Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes

This paper presents the application of chronopotentiometry in the study of membrane electroporation. Chronopotentiometry with a programmable current intensity was used. The experiments were performed on planar bilayer phosphatidylcholine and cholesterol membranes formed by the Mueller-Rudin method. It was demonstrated that a constant-intensity current flow through the bilayer membranes generated voltage fluctuations during electroporation. These fluctuations (following an increase and decrease in membrane conductance) were interpreted as a result of the opening and closing of pores in membrane structures. The decrease in membrane potential to zero did not cause the pore to close immediately. The pore was maintained for about 200 s. The closing of the pore and recovery of the continuous structure of the membrane proceeded not only when the membrane potential equalled zero, but also at membrane potentials up to several tens of millivolts. The fluctuations of the pore were possible at values of membrane potential in the order of at least 100 mV. The size of the pore changed slightly and it closed after some time below this potential value.     

Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes

This paper presents the application of chronopotentiometry in the study of membrane electroporation. Chronopotentiometry with a programmable current intensity was used. The experiments were performed on planar bilayer phosphatidylcholine and cholesterol membranes formed by the Mueller-Rudin method. It was demonstrated that a constant-intensity current flow through the bilayer membranes generated voltage fluctuations during electroporation. These fluctuations (following an increase and decrease in membrane conductance) were interpreted as a result of the opening and closing of pores in membrane structures. The decrease in membrane potential to zero did not cause the pore to close immediately. The pore was maintained for about 200 s. The closing of the pore and recovery of the continuous structure of the membrane proceeded not only when the membrane potential equalled zero, but also at membrane potentials up to several tens of millivolts. The fluctuations of the pore were possible at values of membrane potential in the order of at least 100 mV. The size of the pore changed slightly and it closed after some time below this potential value.     

Modeling electroporation in a single cell. I. Effects Of field strength and rest potential.

This study develops a model for a single cell electroporated by an external electric field and uses it to investigate the effects of shock strength and rest potential on the transmembrane potential V(m) and pore density N around the cell. As compared to the induced potential predicted by resistive-capacitive theory, the model of electroporation predicts a smaller magnitude of V(m) throughout the cell. Both V(m) and N are symmetric about the equator with the same value at both poles of the cell. Larger shocks do not increase the maximum magnitude of V(m) because more pores form to shunt the excess stimulus current across the membrane. In addition, the value of the rest potential does not affect V(m) around the cell because the electroporation current is several orders of magnitude larger than the ionic current that supports the rest potential. Once the field is removed, the shock-induced V(m) discharges within 2 micros, but the pores persist in the membrane for several seconds. Complete resealing to preshock conditions requires approximately 20 s. These results agree qualitatively and quantitatively with the experimental data reported by Kinosita and coworkers for unfertilized sea urchin eggs exposed to large electric fields.     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

Pore disappearance in a cell after electroporation: theoretical simulation and comparison with experiments.

The process of pore disappearance after cell electroporation is analyzed theoretically. On the basis of the kinetic model, in which the formation and annihilation of a metastable hydrophilic pore are considered as random one-step processes, a distribution function of cell resealing times, Fr(t), is derived. Two cases are studied: 1) the rate of pore resealing, k(r), is significantly greater than the rate of pore formation, k(f); and 2) the rate of pore formation, k(f), is comparable with k(r). It is determined that the shape of the distribution function depends on the initial number of pores in a cell, n(i). If in the absence of an external electric field the rate of pore formation, k(f), is significantly less than the rate of pore resealing, k(r) (case 1), pores disappear completely, whereas when k(f) approximately k(r) (case 2), the cell achieves a steady state in which the number of pores is equal to k(f)/k(r). In case 1, when n(i) = 1, the distribution function Fr(t) is exponential. The developed theory is compared with experimental data available in the literature. Increasing the time of incubation at elevated temperature increases the fraction of resealed cells. This indicates that the time necessary for the resealing varies from cell to cell. Although the shape of experimental relationships depends on the electroporation conditions they can be described by theoretical curves quite well. Thus it can be concluded that the disappearance of pores in the cell membrane after electroporation is a random process. It is shown that from the comparison of presented theory with experiments, the following parameters can be estimated: the average number of pores, n(i), that appeared in a cell during an electric pulse; the rate of pore disappearance, k(r); the ratio k(f)/k(r); and the energy barrier to pore disappearance deltaWr(0). Estimated numerical values of the parameters show that increasing the amplitude of an electric pulse increases either the apparent number of pores created during the pulse (the rate of pore resealing remains the same) or the rate of pore resealing (the average number of pores remains the same).     

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.     

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.     

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.     

Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either never greater than 0.5 nm in radius, much smaller in number if they were greater than 0.5 nm in radius, or shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.     

An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.     

Soft perforation of planar bilayer lipid membranes of dipalmitoylphosphatidylcholine at the temperature of the phase transition from the liquid crystalline to the gel state

In contrast to the widely used method of electroporation, the method of soft perforation of lipid bilayers is proposed. It is based on the structural rearrangement of the lipid bilayer formed from disaturated phospholipids at the temperature of the phase transition from the liquid crystalline state to the gel state. This allows us to obtain a lipid pore population without the use of a strong electric field. It is shown that the planar lipid bilayer membrane (pBLM) formed from dipalmitoylphosphatidylcholine in 1 M LiCl aqueous solution exhibits the appearance of up to 50 lipid pores per 1 mm² of membrane surface, with an average single pore conductivity of 31±13 nS. The estimation of a single pore radius carried out with water-soluble poly(ethylene glycol)s (PEGs) showed that the average pore radius ranged between 1.0–1.7 nm. It was found experimentally that PEG-1450, PEG-2000, and PEG-3350 should be in a position to block the single pore conductivity completely, while PEG-6000 fully restored the ionic conductivity. The similarity of these PEG effects to ionic conductivity in protein pores makes it possible to suggest that the partition of the PEG molecules between the pore and the bulk solution does not depend on the nature of the chemical groups located in the pore wall.     

Molecular-Level Characterization of Lipid Membrane Electroporation using Linearly Rising Current

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-μm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.     

Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores.

It has been shown recently that electrically induced DNA transfer into cells is a fast vectorial process with the same direction as DNA electrophoresis in an external electric field (Klenchin, V. A., S. I. Sukharev, S. M. Serov, L. V. Chernomordik, and Y. A. Chizmadzhev. 1991. Biophys. J. 60:804-811). Here we describe the effect of DNA interaction with membrane electropores and provide additional evidences for the key role of DNA electrophoresis in cell electrotransfection. The assay of electrically induced uptake of fluorescent dextrans (FDs) by cells shows that the presence of DNA in the medium during electroporation leads to a sharp increase in membrane permeability to FDs of M(r) < 20,000. The permeability increases with DNA concentration and the effect is seen even if FD is added to the cell suspension a few minutes after pulse application. The longer the DNA fragment, the greater the increase in permeability. The use of a two-pulse technique allows us to separate two effects provided by a pulsed electric field: membrane electroporation and DNA electrophoresis. The first pulse (6 kV/cm, 10 microseconds) creates pores efficiently, whereas transfection efficiency (TE) is low. The second pulse of much lower amplitude, but substantially longer (0.2 kV/cm, 10 ms), does not cause poration and transfection by itself but enhances TE by about one order of magnitude. In two-pulse experiments, TE rises monotonously with the increase of the second pulse duration. By varying the delay duration between the two pulses, we estimate the lifetime of electropores (which are DNA-permeable in conditions of low electric field) as tens of seconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electroporation of the photosynthetic membrane: A study by intrinsic and external optical probes

The study examines the relationship between electric field-induced conductivity and permeability changes in a biological membrane (electroporation) and the amplitude-duration parameters of the externally applied electric field. These reversible changes were characterized in giant photosynthetic membrane vesicles by means of the calibrated response of an intrinsic voltage-sensitive optical probe (electrophotoluminescence) and by the uptake studies of dextran-FITC fluorescent probes of different molecular weights. We quantitatively monitored electric field-induced conductivity changes by translating the electrophotoluminescence changes into conductivity changes. This was carried out by measuring the attenuation of the electrophotoluminescent signal after the addition of known amounts of gramicidin. The results demonstrate that electroporation involves the reversible formation of discrete holes in the membrane having radii <5.8 nm. The total area of the electric field-induced holes was 0.075% of the total surface of the vesicle. The formation of the electropores was affected differently by the electric field strength than by its duration. Increase in electric field strength caused increase in the total area of the vesicle that undergoes electroporation. Increase in the duration of the electric field increases the area of single electropores. Each of the two electric parameters can be rate limiting for the dynamics of electropore formation. These results are in accordance with the model of electroporation based on electric field-induced expansion of transient aqueous holes.     

Self-electroporation as a model for fusion pore formation

The creation of a small opening called the fusion pore is a necessary prerequisite for neurotransmitter release from synaptic vesicles. It is known that high intensity electric fields can create pores in vesicles by a process called electroporation. Due to the presence of charged phosphatidylserine (PS) molecules on the inner leaflet of the cell membrane, an electric field that is strong enough to cause electroporation of a synaptic vesicle might be present. It was shown by K. Rosenheck [K. Rosenheck. Biophys J 75, 1237-1243 (1998)] that in a planar geometry, fields sufficient to cause electroporation can occur at intermembrane separations of less than approximately 3 nm. It is frequently found, however, that the cell membrane is not planar but caves inward at the locations where a vesicle is close to it. Indentation of the cell membrane in the fusion region was modelled as a hemisphere and a theoretical study of the electric field in the vicinity of the cell membrane taking into account the screening effect of dissolved ions in the cytoplasm was performed. It was discovered that fields crossing the electroporation threshold occurred at a distance of 2 nm or less, supporting the claim that electroporation could be a possible mechanism for fusion pore formation.     

Influence of Polyethylene Glycol on the Size of Schizosaccharomyces pombe Electropores

The role of polyethylene glycol (PEG) in the transformation of Schizosaccharomyces pombe by electroporation is investigated by fluorescein isothiocyanate-dextran uptake and transformation studies. It is shown that when S. pombe cells are electroporated in the presence of PEG, the permeability state created is sustained until removal of PEG. In addition, the permeability of electroporated S. pombe envelopes is further increased with longer incubation times in PEG. The increased permeability is apparently a result of enlarged pores (electropores) due to the presence of PEG. Comparison of a heat pulse transformation protocol with electroporation suggests a second role for PEG in the uptake of macromolecules. Since pores are not thought to be created during a heat pulse, the PEG may be facilitating the uptake of plasmid DNA. This facilitation of uptake would also be expected to affect DNA uptake by electroporated cells.     

GM1 asymmetry in the membrane stabilizes pores

Cell membranes are highly asymmetric and their stability against poration is crucial for survival. We investigated the influence of membrane asymmetry on electroporation of giant unilamellar vesicles with membranes doped with GM1, a ganglioside asymmetrically enriched in the outer leaflet of neuronal cell membranes. Compared with symmetric membranes, the lifetimes of micronsized pores are about an order of magnitude longer suggesting that pores are stabilized by GM1. Internal membrane nanotubes caused by the GM1 asymmetry, obstruct and additionally slow down pore closure, effectively reducing pore edge tension and leading to leaky membranes. Our results point to the drastic effects this ganglioside can have on pore resealing in biotechnology applications based on poration as well as on membrane repair processes.     

Electroporative deformation of salt filled lipid vesicles

Membrane electroporation, vesicle shape deformation and aggregation of small, NaCl-filled lipid vesicles (of radius a = 50 nm) in DC electric fields was characterized using conductometric and turbidimetrical data. At pulse durations t_E≤ 55 ± 5 ms the increase in the conductivity of the vesicle suspension is due to the field-induced efflux of electrolyte through membrane electropores. Membrane electroporation and Maxwell stress on the vesicle membrane lead to vesicle elongation concomitant with small volume reduction (up to 0.6% in an electric field of E = 1 MV m^–1). At t_E > 55 ± 5 ms, further increases in the conductivity and the optical density suggest electroaggregation and electrofusion of vesicles. The conductivity changes after the electric pulse termination reflect salt ion efflux through slowly resealing electropores. The analysis of the volume reduction kinetics yields the bending rigidity κ = (4.1 ± 0.3) ⋅ 10^–20 J of the vesicle membrane. If the flow of Na⁺ and Cl^– ions from the vesicle interior is treated in terms of Hagen-Poiseuille's equation, the number of permeable electropores is N = 39 per vesicle with mean pore radius r_p = 0.85 ± 0.05 nm at E = 1 MVm^–1 and t_E≤ 55 ± 5 ms. The turbidimetric and conductometric data suggest that small lipid vesicles (a ≤ 50 nm) are not associated with extensive membrane thermal undulations or superstructures. In particular with respect to membrane curvature, the vesicle results are suggestive for the design and optimization of electroporative delivery of drugs and genes to cell tissue at small field strengths (≤1 MVm^–1) and large pulse durations (≤100 ms). Received: 8 July 1997 / Accepted: 15 September 1997     

Self-electroporation as a Model for Fusion Pore Formation

Abstract

The creation of a small opening called the fusion pore is a necessary prerequisite for neurotransmitter release from synaptic vesicles. It is known that high intensity electric fields can create pores in vesicles by a process called electroporation. Due to the presence of charged phosphatidylserine (PS) molecules on the inner leaflet of the cell membrane, an electric field that is strong enough to cause electroporation of a synaptic vesicle might be present. It was shown by K. Rosenheck [K. Rosenheck. Biophys J 75, 1237–1243 (1998)] that in a planar geometry, fields sufficient to cause electroporation can occur at intermembrane separations of less than ～3 nm. It is frequently found, however, that the cell membrane is not planar but caves inward at the locations where a vesicle is close to it. Indentation of the cell membrane in the fusion region was modelled as a hemisphere and a theoretical study of the electric field in the vicinity of the cell membrane taking into account the screening effect of dissolved ions in the cytoplasm was performed. It was discovered that fields crossing the electroporation threshold occurred at a distance of 2 nm or less, supporting the claim that electroporation could be a possible mechanism for fusion pore formation.