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.     

Single ion channels and macroscopic conductivity of bilayer lipid membranes

The dynamic conductivity of bilayer lipid membranes unmodified by ionophores in current ranges of 10(-12)-10(-10) A was studied. On the current voltage characteristics the jumps of dynamic conductivity in the voltage ranges near zero and disruption value were observed. The lifetime of these jumps was 1-5 s. It was shown that these effects were due to electrostriction phenomena and defects in the bilayer lipid structure correspondingly. Apparently, lipid peroxidation products participate in the building of defects in lipid bilayers.     

Electrical conductivity of bilayer lipid membranes in electrolytic solution

The electrical conductivity of bilayer lipid membranes (BLM) of oxidized cholesterol has been measured separately in bathing solutions of sodium sulphate, sodium chloride, sodium bromide, sodium iodide and also in bathing solutions of iodine and iodine containing these salts. An attempt has been made to explain the conduction of electric current across the membranes.     

Electrical conductivity in lipid bilayer membranes induced by pentachlorophenol.

Electrical conductivity induced in thin lipid bilayer membranes by pentachlorophenol has been studied. The membranes were formed from phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl glycerol and various amounts of cholesterol. The position and the magnitude of the maximum of the conductivity vs. pH curve depend on the type of lipids and cholesterol content. At low pentachlorophenol concentrations and low pH the concentration dependence of conductivity is quadratic and becomes linear at higher pH. Above 10(-5) M of pentachlorophenol the concentration dependence of the membrane conductivity tends to saturate. Presence of pentachlorophenol enhances membrane transport of nonactin-K+ complex. Increase of cholesterol content increases pentachlorophenol induced conductivity in all membranes and shifts the conductivity toward lower pH. For phosphatidyl choline the largest rate of change of membrane conductivity with cholesterol occurs at 1:1 phospholipid to cholesterol molar ratio. Pentachlorophenol is found to be a class II uncoupler and the experimental results are consistent with the hypothesis that the membrane permeable species are dimers formed by combination of neutral and dissociated pentachlorophenol molecules. Several schemes of membrane conduction, including dimer formation in the aqueous phase as well as at the membrane-water interface have been considered. Arguments are given in favor of the formation of dimers within the membrane surface.     

Effect of glycerol on the capacity and conductivity of lipid bilayer membranes

It is shown that glycerol addition to one side of BLM containing cholesterol leads to a significant decrease of its capacity, and the decrease rate is in indirect proportion to glycerol concentration. Washing out or addition of glycerol to another side results in a full or partial reconstitution of the capacity. The effect of glycerol on the BLM conductivity is manifested in its increase for membranes having specific conductivity above 2.10(-7) Om-1 X cm2. The peculiarity of glycerol is manifested in the fact that during definite period of time (20 +/- 30 min) the membrane is under "stress" condition with the characteristic current fluctuations, which may be compared with its medium meaning. Such condition is preserved up to the moment of BLM rupture. It is supposed that such effects are due to the big-scale reconstructions of membrane structure under the influence of glycerol.     

Interfacial tension of phosphatidylcholine-phosphatidylserine system in bilayer lipid membrane

The effect of pH of electrolyte solution on the interfacial tension of lipid membrane formed of phosphatidylcholine (PC, lecithin)–phosphatidylserine (PS) system was studied. In this article, three models describing the H⁺ and OH⁻ ions adsorption in the bilayer lipid surface are presented. In Model I and Model II, the surface is continuous with uniformly distributed functional groups constituting the centres of H⁺ and OH⁻ ions adsorption while in the other the surface is built of lipid molecules, free or with attached H⁺ and OH⁻ ions. In these models contribution of the individual lipid molecule forms to interfacial tension of the bilayer were assumed to be additive. In Model III the adsorption of the H⁺ and OH⁻ ions at the PC–PS bilayer surface was described in terms of the Gibbs isotherm. Theoretical equations are derived to describe this dependence in the whole pH range.     

Interfacial tension of bilayer lipid membrane formed from phosphatidylethanolamine

The dependence of the interfacial tension of a lipid membrane on the pH of the aqueous solution has been studied. Interfacial tension measurements of phosphatidylethanolamine (PE) were carried out. A theoretical equation is derived to describe this dependence in the whole pH range. A maximum corresponding to the isoelectric point appears both in the theoretical equation and in the experimental data.     

Effect of ultrasound on a bilayer lipid membrane.

The effects of continuous wave ultrasound at a frequency of 1 MHz in the intensity range of 0-1.4 W/cm2 on an oxidized cholesterol bilayer lipid membrane (BLM) were observed. Ultrasound at 1.5 W/cm2 broke the membrane; in the range from 0.5 to 1.4 W/cm2, it accelerated the draining of the bulk lipid solution from the annulus to the Teflon support. At all intensities it has no effect on the conductance, the capacitance, or the dependence of each on the voltage applied across the membrane. Electrical parameters were measured in the presence of aqueous solutions of NaCl, KCl, and distilled water. The motivation and results of this project are explained in relation to an overall objective of determining the specific effects of ultrasound on biological membranes.     

Relation between gramicidin D and valinomycin-induced conductivity of lipid bilayer and cholesterol levels

Kinetics of conductance and current-voltage characteristics (VACH) were studied on the bilayer lipid membranes from egg lecithin and cholesterol modified by gramicidin D and valinomycin. There were found significant changes of ionic channels conductance g, lifetime tau and non-linearity coefficient beta of VACH of modified membranes dependent on cholesterol content. In the region of high cholesterol concentrations, more than 20 mol%, there were observed abrupt changes of the ionic channels parameters: tau increased more than ten times, conductance decreased two times and beta greater than 0 in the whole region of electrolyte concentration.     

Effect of bilayer lipid membrane thickness, composition, and tension on gramicidin channel parameters

Dependence of channel parameters formed by gramicidin A (conductivity and mean life time) on thickness, composition and tension of planar bilayer lipid membranes (BLM) was studied. BLM were obtained from solutions of alpha-monoglycerides of fatty acids in n-alkanes. It has been shown that channel conductivity depends on the length of lipid radical hydrocarbon and is insensitive to the isomerization of lipid and to the change of solvent. There was no direct relationship between the life time, thickness and composition of BLM. Logarithm tau for all the systems studied is proportional to BLM tension, which points to a significant role of surface phenomena in the formation by grammicidine A of a conducting pore in the lipid bilayer.     

Resolution of partition coefficients in the transverse plane of the lipid bilayer

The distribution of a small lipid soluble molecule across a lipid bilayer has been determined using fluorescence quenching techniques. The neutral form of the amine, N,N-dimethylaniline (DMA) quenches the fluorescence of a series of n-(9-anthroyloxy) fatty acids (n = 2,6,9,12,16) which place a fluorophore at a graded series of positions from the surface to the centre of the lipid bilayer. A method is described for determining the partition coefficient of a quencher at each transverse position. The results show that DMA is located at all depths within the bilayer leaflet but that it is concentrated at the bilayer centre and to a lesser extent at the bilayer surface.     

Insulating tethered bilayer lipid membranes to study membrane proteins

Tethered bilayer lipid membranes are stable and promising model systems that mimic several properties of biological membranes. They provide an electrically insulating platform for the incorporation and study of functional membrane proteins, especially ion channels. Covalently linked to a solid support, they also offer enhanced stability compared with other model architectures. If the support can be used as an electrode, electrical characterisation of the system is possible and biosensing applications can be envisioned.Here, we will review some tethered bilayer structures developed in the past and show some examples of functional protein incorporation, both on oxide and gold substrates.     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.     

Interaction of gramicidin S analogs with lipid bilayer membrane.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.