Characterization and property of DNA incorporated bilayer lipid membranes

Calf-thymus DNA-incorporated bilayer lipid membranes supported on a glassy carbon (GC) electrode was prepared by making layers of phosphatidylcholine dimyristoyl (DMPC) on GC electrode. DNA in the BLM was characterized by cyclic voltammetry, IR and AFM, and lipid layers formed on the GC electrode were demonstrated to be a bilayer lipid membrane by electrochemical impedance experiment. In IR and AFM experiments the findings indicated that DNA was incorporated into BLM. The ion channel of bilayer lipid membranes incorporated was studied. The result showed that the ion channel was opened in the presence of the stimulus quinacrine. In the absence of quinacrine the channel was switched. The process can repeat itself many times. The impedance spectroscopy measurements demonstrate that the stimulus quinacrine opens the channel for permeation of marker ion. The mechanism of forming an ion channel was investigated.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Capacitive and ionic currents in BLM from phosphatidic acid in Ca2+-induced phase transition

The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm.     

Conductivity and diameter of latrotoxin channels in lipid bilayers

It is shown that approximately 9 A Ca-selective ion channels were induced in bilayer lipid membrane (BLM) from phosphatidylserine by nonpurified spider venom (Latrodectus tredicimguttatus) and by alpha-latrotoxin obtained from it. It is established that channels greatly different in conductivity have the same diameter and nearly the same charge constitution, that evidences for their claster organization. The purification process as well as freezing-thawing and long time keeping influence the channel conductivity without changing its diameter and selectivity.     

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.     

Conductivity and structural transitions in bilayer lipid membranes

5 structural transitions were found in bilayer lipid membranes (BLM) from egg lecithin (EL) within the temperature range 14-44 degrees C. In the transition zone BLM conductivity abruptly increases, in some cases current fluctuations of the order 150 pC of the channel type are initiated. The transition temperatures observed in BLM from EL coincide with those in biological membranes. The cause of this phenomenon is discussed, as well as possible use of these BLM in the region of structural transition as a model of cellular receptor to electromagnetic fields.     

Low molecular channel-former protein from the mitochondrial membrane

Individual low molecular weight protein component was isolated by gel-filtration method from the inner mitochondrial membrane. This membrane component increases the conductivity of bilayer lipid membranes (BLM) in the presence of K+ and Ca2+-ions. This phenomenon may be explained by the formation of single conductivity channels. The voltage - current characteristics of this channel is nonlinear, which may be the result of asymmetrical operation of the channel - former in the polarized membrane, in spite of equal concentration of protein on both sides of the membrane.     

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.     

Channel properties of the purified glutamate receptor from rat brain reconstituted in planar lipid bilayer membrane

The properties of the purified rat brain glutamate receptor (GluR), reconstituted in planar lipid bilayer (BLM) were characterised. The single channel currents activated by glutamate and aspartate were similar. The different kinetics of current fluctuation were observed. Paroxysms of channel activity seems to be resulted from the transit of GluR through its active conformation from which it can open several times before desensitising. The effect of concanovaline A (Con A) as an agent blocking desensitisation of glutamatergic synapses was investigated. It was shown that Con A evokes high levels of conductivity and prolonged opening events of channels. Another agent, which stabilises glutamate activated conductivity, dithiothreitol (DTT), evokes "chronic" channel activity. This study demonstrates that purified GluR reconstituted in planar lipid bilayers exhibits the ion-conductivity properties that are associated with the postsynaptic membrane.     

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.     

Rimantadine effects on the elasticity of bilayer lipid membranes and on ion transport through gramicidin D channels.

The effect of the antiviral preparation rimantadine on lipid bilayer membranes (BLM) was studied by measuring the modulus of elasticity in the direction normal to the surface (E perpendicular) and by estimating the conductance lambda, the lifetime tau of single gramicidin D channels (GRD), and the coefficient of nonlinearity beta of current voltage characteristics (IVC) of GRD-modified BLM. Rimantadine induced a nonmonotonic change in E perpendicular of BLM prepared from a mixture of egg lecithin with cholesterol: at relatively low rimantadine concentrations (0-40 micrograms/ml) E perpendicular first increased, reached a maximum and started to decrease. The effectivity of rimantadine was dependent on the cholesterol concentration in the BLM. Changes in E perpendicular suggest an increased ordering of the lipid bilayer at low rimantadine concentrations and formation of clusters of the preparation at concentrations exceeding those necessary to obtain maximal values of E perpendicular for the given BLM lipid composition. Rimantadine concentrations lifetime by approximately 20 percent, affected the degree of IVC nonlinearity and superlinearity of GRD-modified membranes, which suggests some effect on the height of the barrier at the ionic channel mouth and in its centre.     

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.     

A quantitative explanation of the effects of some alcohols on gramicidin single-channel lifetime

The effects of n-decanol, n-hexadecanol, n-octyl(oxyethylene)3 alcohol and cholesterol on gramicidin single-channel lifetime in planar lipid bilayers have been determined. The bilayers used were formed from a solution of monoolein in squalene. Measurements have also been made of the above compounds' effects on membrane thickness (as measured by electrical capacity and optical reflectance technique) and surface tension (as derived from bulk interfacial tension and bilayer-lens contact angle measurements). The reduction in single-channel lifetime caused by the n-alkanols may be accounted for quantitatively in terms of the effects of these compounds on bilayer thickness and surface tension. The n-octyl(oxyethylene)3 alcohol caused an increase in single-channel lifetime which is also consistent with the thickness/tension theory. The reduction in channel lifetime caused by cholesterol, however, was much larger than would be predicted from its effects on bilayer thickness and surface tension.     

Structure and permeability of the contact zone of 2 artificial lipid-lipoprotein membranes

Contact interaction of two flat artificial lipid-proteolipid membranes (LPLM) obtained from lipid-proteolipid mixture of cattle brain white matter was studied. Investigation of the optical structure of the contact region revealed existence of fused proteolipid (PL) nodes and bilayer lipid areas. The lipid bilayers are fixed at stationary distance--30-40 nm, irrespective of ionic composition and ionic strength of the aqueous medium. Apparently, the maximal thickness of proteolipid regions at the two sides of the lipid framework of single LPLM reaches 15-20 nm, and LPLM after putting into contact fused in the region of PL nodes. It prevents the lipid areas from further drawing together. Conductance of the contact zone of two LPLM in NaCl solutions (10 and 100 mM) corresponds to that of single LPLM, but in KCl solutions it decreases 3.5 times, remaining in all the cases 1-2 orders above the conductance of single bilayer lipid membrane (BLM) or the contact zone of two BLM. Conductivity of single as well as of two contacting LPLM is higher for K+, than for Na+. The data obtained suggest that PL areas of the contact zone of two LPLM, likewise a single LPLM, are adjacent to the aqueous medium forming together with the bound lipids the zones of high conductivity.     

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.     

Deformation free energy of bilayer membrane and its effect on gramicidin channel lifetime.

The deformation free energy of a lipid bilayer is presented based on the principle of a continuum theory. For small deformations, the free energy consists of a layer-compression term, a splay-distortion term, and a surface-tension term, equivalent to the elastic free energy of a two-layer smectic liquid crystal with surface tension. Minimization of the free energy leads to a differential equation that, with boundary conditions, determines the elastic deformation of a bilayer membrane. When a dimeric gramicidin channel is formed in a membrane of thickness greater than the length of the channel, the membrane deformation reduces the stability of the channel. Previously this effect was studied by comparing the variation of channel lifetime with the surface tension of bilayers (Elliott, J. R., D. Needham, J. P. Dilger, and D. A. Hayden, 1983, Biochim. Biophys. Acta, 735:95-103). The tension was assumed to pull a dimer for a distance z before the channel loses ion conductivity. To account for the data, z was found to be 18 A. With the deformation free energy, the data can be accounted for with z less than or approximately to 1 A, which is consistent with the breaking of hydrogen bonds in a dimer dissociation. Increasing the strength of lipid-protein interactions is not the only consequence of the complete free energy compared with the previous discussions. It also changes the shape of membrane deformation around an embedded channel from convex to concave, and increases the range of deformation from less than 10 A to greater than 20 A. Clearly these will be important factors in the general considerations of lipid-protein interactions and membrane-mediated interactions between proteins. In addition, thermal fluctuations of a membrane are calculated; in particular, we calculate the relations between the intrinsic thickness and the experimentally measured values. The experimental parameters of monoolein-squalene membranes are used for quantitative analyses.     

Measurements of local pH changes near bilayer lipid membrane by means of a pH microelectrode and a protonophore-dependent membrane potential. Comparison of the methods.

Shifts of pH near the bilayer lipid membrane (BLM) were measured in the absence of pH difference between bulk solutions by two methods, i.e. pH microelectrode and membrane potential recordings in the presence of a protonophore. A quantitative agreement of the results of both methods was obtained. The kinetics of the generation of potential induced by the addition of ammonium chloride was accounted for by the time of the diffusion through the unstirred layers. The thickness of the unstirred BLM layers was determined in the experiment.     

Porins from marine bacteria of the genus Pseudoalteromonas (Gammaproteobacteria: Pseudoalteromonadaceae)

Outer membrane (OM) fractions were isolated from marine bacteria of the genus Pseudoalteromonas (P. haloplanktis, P. tetraodonis, and Pseudoalteromonas sp. KMM 223). The purity of OM fractions was confirmed by ultracentrifugation in a sucrose gradient. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the bilayer lipid membrane (BLM) technique, heat-modifiable porin-like proteins were identified among the OM proteins of marine pseudomonads. The pore-forming P-1 and P-2 proteins with molecular masses of 43 and 39 kDa, respectively, were obtained from the marine bacterium P. haloplanktis. The nature of current fluctuations in the BLM and the conductivity of pores formed by these proteins suggest that these isolated porins are not identical in their functional properties. A nonlinear dependence of channel conductivity on salt concentration in the aqueous phase was found for the P-2 protein, which is typical of marine bacterial porins.     

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.     

The dependence of the conductance and lifetime of gramicidin channels on the thickness and tension of lipid bilayers

The lifetimes of channels formed by natural gramicidin and its dimeric analog in monoglyceride lipid bilayers of various compositions were investigated. The bilayer surface tension was altered by changing the length of the monoglycerides' fatty acid chain or the chain length of hydrocarbon solvent by isomerization or saturation of the lipid, by varying the amount of solvent in the bilayer, and by changing the salt composition of the aqueous solutions. The logarithms of mean channel lifetimes were found to be proportional to the surface tension of the membrane irrespective of how the surface tension was changed. In contrast, no simple relationship between channel conductance and surface tension or bilayer thickness was found.