Electro-mechanical permeabilization of lipid vesicles. Role of membrane tension and compressibility.

A simple micropipet technique was used to determine the critical electric field strength for membrane breakdown as a function of the applied membrane tension for three different reconstituted membranes: stearoyloleoylphosphatidylcholine (SOPC), red blood cell (RBC) lipid extract, and SOPC cholesterol (CHOL), 1:1. For these membranes the elastic area expansivity modulus increases from approximately 200 to 600 dyn/cm, and the tension at lysis increases from 5.7 to 13.2 dyn/cm, i.e., the membranes become more cohesive with increasing cholesterol content. The critical membrane voltage, Vc, required for breakdown was also found to increase with increasing cholesterol from 1.1 to 1.8 V at zero membrane tension. We have modeled the behavior in terms of the bilayer expansivity. Membrane area can be increased by either tensile or electrocompressive stresses. Both can store elastic energy in the membrane and eventually cause breakdown at a critical area dilation or critical energy. The model predicts a relation between tension and voltage at breakdown and this relation is verified experimentally for the three reconstituted membrane systems studied here.     

Dependence of the electrical breakdown voltage on the charging time inValonia utricularis

Summary Charge-pulse experiments were performed on giant algal cells ofValonia utricularis. For a charging time of 420 sec the breakdown voltage is about 750 mV (18°C), a value that is in close agreement with earlier results obtained with current pulses (Coster & Zimmermann, 1975;J. Membrane Biol. 22:73). If the membrane is charged to the breakdown voltage in a shorter time, the breakdown voltage is found to be a function of the duration of the charge pulses. Whereas towards smaller pulse lengths down to 10 sec only a small, but significant, increase in the breakdown voltage is observed (1.1 V at 10 sec pulse length and 18°C), a strong increase in the breakdown voltage is found for even shorter charging times. For a pulse length of 800 nsec the breakdown voltage has a value of about 2.4 V (18°C) and a plateau seems to be reached for a pulse duration of 500 nsec. The influence of temperature on the breakdown voltage as observed for short charging times is very similar to that reported earlier for current pulses of 500 sec duration. For charge pulses of 1 to 2 sec duration the breakdown voltage decreases from 3.6 V at 3°C to 1.6 V at 25°C by more than a factor of two.Voltage relaxation studies in the low-field range suggest that the time constants of the two membranes arranged in series, tonoplast and plasmalemma, are similar. From this, it is suggested that both membranes show electrical breakdown, whereby the breakdown voltage of a single membrane is probably half the value of the total breakdown voltage. Its dependence on pulse length is therefore considered to be an intrinsic property of one single membrane. The strong dependence of the breakdown voltage on the charging time of the membrane further supports the interpretation of the breakdown phenomenon on the basis of the electro-mechanical model proposed earlier. In this model it is assumed that the electrical and mechanical compressive forces are counter balanced by elastic restoring forces within the membrane. However, towards very short pulses (less than 800 nsec), where a plateau seems to be reached, other processes may be generated by the application of the electric field. We discuss whether one of these processes is the ion movement through the membranes induced by a high electric field (Born energy).     

Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation.

Membrane patches from chick skeletal muscle were stretched by applying controlled suction or pressure to the pipette. From images of the patch, the patch dimensions (area and radius of curvature) were computed by nonlinear regression of the images to a geometric model. With no applied pressure, patch membranes are nearly planar and normal to the wall of the pipette. With increasing pressure gradients, the patch bulges, the radius of curvature decreases, and the area increases. The patch capacitance changes in exact proportion to the change in area at a rate of 0.7 microF/cm2. The increase in area is due to a flow of lipid (with perhaps small amounts of diffusible protein) along the walls of the pipette into the patch. The flow is reversible with a relaxation of the pressure gradient. The area elastic constant of the membrane is approximately 50 dyn/cm, insensitive to cytochalasin B and probably represents the elasticity of the underlying spectrin/dystrophin network. Simultaneous measurements of stretch activated (SA) ion channel activity in the patch showed that the sensitivity of channels from different patches, although different when calculated as a function of applied pressure, was the same when calculated as a function of tension. Because patch lipid is free to flow, and hence stress-free in the steady state, SA channels must be activated by tension in the cytoskeleton.     

Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm² in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.     

Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study

Summary Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50–200 sec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10⁸ to 0.1 cm²), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential differenceV _c. For 1m alkali ion chloridesV _c was about 1 V. The temperature dependence of the electrical breakdown voltageV _c is comparable to that being observed with cell membranes.V _c decreases between 2 and 48°C from 1.5 to 0.6 V in the presence of 1m KCl.Breakdown experiments were also performed with lipid bilayer membranes composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability.The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).     

The resealing process of lipid bilayers after reversible electrical breakdown

The resealing process of lipid bilayer membranes after reversible electrical breakdown was investigated using two voltage pulses switched on together. Electrical breakdown of the membranes was induced with a voltage pulse of high intensity and short duration. The time course of the change in membrane conductance after the application of the high (short) voltage pulse was measured with a longer voltage pulse of low amplitude. The decrease in membrane conductance during the resealing process could be fitted to a single exponential curve with a time constant of 10-2 μs in the temperature range between 2 and 20°C. The activation energy for this exponential decay process was found to be about 50 kJ/mol, which might indicate a diffusion process. Above 25°C the resealing process is controlled by two exponential processes.The data obtained for the time course of the resealing process can be explained in terms of pore formation in the membranes in response to the high electrical field strength. A radius of about 4 nm is calculated for the initial pore size. From the assumed exponential change of the pore area with progressive resealing time a diffusion constant of 10^?8 cm²/s for lateral lipid diffusion can be estimated.     

Comparison of Na+/K(+)-ATPase pump currents activated by ATP concentration or voltage jumps.

Using the giant patch technique, we combined two fast relaxation methods on excised patches from guinea pig cardiomyocytes to compare the rate constants of the involved reaction steps. Experiments were done in the absence of intra- or extracellular K+. Fast ATP concentration jumps were generated by photolysis of caged ATP at pH 6.3 with laser flash irradiation at a wavelength of 308 nm and 10 ns duration, as described previously. Transient outward currents with a fast rising phase, followed by a slower decay and a small stationary current, were obtained. Voltage pulses were applied to the same patch in the presence or absence of intracellular ATP. Subtraction of the voltage jump-induced currents in the absence of ATP from those taken in the presence of ATP yielded monoexponential transient current signals, which were dependent on external Na+ but did not differ between intracellular pH (pHi) values 6.3 or 7.4. Rate constants showed a characteristic voltage dependence, i.e., saturating at positive potentials (approximately 200 s-1, 24 degrees C) and exponentially rising with increasing negative potentials. Rate constants of the fast component from transient currents obtained after an ATP concentration jump agree well with rate constants from currents obtained after a voltage jump to zero or positive potentials (pHi 6.3), and the two exhibit the same activation energy of approximately 80 kJ.mol-1. For a given membrane patch, the amount of charge that is moved across the plasma membrane is roughly the same for each of the two relaxation techniques.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.     

Voltage-independent Adaptation of Mechanosensitive Channels in Escherichia coli Protoplasts

Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses, which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude, providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation. Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated protein. Received: 29 January 1998/Revised: 16 April 1998     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

Kinetics of cell detachment: peeling of discrete receptor clusters.

Clustering of cell surface adhesion receptors is an essential step in the development of focal contacts, specialized cell-substrate attachment sites where receptors are simultaneously linked to extracellular ligand and cytoskeletal proteins. Previously, we examined the effect of receptor clustering on attachment strength. Here, we employ the numerical methodology developed by Dembo and colleagues (Dembo, M., D.C. Torney, K. Saxman, and D. Hammer. 1988. Proc. R. Soc. Lond. B. 234:55-83) to investigate the kinetics of cell detachment when receptors are clustered into discrete patches. We show that the membrane peeling velocity decreases if receptors are clustered within a patch located inside the contact region. Peeling of clusters is influenced by the chemistry and mechanics of receptor-ligand bonds within the patch. Detachment is also prohibited if the applied tension equals the critical tension of the patch, unless the patch length is small compared with the boundary length over which membrane bending occurs, in which case the patch will peel. Peeling of these short patches only occurs when the mechanical stiffness of clustered bonds is within an optimal range. We compare our model predictions with experimental measurements of T lymphocyte detachment from ICAM-1 substrates. We demonstrate that if discrete patches of receptors are present, detachment occurs through intervals of slow and fast peeling, similar to the dynamics of T lymphocyte peeling, indicating that clustering of LFA-1 receptors is one possible explanation for the observed detachment kinetics in this system.     

Electro-deformation and poration of giant vesicles viewed with high temporal resolution

Fast digital imaging was used to study the deformation and poration of giant unilamellar vesicles subjected to electric pulses. For the first time the dynamics of response and relaxation of the membrane at micron-scale level is revealed at a time resolution of 30 micros. Above a critical transmembrane potential the lipid bilayer ruptures. Formation of macropores (diameter approximately 2 microm) with pore lifetime of approximately 10 ms has been detected. The pore lifetime has been interpreted as interplay between the pore edge tension and the membrane viscosity. The reported data, covering six decades of time, show the following regimes in the relaxation dynamics of the membrane. Tensed vesicles first relax to release the acquired stress due to stretching, approximately 100 micros. In the case of poration, membrane resealing occurs with a characteristic time of approximately 10 ms. Finally, for vesicles with excess area an additional slow regime was observed, approximately 1 s, which we associate with relaxation of membrane curvature. Dimensional analysis can reasonably well explain the corresponding characteristic timescales. Being performed on cell-sized giant unilamellar vesicles, this study brings insight to cell electroporation. The latter is widely used for gene transfection and drug transport across the membrane where processes occurring at different timescales may influence the efficiency.     

Relation between Membrane Potential Changes and Tension in Barnacle Muscle Fibers

Constant current pulses have been applied to single muscle fibers of the barnacle, Balanus nubilus Darwin, with an axial metal electrode. The membrane potential change, which took place over a large part of the muscle fiber, was measured with a similar electrode. Depolarizing pulses, if the voltage was greater than threshold, produced tension. The size of the tension was a function of the magnitude and the duration of the depolarizing pulses. The latency between the onset of depolarization and tension can be only in part attributable to mechanical factors. AC stimulation produced tension, but 5 to 10 seconds were required for the steady-state level of the tension to be reached. Muscles were depolarized in elevated K and studied after the contracture had terminated. If not too depolarized, further depolarization produced tension. Termination of hyperpolarizing pulses also produced tension, which decayed quite slowly. Hyperpolarizing pulses reduced, or abolished, any preexisting tension. Thus, it appears that at certain values of the membrane potential tension is set up, but there is also a slow process of accommodation present.     

Lipid bilayer mechanics in a pipette with glass-bilayer adhesion

Electrophysiology is a central tool for measuring how different driving forces (e.g., ligand concentration, transmembrane voltage, or lateral tension) cause a channel protein to gate. Upon formation of the high resistance seal between a lipid bilayer and a glass pipette, the so-called “giga-seal”, channel activity can be recorded electrically. In this article, we explore the implications of giga-seal formation on the mechanical state of a lipid bilayer patch. We use a mechanical model for the free energy of bilayer geometry in the presence of glass-bilayer adhesion to draw three potentially important conclusions. First, we use our adhesion model to derive an explicit relationship between applied pressure and patch shape that is consistent with the Laplace-Young Law, giving an alternative method of calculating patch tension under pressure. With knowledge of the adhesion constant, which we find to be in the range ∼0.4–4 mN/m, and the pipette size, one can precisely calculate the patch tension as a function of pressure, without the difficultly of obtaining an optical measurement of the bilayer radius of curvature. Second, we use data from previous electrophysiological experiments to show that over a wide range of lipids, the resting tension on a electrophysiological patch is highly variable and can be 10–100 times higher than estimates of the tension in a typical cell membrane. This suggests that electrophysiological experiments may be systematically altering channel-gating characteristics and querying the channels under conditions that are not the same as their physiological counterparts. Third, we show that reversible adhesion leads to a predictable change in the population response of gating channels in a bilayer patch.     

Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields.

Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue electroporation. Observation of reversible electrical breakdown in viable frog skin.

Experiments by others have used isolated cell or bilayer membrane preparations to study the dramatic phenomena associated with electroporation. The present study observes electroporation behavior in an intact tissue. Viable samples of frog skin (Rana pipiens) were exposed to short electrical pulses of varying width and magnitude under "charge injection" conditions. After a pulse, the transtissue potential decayed with two distinct time constants, one short (tau approximately 0.3 ms) and the other longer (tau L approximately 2 ms). Above thresholds for the pulse magnitude and for the pulse width tau L decreased significantly, with progressively smaller tau L as the pulse magnitude and width increased. The postpulse potential, delta Utissue (t), and resistance, Rtissue, also decreased progressively. The tissue subsequently recovered to its original resistance and open circuit potential, delta U tissue,oc, within 2-3 min after a pulse. At that time another pulse experiment could be carried out, demonstrating repeatability and reversibility. No significant permanent changes in Rtissue and delta Utissue,oc were found. This is interpreted as avoidance of significant tissue damage. Taken together, these dramatic phenomena are characteristic of the reversible electrical breakdown previously observed in charge injection experiments with artificial planar bilayer membranes and with isolated cell membranes by similar very short pulses. The present experiments therefore demonstrate that electroporation can be repeatedly caused and observed in a viable tissue without apparent damage.     

Elastic area compressibility modulus of red cell membrane.

Micropipette measurements of isotropic tension vs. area expansion in pre-swollen single human red cells gave a value of 288 +/- 50 SD dyn/cm for the elastic, area compressibility modulus of the total membrane at 25 degrees C. This elastic constant, characterizing the resistance to area expansion or compression, is about 4 X 10(4) times greater than the elastic modulus for shear rigidity; therefore, in situations where deformation of the membrane does not require large isotropic tensions (e.g., in passage through normal capillaries), the membrane can be treated by a simple constitutive relation for a two-dimensionally, incompressible material (i.e. fixed area). The tension was found to be linear and reversible for the range of area changes observed (within the experimental system resolution of 10%). The maximum fractional area expansion required to produce lysis was uniformly distributed between 2 and 4% with 3% average and 0.7% SD. By heating the cells to 50 degrees C, it appears that the structural matrix (responsible for the shear rigidity and most of the strength in isotropic tension) is disrupted and primarily the lipid bilayer resists lysis. Therefore, the relative contributions of the structural matrix and lipid bilayer to the elastic, area compressibility could be estimated. The maximum isotropic tension at 25 degrees C is 10-12 dyn/cm and at 50 degrees C is between 3 and 4 dyn/cm. From this data, the respective compressibilities are estimated at 193 dyn/cm and 95 dyn/cm for structural network and bilayer. The latter value correlates well with data on in vitro, monolayer surface pressure versus area curves at oil-water interfaces.     

An investigation of electrical breakdown of bimolecular lipid membranes

The investigation is concerned with the irreversible electrical breakdown of bimolecular lipid membranes, depending on the velocity of linear voltage scanning. It was found that the membrane breakdown potential depended on the velocity of electric field variation. For instance, at voltage scanning velocities of up to 0.1 V/s, the rupture of membrane from glycerol monooleate occurs at 0.20–0.25 V and, at velocities higher than 1 V/s, at 0.5–0.6 V. Then the film breakdown depending on lipid phase transition was studied. At high velocities of imposed voltage scanning, the disruption of the bimolecular lipid membranes was shown not to depend on their phase states; at the same time, at low velocities, one could note a slight difference in the stability of the films at temperatures higher and lower than those of the phase transition. Whereas transition from gel to liquid-crystalline state involves transition from an ordered to a less ordered membrane structure with a sharp increase in the number of defects in the membrane, the authors, conclude that the film breakdown in the second case occurs by the ‘defect’ mechanism suggested earlier. It was also assumed that, in certain cases involving low velocities of voltage scanning, membrane breakdown may occur because of variation in the interfacial tension and in the contact angle between the film and torus. Possible mechanisms of the membrane irreversible electrical breakdown at high velocities of voltage variation are discussed. It was shown that breakdown should occur as a result of membrane compression in an electric field by a mechanism previously examined. The elastic moduli of a number of membranes were calculated by the breakdown criterion suggested earlier. They were found to coincide with the results of other investigators and, depending on the type of lipid, to equal 10⁵–10⁶ Pa.