Electrical capacitance of lipid bilayer membranes of hydrogenated egg lecithin at the temperature phase transition

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.     

Elasticity, strength and stability of bilayer lipid membranes and their changes due to phospholipid modification

Elasticity measurements of bilayer lipid membranes (BLM) based on registration of the third harmonic of the membrane current during the application of a periodic tension to the membrane was used to study the effects of lipid peroxidation (LPO) and phospholipase A on BLM. LPO resulted in decreased values of the Young modulus for BLM, while some products of LPO and phospholipid hydrolysis (linolenic acid) were able to increase drastically the modulus. The presence of individual products of LPO and phospholipid hydrolysis in BLM produced non-additive effects on the elasticity, strength and stability of BLM. Lysolecithine strongly affected both the strength and stability of BLM. without changing its elasticity modulus. It was found that the lower the rate of structural changes in lecithine BLM, the longer its lifetime. Membranes having a heterogeneous polar composition form more stable BLM as compared to chemically homogeneous membranes.     

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.     

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 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.     

Ion permeability of bilayer membranes formed from synthetic phospholipids in the phase transition region

Ion permeability of black lipid membranes formed from synthetic phospholipids has been studied. The resistance of BLM formed from phosphatidylcholine, tiophosphatidylcholine, threealkylphosphate and threealkyltiophosphate was 10(7)--10(8) Ohm.cm2. It was shown that the membrane potential of the 10--30 mV arised in KCl gradient indicating the preference cation conductance in synthetic lipid membranes. A sharp decrease of the membrane conductance near to the phase transition temperature was discovered. The change of conductance by phase transition temperature was sensitive to chemical nature of the polar head of phospholipids used.     

Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388–400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.     

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.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

Conjugation of bleomycin with concanavalin A or immunoglobulin G increases its ability to destroy cell membranes.

Hybrid molecules consisting of an address peptide and an active oxygen-generating fragment may be used for selective destruction of cells. We tested the possibility of using the antibiotic bleomycin (BLM) as an active oxygen-generating fragment of such a molecule. It was found that bleomycin can induce destruction of cell membranes. BLM-mediated cell destruction was inhibited by addition of catalase, superoxide dismutase, and OH. scavangers (mannitol and ethanol), suggesting that hydroxy radical is involved in the process. BLM can induce membrane impairment at the expense of electrons supplied by NADPH-cytochrome P450 reductase. Covalent binding of BLM to an address fragment (concanavalin A, immunoglobulin G) increases the ability of BLM to destroy erythrocyte membranes. The data obtained lead to the conclusion that BLM can be used as an active oxygen-generating fragment of a proposed cell-destroying hybrid molecule.     

Interaction between plasma lipoproteins and bilayer lipid membranes. The role of surface charge

Interactions of planar BLM with different thickness and surface charge were analysed theoretically. Drawing together of the membranes is accompanied with the appearance of intramembrane potential jumps which may cause destruction and breakdown of the membranes. The theory is extrapolated to the interaction between spherical lipoprotein particles and planar BLM. Experimentally calculated (by means of ESR) surface charges of lipoproteins of low density (LLD) (--0,3 . 10(-2) C/m2) and lipoproteins of high density (LHD) (--2 . 10(-2) C/m2) enabled calculation of the interaction energy between the particles and BLM as well as of the values of intramembrane potential jumps. The latter cause local reconstructions of the membranes in the contact region and fusion of the particles with them. The earlier obtained experimental data were proved by the finding that LHD adsorption as compared with LLD is impeded due to the existence of a high energetic barrier. These peculiarities of the particles manifested during their interactions with BLM seem to be one of the factors responsible for atherogenic function of LLD and antiatherogenic one of LHD.     

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.     

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.     

Conducting polymer polypyrrole supported bilayer lipid membranes

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayer lipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidyl-L-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes (s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than the PPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopy measurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLM structures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film after BLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining at its two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expected protein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogen peroxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.     

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.     

Bilayer lipid membranes (BLM): Study of antigen-antibody interactions

Previous work of del Castillo and co-workers has shown that bilayer lipid membranes (BLM) can be used as transducers for detection of antigen-antibody reactions. The present experiments extend the previous work by incorporating complement into the BLM system. The results indicate that the antigen-antibody complex or the complement has no ability to affect the BLM system separately, but when carefully combined they will destabilize the BLM even at a much reduced concentration. Further development using the BLM as a tool for investigating immunological reactions is suggested.     

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.     

Interaction of plasma lipoproteins and apolipoproteins with lipid bilayer membranes

It was shown that the interaction of lipoproteins (LP) with bilayer lipid membranes (BLM) resulted in some changes in the physical-chemical properties of the membranes. Adsorption of very low and low density lipoproteins (VLDL and LDL) at concentrations of 5-8 g protein/ml increased the surface potential difference and decreased transversal elasticity module of the bilayer. LP concentrations higher than the mentioned ones increased BLM conductance and caused instability and disruption of the membranes. The same effects were revealed for high density lipoproteins (HDL) at higher concentrations--15-20 micrograms protein/ml. The effect of apolipoproteins in the interaction of LP with BLM was investigated. It is proposed that apolipoproteins and especially apo B are the main factor which affects the nonreceptor interactions of LP with the membranes.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles.

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Effect of a permanent magnetic field on the formation of lipid bilayers

Effect of the constant magnetic field (MF) by the induction of 1.1 T on formation kinetics of bilayer lipid membranes (BLM) from egg lecithin in decane was discovered. Under the effect of MF oriented in parallel to the lipid film place the rate of BLM formation decreases, while at its perpendicular orientation it is accelerated. The stationary value of BLM capacity decreases under MF effect at both orientations. The discovered changes in the rate of BLM formation under MF effect seem to be related to the effect of MF on liquid crystalline structure of colour lipid film.