Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential.

Changes in the membrane conductance of sea urchin eggs, during the course of electroporation, were investigated over the time range of 0.5 microsecond to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a rectangular electric pulse of moderate intensity was applied across an egg, a position-dependent potential developed synchronously with the pulse, as theory predicts for a cell with an insulating membrane. From the rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 microF/cm2 and the intracellular conductance 220 omega.cm. Under an electric pulse of much higher intensity, the rise of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequent reversal of the potential development was ascribed to the introduction of finite membrane conductance, or permeabilization of the membrane, by the action of the intense pulse (electroporation). Detailed analysis indicated the following: already at 0.5 microsecond in the rectangular electric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the order of 1 S/cm2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estimated in a second electric pulse of a small amplitude. At least two recovery processes were distinguished, one with a time constant of 7 microseconds and the other 0.5 ms, at the end of which the membrane conductance was already < 0.1 S/cm2.     

Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.

Transmembrane potential was induced in a sea urchin egg by applying a microsecond electric pulse across the cell. The potential was imaged at a submicrosecond time resolution by staining the cell membrane with the voltage-sensitive fluorescent dye RH292. Under moderate electric fields, the spatial distribution of the induced potential as well as its time dependence were in accord with the theoretical prediction in which the cell membrane was regarded as an insulator. At higher field intensities, however, the potential apparently did not fully develop and tended to saturate above a certain level. The saturation is ascribed to the introduction of a large electrical conductance, in the form of aqueous openings, in the membrane by the action of the induced potential (electroporation). Comparison of the experimental and theoretical potential profiles indicates that the two regions of the membrane that opposed the electrodes acquired a high membrane conductance of the order of 1 S/cm2 within 2 microseconds from the onset of the external field. The conductance was similar in the two regions, although permeability in the two regions of the membrane long after the pulse treatment appeared quite different.     

The generation of reactive-oxygen species associated with long-lasting pulse-induced electropermeabilisation of mammalian cells is based on a non-destructive alteration of the plasma membrane.

Chinese hamster ovary (CHO) cells in suspension were subjected to pulsed electric fields suitable for electrically mediated gene transfer (pulse duration longer than 1 ms). Using the chemiluminescence probe lucigenin, we showed that a generation of reactive-oxygen species (oxidative jump) was present when the cells were electropermeabilised using millisecond pulses. The oxidative jump yield was controlled by the extent of alterations allowing permeabilisation within the electrically affected cell area, but showed a saturating dependence on the pulse duration over 1 ms. Cell electropulsation induced reversible and irreversible alterations of the membrane assembly. The oxidative stress was only present when the membrane permeabilisation was reversible. Irreversible electrical membrane disruption inhibited the oxidative jump. The oxidative jump was not a simple feedback effect of membrane electropermeabilisation. It strongly controlled long-term cell survival. This had to be associated with the cell-damaging action of reactive-oxygen species. However, for millisecond-cumulated pulse duration, an accumulation of a large number of short pulses (microsecond) was extremely lethal for cells, while no correlation with an increased oxidative jump was found. Cell responses, such as the production of free radicals, were present during electropermeabilisation of living cells and controlled partially the long-term behaviour of the pulsed cell.     

Reversible and irreversible modification of erythrocyte membrane permeability by electric field

Electric fields of a few kV/cm and of duration in microseconds are known to implant pores of limited size in cell membranes. We report here a study of kinetics of pore formation and reversibility of pores. Loading of biologically active molecules was also attempted. For human erythrocytes in an isotonic saline, pores allowed passive Rb+ entry formed within 0.5 microsecond when a 4 kV/cm electric pulse was used. Pores that admitted oligosaccharides were introduced with an electric pulse of a longer duration in an isosmotic mixture of NaCl and sucrose. These pores were irreversible under most circumstances, but they could be resealed in an osmotically balanced medium. A complete resealing of pores that admitted Rb+ took approximately 40 min at 37 degrees C. Resealing of pores that admitted sucrose took much longer, 20 h, under similar conditions. In other cell types, resealing step may be omitted due to stronger membrane structures. Experimental protocols for loading small molecules into cells without losing cytoplasmic macromolecules are discussed.     

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.     

The effects of intense submicrosecond electrical pulses on cells

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.     

Breakdown of lipid bilayer membranes in an electric field

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO₂²⁺-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO₂²⁺-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.     

Modeling electroporation in a single cell

Electroporation uses electric pulses to promote delivery of DNA and drugs into cells. This study presents a model of electroporation in a spherical cell exposed to an electric field. The model determines transmembrane potential, number of pores, and distribution of pore radii as functions of time and position on the cell surface. For a 1-ms, 40 kV/m pulse, electroporation consists of three stages: charging of the cell membrane (0-0.51 micros), creation of pores (0.51-1.43 micros), and evolution of pore radii (1.43 micros to 1 ms). This pulse creates approximately 341,000 pores, of which 97.8% are small ( approximately 1 nm radius) and 2.2% are large. The average radius of large pores is 22.8 +/- 18.7 nm, although some pores grow to 419 nm. The highest pore density occurs on the depolarized and hyperpolarized poles but the largest pores are on the border of the electroporated regions of the cell. Despite their much smaller number, large pores comprise 95.3% of the total pore area and contribute 66% to the increased cell conductance. For stronger pulses, pore area and cell conductance increase, but these increases are due to the creation of small pores; the number and size of large pores do not increase.     

Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation

Extremely large but very short (20 kV/cm, 300 ns) electric field pulses were reported recently to non-thermally destroy melanoma tumors. The stated mechanism for field penetration into cells is pulse characteristic times faster than charge redistribution (displacement currents). Here we use a multicellular model with irregularly shaped, closely spaced cells to show that instead overwhelming pore creation (supra-electroporation) is dominant, with field penetration due to pores (ionic conduction currents) during most of the pulse. Moreover, the model's maximum membrane potential (about 1.2 V) is consistent with recent experimental observations on isolated cells. We also use the model to show that conventional electroporation resulting from 100 microsecond, 1 kV/cm pulses yields a spatially heterogeneous electroporation distribution. In contrast, the melanoma-destroying pulses cause nearly homogeneous electroporation of cells and their nuclear membranes. Electropores can persist for times much longer than the pulses, and are likely to be an important mechanism contributing to cell death.     

Electroporation by subnanosecond pulses

Electropermeabilization of cell membranes by micro- and nanosecond-duration stimuli has been studied extensively, whereas effects of picosecond electric pulses (psEP) remain essentially unexplored. We utilized whole-cell patch clamp and Di-8-ANEPPS voltage-sensitive dye measurements to characterize plasma membrane effects of 500 ps stimuli in rat hippocampal neurons (RHN), NG108, and CHO cells. Even a single 500-ps pulse at 190 kV/cm increased membrane conductance and depolarized cells. These effects were augmented by applying brief psEP bursts (5–125 pulses), whereas the rate of pulse delivery (8 Hz–1 kHz) played little role. psEP-treated cells displayed large inward current at negative membrane potentials but modest or no conductance changes at positive potentials. A 1-kHz burst of 25 pulses increased the whole-cell conductance in the range (?100)–(?60) mV to 22–26 nS in RHN and NG108 cells (from 3 and 0.7 nS, respectively), but only to 5 nS in CHO (from 0.3 nS). The conductance increase was reversible within about 2 min. Such pattern of cell permeabilization, with characteristic inward rectification and slow recovery, was similar to earlier reported effects of 60- and 600-ns pulses, pointing to the similarity of structural membrane rearrangements in spite of a different membrane charging mechanism.     

Electric Field Pulses Can Induce Apoptosis

Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane. Here we demonstrate that application of supercritical field pulses between 4.5 and 8.1 kV/cm strength and 40 μsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD. Received: 16 December 1998/Revised: 24 February 1999     

Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse

Time courses of electropermeabilization were analyzed during the electric field application using a rapid fluorescent imaging system. Exchanges of calcium ions through electropermeabilized membrane of Chinese hamster ovary cells were found to be asymmetrical. Entry of calcium ions during a millisecond pulse occurred on the anode-facing cell hemisphere. Entry through the region facing the cathode was observed only after the pulse. Leakage of intracellular calcium ions from electropermeabilized cell in low-calcium content medium was observed only from the anode-facing side. The exchanges during the pulse were mostly due to diffusion-driven processes, i.e., governed by the concentration gradient. Interaction of propidium iodide, a dye sensitive to the structural alteration of membrane, with cell membrane was asymmetrical during electropermeabilization. Localized enhancement of the dye fluorescence was observed during and after the pulsation on the cell surface. Specific staining of a limited anode-facing part of the membrane was observed as soon as the pulse was applied. The membrane fluorescence level increased during and immediately after the pulse whereas the geometry of the staining was unchanged. The membrane regions stained by propidium iodide were the same as those where calcium exchanges occurred. The fraction of the membrane on which structural alterations occurred was defined by the field strength. The density of defects was governed by the pulse duration. Electropermeabilization is a localized but asymmetrical process. The membrane defects are created unequally on the two cell sides during the pulse, implying a vectorial effect of the electric field on the membrane.     

Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field

We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca²⁺. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca²⁺ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca²⁺ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells.     

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field‐induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Bioelectromagnetics 34:253–263, 2013. © 2012 Wiley Periodicals, Inc.     

Biological effects of oscillating electric fields: Role of voltage-sensitive ion channels

An alternating component of potential across the membrane of an excitable cell may change the membrane conductance by interacting with the voltagesensing charged groups of the protein macromolecules that form voltage-sensitive ion channels. Because the probability that a voltage sensor is in a given state is a highly nonlinear function of the applied electric field, the average occupancy of a particular state will change in an oscillating electric field of sufficient magnitude. This “rectification” at the level of the voltage sensors could result in conformational changes (gating) that would modify channel conductance. A simplified two-state model is examined where the relaxation time of the voltage sensor is assumed to be considerably faster than the fastest changes of ionic conductance. Significant changes in the occupancy of voltage sensor states in response to an applied oscillating electric field are predicted by the model.     

Nanoelectropulse-induced phosphatidylserine translocation

Nanosecond, megavolt-per-meter, pulsed electric fields induce phosphatidylserine (PS) externalization, intracellular calcium redistribution, and apoptosis in Jurkat T-lymphoblasts, without causing immediately apparent physical damage to the cells. Intracellular calcium mobilization occurs within milliseconds of pulse exposure, and membrane phospholipid translocation is observed within minutes. Pulsed cells maintain cytoplasmic membrane integrity, blocking propidium iodide and Trypan blue. Indicators of apoptosis-caspase activation and loss of mitochondrial membrane potential-appear in nanoelectropulsed cells at later times. Although a theoretical framework has been established, specific mechanisms through which external nanosecond pulsed electric fields trigger intracellular responses in actively growing cells have not yet been experimentally characterized. This report focuses on the membrane phospholipid rearrangement that appears after ultrashort pulse exposure. We present evidence that the minimum field strength required for PS externalization in actively metabolizing Jurkat cells with 7-ns pulses produces transmembrane potentials associated with increased membrane conductance when pulse widths are microseconds rather than nanoseconds. We also show that nanoelectropulse trains delivered at repetition rates from 2 to 2000 Hz have similar effects, that nanoelectropulse-induced PS externalization does not require calcium in the external medium, and that the pulse regimens used in these experiments do not cause significant intra- or extracellular Joule heating.     

Increased sensitivity to epinephrine stimulated lipolysis during starvation: tighter coupling of the adenylate cyclase complex

Histamine, bombesin, and pentagastrin produced different patterns of changes in short circuit current, electric conductance, potential difference, and acid secretion in isolated bullfrog gastric mucosa. Histamine produced a gradual increase in electric conductance, parallel to the increase in acid secretion, and a transient rise in short circuit current. Bombesin induced an abrupt increase in electric conductance and in short circuit current, which peaked after 8 minutes. Pentagastrin also produced an increase in short circuit current, which peaked after 8 minutes; electric conductance, however, rose more gradually. Bombesin produced only a short term increase in acid secretion. These experiments show that histamine, bombesin, and pentagastrin affect gastric mucosa by different mechanisms. Histamine may have a more pronounced effect on the fusion process and activation of the tubulovesicular system of the parietal cell; bombesin may act by transiently increasing the permeability of the basolateral membrane. Pentagastrin seems to have an effect on both the basolateral membrane and the tubulovesicular acid secretory apparatus. These observations are not consistent with the hypothesis that histamine is the final common mediator for the effects of other secretagogues.     

Unique after-hyperpolarization accompanying action potential inChara globularis

A unique after-hyperpolarization was found in internodal cells ofChara globularis. The cells generated an ordinary action potential due to regenerative depolarization induced by the outward electric current pulse larger than a threshold stimulus. After reaching a depolarizing peak, the membrane potential repolarized and overshooted the resting potential to a value which was somehow 40 mV more negative than the resting potential before stimulation (after-hyperpolarization). Since the membrane resistance increased during the after-hyperpolarization, the after-hyperpolarization is thought to be caused by an increase in the resistance (decrease in the conductance) of the passive diffusion channel.     

A Theoretical Study of Single-Cell Electroporation in a Microchannel

Electroporation of a single cell in a microchannel was studied. The effects of electrical (e.g., strength of the electric pulse) and geometrical (e.g., microchannel height, electrode size and position) parameters on cell membrane permeabilization were investigated. The electrodes were assumed to be embedded in the walls of the microchannel; the cell was suspended between these two electrodes. By keeping the electric pulse constant, increasing the microchannel height reduces the number and the radius of the biggest nanopores, as well as the electroporated area of the cell membrane. If the width of the electrodes is bigger than the cell diameter, the transmembrane potential will be centralized and have a sinusoidal distribution around the cell if nanopores are not generated. As the width of the electrode decreases and becomes smaller than the cell diameter, the local transmembrane potential decreases; in the nonelectroporative area, the transmembrane potential distribution deviates from the sinusoidal behavior; the induced transmembrane potential also concentrates around the poles of the cell membrane (the nearest points of the cell membrane to the electrodes). During cell membrane permeabilization, the biggest nanopores are initially created at the poles and then the nanopore population expands toward the equator. The number of the created nanopores reaches its maximal value within a few microseconds; further presence of the electric pulse may not influence the number and location of the created nanopores anymore but will develop the generated nanopores. Strengthening the electric pulse intensifies the size and number of the created nanopores as well as the electroporated area on the cell membrane.     

Fusion of a single influenza virion particle with BLM

One of the key stages of cell infection with influenza virus is the enveloped virus fusion with the cell endosome membrane. To study fusion of single fluorescently-labeled influenza virions with a model bilayer membrane (BLM), a special model system was developed. A small patch of BLM with several adsorbed virions was localized upon a contact with a glass micropipette. Low pH of solution inside the pipette triggered fusion that could be registered by a change in the conductance and integral fluorescence of the BLM patch. It has been shown that the fusion initiation is followed by an increase of fluorescence signal due to the probe redistribution from the virus membrane to the BLM fragment. The increase in fluorescence was accompanied by changes in conductance. Usually, from two to five periods of the channel activity were observed, each of which probably corresponded to fusion of a single virion. It has been found that electric activity was completely inhibited by amantadine known as a blocking agent of M2 channels. This allows one to suggest that the observed changes in conductance are connected with the activity of M2 channels in the virus membrane, whose electric accessibility was the result of fusion of single virions with BLM.