrane composition and have different signs. The results may be explained by the rimantadine action on the lipid bilayer structure: "rigidification" of A-membranes and "fluidization" of BLM from DOL and CL. Structural reorganization of RBC membranes as investigated by the ability of the cells to enter a micropipette (inner diameter greater than or equal to 3 microns) thereby undergoing deformation. It was shown that rimantadine influences RBC deformability due to drug induced inhomogenous mechanical membrane properties. Also, rimantadine accelerated the process of artificially induced aggregation of erythrocytes. The relation of the effects on artificial and biological membranes, and the structural changes in the lipid phase of membrane are discussed.     

Study of the interaction of myoglobin with lipid bilayer membranes by potentiodynamic method

For modeling the interaction of myoglobin with mitochondrial membranes, the adsorption of different ligand forms, the physiologically active reduced MbO2 and inactive oxidized met-Mb, on one of the surfaces of artificial bilayer lipid membrane (BLM) was studied using potentiodynamic technique known as the "capacity minimization" method. As mitochondrial membranes are negatively charged, BLM from the negatively charged palmitoyl-2-oleil-phosphatidyl glycerol (POPG) and neutral soybean phosphatidylcholine (lecithin) were used. It is shown that both myoglobins strongly interact with BLM in the pH range 6-8. The dependence of the potential difference between cis-and trans-surfaces of the lipid membrane (deltaE, mV) on the protein concentration is characteristic for the Langmuir adsorption isotherm, and the saturation level (deltaEmax, mV) corresponds to monolayer of myoglobin. The protein adsorption is essentially electrostatic in nature, as adsorption activity increases sharply in the case of the membrane from POPG: in a approximately 15-fold in the case of MbO2 and in a approximately 2.5 times for the met-Mb. The parameters of the MbO2 and met-Mb adsorption on BLM from lecithin and POPG do not change in the pH 6-8 range. It can be assumed that the anionic groups of phospholipids associate with the cationic groups of the protein, the charge state of those does not change in the pH 6-8 range. The most likely candidates for interaction with phospholipids of BLM are invariant lysines and arginines in the environment of the myoglobin heme cavity.     

Action of natural and synthetic antioxidants on the electrical parameters and stability of natural and artificial membranes

The effect of antioxidants alpha-tocopherol and ionol on membranes of human red cells and bilayer lipid membrane (BLM) from azolektin has been studied. Ionol at concentration 4-10 mM induces the hemolysis of erythrocytes, the cells form changes are observed at concentration 2 mM alpha-tocopherol doesn't show the hemolytic properties at concentration 23 mM. The ionol concentration 1 mM doesn't change the form of the cells, but influence the passive electric parameters: the capacity (Cs) of erythrocytic membrane increases and the intracellular conductance (chi i) decreases. Tocopherol (3 mM) induces the decrease both Cs and chi i. The fast increase of membrane conductance is almost immediately registered on one side of BLM at addition of ionol (0,2-0,4 g/ml). Phosphatidylionol synthesized from ionol and contining the acyl chains C15H31 and C17H35 doesn't influence the electrical properties of BLM.     

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.     

Interaction of all-<Emphasis Type="Italic">trans</Emphasis>-Retinal with bilayer lipid membranes

The interaction of all-trans-retinal (hereinafter referred to as retinal) with planar bilayer lipid membranes has been studied. Addition of retinal into aqueous solutions on both sides of the membrane formed from diphytanoilphosphatidylcholine (DPhPC) or its mixture with diphytanoilphosphatidylethanolamine (DPhPC/DPhPE in w/w proportion of 3: 5) led to a change of conductance induced by ionophores nonactin (increase of conductance) or pentachlorophenol (decrease). Increase of nonactin-induced conductance was dependent on the membrane lipid composition and was two times higher in the case of DPhPC/DPhPE mixture. The change of conductance caused by ionophores of different signs (plus or minus) had different direction suggesting the influence of the retinal on the dipole potential upon its incorporation into BLM. The boundary potentials difference measured by the intramembrane field compensation method (IFC) after the retinal addition on one side of the membrane did not exceed 2.5 mV suggesting that its distribution in the bilayer is almost symmetrical. The illumination of the retinal-containing BLM caused a decrease in its lifetime when the membranes were formed from unsaturated lipids. Retinal incorporated into BLM led also to photoinactivation of the gramicidin channels. The process was completely inhibited by a singlet oxygen quencher (sodium azide). These results indicate that retinal accumulated in the membrane can affect both membrane proteins and the unsaturated lipids by their oxidation by the singlet oxygen.     

Effect of beta-lactam antibiotics on lipid bilayer membranes. I. Penicillin antibiotics

Interaction between penicillins and model membrane systems, flat black bilayer lipid membranes (BLM) composed of vegetable or bacterial phospholipids was studied with an account of the complicated structure of bacterial cell membranes and possible presence in them of "pure" bilayer lipid areas. By their effect on electroconductivity of the BLM the antibiotics could be divided into three groups: those having no effect on the BLM electroconductivity at the maximum concentrations i.e. benzylpenicillin, carbenicillin, piperacillin (at pH 6.0 and 7.0) and ampicillin (at pH 6.0), those insignificantly changing electroconductivity of the BLM i.e. carfecillin and azlocillin and those having a significant effect on the BLM electroconductivity i.e. ampicillin N-acyl derivatives and 6-APA. The effect of ampicillin on the BLM conductivity markedly depended on the electrolite pH. The penicillins bound to the bilayer and induced changes in the transmembrane potential (evident from the changes in the second harmonic of the capacitive current) and the BLM elasticity-capacitance parameters (evident from the changes in the ratio of the amplitudes of the first and third harmonics). It was shown that all the penicillins penetrated through the BLM composed of either vegetable or bacterial phospholipids. The capacity for the transmembrane transfer without changing of the bilayer conductivity must be connected with the fact that the penetrating antibiotics did not induce any changes in the BLM structure. The effect on the conductivity probably depended in its turn on the form of the molecule and the ratio of the hydrophilic and hydrophobic parts in it.     

Angiotensin II-induced formation of ionic channels in bilayer lipid membranes.

The interaction of angiotensin II (ANG II) with membrane was studied by measuring conductance and current-voltage characteristics (IVC) of bilayer lipid membranes (BLM) prepared of a mixture of egg lecithin with cholesterol, and of gramicidin D-modified membranes of the same composition. Addition of physiological concentrations of ANG II (approx. 15 mumol/l) into the electrolyte (1 mol/l KCl, pH = 7) in contact with one side of BLM resulted in the appearance of discrete membrane conductance (symbol; see text) = (39.5 +/- 1.07) pS with a duration of the conductivity state tau = (52.15 +/- 6.44) s. Raising ANG II concentration to 75 mumol/l resulted in an additional conductance level of approx. 130 pS with a lifetime of approx. 1s. The electrolyte pH markedly influenced ANG II modified BLM conductance. A decrease of the electrolyte pH to 2.8 resulted in a reduction of the discrete conductance level to approx. 14 pS, whereas ANG did not induce any conductivity at pH = 11.5. The results obtained suggest that ion channels are formed consisting at least of two ANG II molecules. IVC of ANG II-modified BLM are superlinear within the range of electrolyte concentrations studied (between 0.01 and 3 mol/l KCl), i.e, the limiting stage of ion transport is the internal area of the conducting pore. ANG II affects in a cooperative manner the gramicidin D (GRD)-mediated transport, most likely by forming ANG II aggregates in the area of local inhomogeneities in the BLM structure of GRD channels.     

Lipid-polypeptide interactions in bilayer lipid membranes

Summary The modifications of the electrical properties of bilayer lipid membranes (BLM) composed of cholesterol and an ionic surfactant upon interaction with charged polypeptides were studied. The addition of 10^–8 m polylysine (Ps⁺) to one side of anionic cholesterol dodecylphosphate BLM increases the specific membrane conductance over 1000-fold (from 10^–8 to 10^–5 mho/cm²) and develops a cationic transmembrane potential larger than 50 mV. This potential is reverted by addition of polyanions such as RNA, polyglutamic or polyadenilic acid to the same side on which Ps⁺ is present, by addition of Ps⁺ to the opposite side, or by addition of trypsin to either side. Both conductance and potential changes are hindered by increasing the ionic strength or by raising the pH of the bathing medium, disappearing above pH 11.5 where it is known that Ps⁺ folds into an -helix. The interaction of polyglutamic acid (PGA) with a cationic cholesterol-hexadecyltrimethylammonium bromide BLM results in increased membrane conductance and development of an anionic transmembrane potential which is reverted by addition of polycations to the same aqueous phase where PGA is present. Addition of either Ps⁺ or PGA to one or both sides of a neutral BLM composed of 7-dehydrocholesterol induces no significant change. The observations suggest the formation of a lipid polymer membrane resultant from the interaction, predominantly electrostatic, of the isolated components. The implications of these results are discussed in terms of the current models of membrane structure.     

Modeling of damage to cell membranes during photodynamic therapy: photosensitization of planar lipid bilayers by hematoporphyrin dimethyl ether

The variations of electrical conductance of planar bilayer lipid membranes (BLM) sensitized by hematoporphyrin dimethyl ether under visible light illumination were studied. The conductance of BLM does not change for some period after switching on the light, then an increase in the conductance starts and the membrane breaks. This "induction" time does not depend on addition of azide or ferricyanide to the solution, on addition of BHT to the lipid and on substitution of air for argon in the cell. The induction time for any new BLM, formed in the same cell immediately after the previous membrane was broken, is shorter. The variation of BLM boundary potentials during induction time was not observed. The results obtained suggest that the photodamage of BLM sensitized by HPD leads to accumulation of uncharged reaction products and oxygen does not take part in this process.     

Effect of changes in cation concentration near bilayer lipid membrane on the rate of carrier-mediated cation fluxes and on the carrier apparent selectivity

A new approach was applied for the measurements of ion transport through bilayer lipid membranes (BLM) induced by electrically neutral cation/H+ exchangers. This is an improved version of the method of the measurements of the cation/H+ exchange rate based on recording pH shifts in the unstirred layers near the BLM. Using this approach, the pH gradient in the unstirred layers induced by the cation/H+ exchanger was reduced by successive addition of the acetate on one side of the BLM until the pH shift reached zero. The difference in acetate concentration across the membrane is a measure of the cation/H+ exchange rate. In the second part of the work we found that the changes in cation concentration in the unstirred layers under the conditions imposed when measuring cation selectivity (according to Antonenko, Yu.N. and Yaguzhinsky, L.S., Biochim. Biophys. Acta 1988; 938, 125-130) can significantly decrease the apparent value of cation selectivity. It was shown that more accurate results can be obtained if low concentrations of the carrier are used. The values of nigericin cation selectivity for the alkali metals were measured (K+/Rb+ 19 +/- 1, Rb+/Na+ 1.9 +/- 0.2, Na+/Cs+ 8 +/- 0.5, Cs+/Li+ 1.8 +/- 0.3).     

Interaction of dopamine with artificial phospholipid membranes

The interaction of dopamine (DA) with phospholipid membranes has been investigated. The membrane current in planar bilipid membrane (BLM) modified by amphotericin B in voltage clamp conditions under alternating polarity was shown to symmetrically increase 1.2 times when DA was added outside the BLM. This implies a uniform change of charge on each membrane surface and hence the diffusion of DA within the BLM and its exposure on the internal side. The appearance of single threads and bundles of filaments within the internal liposomal cavities was observed in the ultrastructure of suspended thin-walled liposomes filled with globular actin after the introduction of DA into external solution. This reshaped liposomes into rod-like, spindle-shaped or angular structures. Actin serves as a marker for DA due to its property to polymerize itself under the influence of DA. Thus, the structural reorganization of liposomes manifests the presence of DA inside them and the induction of actin polymerization.     

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: Role of cholesterol

Summary Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104vs. 0.60,P<0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35°C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r ) (0.212vs. 0.154,P<0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-As probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids. Results obtained using the surface membrane probes trimethylammonium-DPH (TMA-DPH) and 2-AS suggested a fluidity gradient existed in both BBM and BLM bilayers with the inner core being more fluid in both membranes. These data indicate cholesterol is in large part responsible for fluidity differences between BBM and BLM and that these membranes, while clearly differing in the order component of membrane fluidity, may also difer in the dynamic component as well.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Electric capacitance of bilayer lipid membranes from hydrogenated egg lecithin during phase transition from liquid crystalline state to gel

The electrical capacity of planar bilayer lipid membranes (BLM) from natural hydrogenated egg lecithin (HEL) in n-decane at a temperature of phase transition was measured. The temperature of phase transition was determined calorimetrically to be 51 degrees C. The data obtained revealed a phase separation of HEL in BLM into two fractions, one freezing at 42-44 degrees C and one that is converted to a liquid-crystal state at 51-59 degrees C. It was assumed that the first fraction is rich in dipalmitoyl lecithin, and the second fraction is rich in distearoyl lecithin. Freezing and the transition to the liquid-crystal state were accompanied by an increase and decrease in membrane thickness, respectively, in part due to a displacement of the solvent from the torus to the planar part of the bilayer. The displacement of the solvent is explained by changes in the disjoining pressure in BLM, which arises across the lipid bilayer due to van der Waals forces of attraction between water layers on both sides of the BLM.     

Interaction of cisplatin with planar model membranes - dose dependent change in electrical characteristics

The drug cisplatin has broad antineoplastic activity against advanced testicular and ovarian cancers, epithelial malignancies, cancers of the head, neck, bladder, oesophagus and lungs. Peripheral neurotoxicity, ototoxicity and nephrotoxicity are its major side effects. The nonspecific action of this drug on the lipid bilayer architecture of membranes has been studied by following the effects produced on the electrical characteristics of model planar bilayer lipid membranes (BLM). The results confirm that the drug has a strong surface interaction with the zwitterionic polar head groups of the amphipathic phospholipids constituting the BLM. The permeability characteristics of cisplatin through the hydrophobic core are limited. Cisplatin does not fluidise the membrane sufficiently to cause its breakdown but creates small ion conducting defects on the membrane bilayer resulting in a marginal increase in ion conductivity. These results indicate that cisplatin exhibits a non-specific action on the lipid bilayer component of the membrane which might be partly responsible for its neurotoxic side effects.     

Ca2+ ions induce successive capacity and ion currents in BLM from phosphatidic acid as a result of phase transition

Time dependence of Ca2+-induced electric current in BLM formed from DPPA was studied at constant temperature and pH. The phase transition in BLM is accompanied by capacity current and following appearance of single ionic channels. It was shown that transferred charge was 5 nC/F, conductivity of single ionic channels--500-100 pSm.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Protein from beef heart mitochondria inducing the potassium channel conductivity of bilayer lipid membrane

Protein (M. m. 60 000) inducing selective potassium conductance of bilayer lipid membranes (BLM) was isolated from mitochondria and homogenate of the beef heart. This protein was obtained by means of alcohol (ethanol) extraction and was purified by gel-filtration on Sephadex G-15 and G-50 followed by electrophoresis in the 10% polyacrylamide gel. 6-10 g/ml of the protein produced the conductivity channels on BLM with amplitude divisible of 24 +/- 4 pmho. The channels of 175 +/- 7 pmho were the most typical ones. The modification of BLM by K+-transport in protein under the conditions of potassium gradient resulted in the appearance of the membrane potential close to the theoretical Nernst potential.     

Study of conductance changes of bilayer lipid membrane induced by electric field

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.