Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

Osmotic hemolysis and fragility A new model based on membrane disruption,and a potential clinical test

Red cell osmotic hemolysis has traditionally been defined by the loss of hemoglobin, in response to reduced osmotic pressure, as measured spectroscopically. Previous work from this laboratory using resistive pulse spectroscopy (RPS) has shown that in a mixed population of hemolyzing cell, ghosts can be detected as being more deformable, and hence appearing distinctly smaller, than the remaining intact cells. Other researchers using similar methods have reported detection of ghosts as apparently smaller objects, resulting from their greater sensitivity to dielectric breakdown. We now confirm both of these results, and demonstrate by kinetic studies that changes which occur in the rheological and electrical properties of ghosts are independent phenomena. We include in our analysis the explicit calculation of ghost and intact spherocyte resistivity after dielectric breakdown. The two different characterizations for ghosts are integrated into a proposed model of osmotic hemolysis based on known red blood cell membrane and cytoplasmic properties. This work provides both a theoretical and a practical foundation for RPS-based measures of osmotic fragility, including a potential new clinical test, measures which provide very early detection of the ultimate fate of osmotically stressed red cells.     

Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter.Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar dependence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown.The results are consistent with the electromechanical model suggested for the dielectric breakdown mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

Cell poration and cell fusion using an oscillating electric field.

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.     

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.     

Schwan equation and transmembrane potential induced by alternating electric field.

The transmembrane potential generated by an alternating electric field (ac) depends strongly on the frequency of the field and can be calculated using the Schwan Equation. We have measured the critical electric breakdown potential, delta psi crit, of the plasma membrane of murine myeloma cell line (Tib9) using ac fields, by monitoring the entry of a fluorescence probe, propidium iodide, into the cells. This dye is weakly fluorescent in solution but becomes strongly fluorescent when it binds to DNA. Experiments were done under a microscope by direct visual examination of single cells or by examining photographic prints. When an ac field reached the intensity, Ecrit, that generated a maximal membrane potential delta psi max, equal to or greater than the delta psi crit, the membrane was perforated at the two loci facing the electrodes. The dye diffused into the cell, giving rise to two bright, narrow bands, which expanded to the whole cell in 1-3 min. delta psi crit's were measured in three media of different resistivities, rho ext, (52,600, 7,050, and 2,380 omega cm), over the range of 0.1-300 kHz, with the field duration of 200 ms. Regression analysis based on the Schwan Equation showed that in a medium of given resistivity, the delta psi crit was constant over the frequency range studied. When the capacitance of the membrane, Cmembr, was taken to be 0.90 microF cm-2, the resistivity of the cytoplasmic medium, rho int, was determined to be 910-1,100 omega cm. The delta psi crit were 0.33, 0.48, and 0.53 V, respectively, for the three media in decreasing resistivities.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effect of Electrical Deformation Forces on the Electropermeabilization of Erythrocyte Membranes in Low- and High-Conductivity Media

Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks' in the plasma membrane generated by electric breakdown. Received: 12 December 1997/Revised: 13 March 1998     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Electrical hemolysis of human and bovine red blood cells

Summary The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50% hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50% hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 Ω - cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

Electrophotoluminescence and the electrical properties of the photosynthetic membrane. II. Electric field-induced electrical breakdown of the photosynthetic membrane and its recovery

Preilluminated suspensions of swollen thylakoid vesicles (‘blebs’) were exposed to uni- and bipolar pairs of identical electric field pulses of variable duration, intensity and spacing. The resulting field-stimulated luminescence (electrophotoluminescence) was used as an intrinsic, voltage-sensitive optical probe to monitor electrical phenomena at the membrane level. The application of a pair of voltage pulses of opposite polarity made it possible to produce electric changes in the membrane by the first pulse and to analyse these effects by a second pulse of opposite polarity. It was found that the relative amplitudes of the two electrophoto-luminescence signals depended on the intensity of the applied electric field and on the time interval (t*) between the two pulses. When t* varied from 0.4 to 12 ms, the second stimulated luminescence signal was at first much smaller than the first one and then increased exponentially until the two signals were equal for t* ≥ 3 ms. We analysed these differences between the two field-stimulated luminescence signals as a measure of the electrical breakdown of the membrane, induced during the first pulse. In this way a distinction between irreversible and reversible breakdown could be made with an estimation of the recovery kinetics of the reversible breakdown, which was found to be complete within 3 ms. Irreversible breakdown of the membrane was found to increase with lengthening the exposure time from 0.1 to 1.3 ms especially when applying high electric field of at least 2000 V/cm.     

Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability

By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 s/1.5 kV*cm^–1 and 200 s/0.5 kV*cm^–1, hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), as sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7 +/- 7.49 ohm-cm) compared to the AA (147.6 +/- 1.98 ohm-cm) or AS cells (148.7 +/- 1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2 +/- 5.25 ohm-cm, compared to an average of 153.5 +/- 3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

High frequency fusion of plant protoplasts by electric fields

Mesophyll cell protoplasts of Vicia faba were collected by dielectrophoresis in a highly inhomogeneous alternating electric field (sine wave, 5 to 10 V peak-to-peak value, 500 kHz, electrode distance 200 m). Under these conditions, the cells formed aggregates of two or three on the electrodes or bridges consisting of 4 to 6 protoplasts between the electrodes. This pearl chain arrangement of the cells was only stable for the duration of the applied field. By the additional application of a high single field pulse (square wave, 15 V, 50 s), it was possible to induce cell fusion within the aggregates or bridges. This electrically stimulated fusion of cells proceeded at room temperature and under physiological pH-conditions, without the use of chemical reagents, and gave a high yield. Smaller fused aggregates formed spheres within a few minutes. During the dielectrophoretically induced adhesion of the protoplasts to one another, the field strength must be chosen such that dielectric breakdown of the membrane is avoided, but at the same time, the strength of the subsequently applied single field pulse must be high enough to induce dielectric breakdown at the sites of contact between the protoplast membranes. From these results, one can conclude that in addition to close contact between membranes, the prerequisite for electrically stimulated cell fusion is dielectric breakdown which leads to changes in the membrane conductance, permeability, and probably fluidity.Presented at II Congress FESPP, Santiago de Compostela, Spain, 27.7.–1.8.1980, and Gordon Research Conference of Bioelectrochemistry, Tilton, New Hampshire, USA, 4.8.–8.8.1980     

Stochastic model for electric field-induced membrane pores. Electroporation

Electric impulses (1-20 kV cm-1, 1-5 microseconds) cause transient structural changes in biological membranes and lipid bilayers, leading to apparently reversible pore formation ( electroporation ) with cross-membrane material flow and, if two membranes are in contact, to irreversible membrane fusion ( electrofusion ). The fundamental process operative in electroporation and electrofusion is treated in terms of a periodic lipid block model, a block being a nearest-neighbour pair of lipid molecules in either of two states: (i) the polar head group in the bilayer plane or (ii) facing the centre of a pore (or defect site). The number of blocks in the pore wall is the stochastic variable of the model describing pore size and stability. The Helmholtz free energy function characterizing the transition probabilities of the various pore states contains the surface energies of the pore wall and the planar bilayer and, if an electric field is present, also a dielectric polarization term (dominated by the polarization of the water layer adjacent to the pore wall). Assuming a Poisson process the average number of blocks in a pore wall is given by the solution of a non-linear differential equation. At subcritical electric fields the average pore size is stationary and very small. At supercritical field strengths the pore radius increases and, reaching a critical pore size, the membrane ruptures (dielectric breakdown). If, however, the electric field is switched off, before the critical pore radius is reached, the pore apparently completely reseals to the closed bilayer configuration (reversible electroporation ).     

Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown

Leaks were induced in human erythrocytes by brief (tau = 1-40 microseconds) discharges of high electric fields (3-20 kV/cm). Leak permeabilities were characterized by measuring (a) net and tracer fluxes of K+ and nonelectrolytes under protection of the cells against colloid-osmotic lysis, or (b) rates of colloid osmotic lysis in various salt solutions. The induced permeabilities are essentially stable for hours at 0-2 degrees C. Leak permeability P increases exponentially with the breakdown voltage ED according to a function of the general type P = bED. The basis b varies with the pulse length. A log-linear presentation reveals a biphasic linear relationship with a break at which the slope (= log b) decreases markedly. Elevated ionic strengths of the suspension medium during the electric discharge enhance leak formation. Leak permeability exhibits an apparent activation energy of 29 +/- 5 kJ/mol, indicative of diffusion through aqueous pathways. Somewhat differing equivalent pore radii emerge from measurements with different probes: 0.6-0.8 nm from tracer fluxes of polyols (Mr = 3600, ED = 4-7 kV/cm) and 0.8-1.9 nm from osmotic protection studies with polyethylene glycols (Mr = 200-3300, ED = 6-10 kV/cm). These numbers and the non-monoexponential increase of leak permeability with the field strength suggest a dual mechanism for the increase of leak permeability: an increase of the number of pores at low breakdown voltage and an additional increase of pore size at higher voltage. Estimated numbers of pores range from 1 to 10 per cell, which suggests dynamic fluctuating structural defects to be involved. The leaks discriminate small monovalent inorganic ions in the sequence of free solution mobility. Organic anions are discriminated according to size and charge. Common properties of these electrically induced defects and of chemically induced leaks (diamide, periodate, t-butylhydroperoxide) in the erythrocyte membrane suggest close similarities in the molecular organization.