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1.
Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. 总被引:22,自引:10,他引:12
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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. 相似文献
2.
Pore disappearance in a cell after electroporation: theoretical simulation and comparison with experiments.
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G Saulis 《Biophysical journal》1997,73(3):1299-1309
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). 相似文献
3.
The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of the lipid headgroups could impede pore resealing. 相似文献
4.
Life Cycle of an Electropore: Field-Dependent and Field-Independent Steps in Pore Creation and Annihilation 总被引:1,自引:0,他引:1
Electropermeabilization, an electric field-induced modification of the barrier functions of the cell membrane, is widely used
in laboratories and increasingly in the clinic; but the mechanisms and physical structures associated with the electromanipulation
of membrane permeability have not been definitively characterized. Indirect experimental observations of electrical conductance
and small molecule transport as well as molecular dynamics simulations have led to models in which hydrophilic pores form
in phospholipid bilayers with increased probability in the presence of an electric field. Presently available methods do not
permit the direct, nanoscale examination of electroporated membranes that would confirm the existence of these structures.
To facilitate the reconciliation of poration models with the observed properties of electropermeabilized lipid bilayers and
cell membranes, we propose a scheme for characterizing the stages of electropore formation and resealing. This electropore
life cycle, based on molecular dynamics simulations of phospholipid bilayers, defines a sequence of discrete steps in the
electric field-driven restructuring of the membrane that leads to the formation of a head group-lined, aqueous pore and then,
after the field is removed, to the dismantling of the pore and reassembly of the intact bilayer. Utilizing this scheme we
can systematically analyze the interactions between the electric field and the bilayer components involved in pore initiation,
construction and resealing. We find that the pore creation time depends strongly on the electric field gradient across the
membrane interface and that the pore annihilation time is at least weakly dependent on the magnitude of the pore-initiating
electric field and, in general, much longer than the pore creation time. 相似文献
5.
《生物化学与生物物理学报:生物膜》1985,815(1):128-134
Following lysis in hypotonic media, human erythrocyte membranes will spontaneously reseal and regain their original low permeability for polar solutes. It is generally accepted that resealing will only occur when the membranes are heated above a critical temperature, and that the membrane lesions are stable under cold conditions. Contrary to these prevailing notions, a detailed investigation of the temperature dependence of resealing kinetics over the temperature range 0–22°C revealed that resealing occurs at measurable rates at temperatures as low as 0°C, even in buffers of low ionic strength. At all temperatures studied, initial resealing rates were approximately first-order, and Arrhenius plots of these rates revealed a sharp, singular discontinuity at approx. 7°C. 相似文献
6.
Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells. 总被引:1,自引:0,他引:1
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When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells. 相似文献
7.
Strain‐Based In Situ Study of Anion and Cation Insertion into Porous Carbon Electrodes with Different Pore Sizes
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Jennifer M. Black Guang Feng Pasquale F. Fulvio Patrick C. Hillesheim Sheng Dai Yury Gogotsi Peter T. Cummings Sergei V. Kalinin Nina Balke 《Liver Transplantation》2014,4(3)
The expansion of porous carbon electrodes in a room temperature ionic liquid (RTIL) is studied using in situ atomic force microscopy (AFM). The effect of carbon surface area and pore size/pore size distribution on the observed strain profile and ion kinetics is examined. Additionally, the influence of the potential scan rate on the strain response is investigated. By analyzing the strain data at various potential scan rates, information on ion kinetics in the different carbon materials is obtained. Molecular dynamics (MD) simulations are performed to compare with and provide molecular insights into the experimental results; this is the first MD work investigating the pressure exerted on porous electrodes under applied potential in a RTIL electrolyte. Using MD, the pressure exerted on the pore wall is calculated as a function of potential/charge for both a micropore (1.2 nm) and a mesopore (7.0 nm). The shape of the calculated pressure profile matches closely with the strain profiles observed experimentally. 相似文献
8.
Sun S Yin G Lee YK Wong JT Zhang TY 《Biochemical and biophysical research communications》2011,(2):684-688
Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(mol Å2) in the external electric field of 1.4 kcal/(mol Å e), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(mol Å2) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(mol Å e), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease. 相似文献
9.
In this paper, we report the results of a systematic attempt to relate the intrinsic plasma membrane fluidity of three different
cell lines to their electroporation behaviour, which consists of reversible and irreversible electroporation. Apart from electroporation
behaviour of given cell lines the time course required for membrane resealing was determined in order to distinguish the effect
of resealing time from the cell’s ability to survive given electric pulse parameters. Reversible, irreversible electroporation
and membrane resealing were then related to cell membrane fluidity as determined by electron paramagnetic resonance spectroscopy
and computer characterization of membrane domains. We found that cell membrane fluidity does not have significant effect on
reversible electroporation although there is a tendency for the voltage required for reversible electroporation to increase
with increased membrane fluidity. Cell membrane fluidity, however, may affect irreversible electroporation. Nevertheless,
this effect, if present, is masked with different time courses of membrane resealing found for the different cell lines studied.
The time course of cell membrane resealing itself could be related to the cell’s ability to survive. 相似文献
10.
Ming-Chak Ho Zachary A. Levine P. Thomas Vernier 《The Journal of membrane biology》2013,246(11):793-801
Formation of a water bridge across the lipid bilayer is the first stage of pore formation in molecular dynamic (MD) simulations of electroporation, suggesting that the intrusion of individual water molecules into the membrane interior is the initiation event in a sequence that leads to the formation of a conductive membrane pore. To delineate more clearly the role of water in membrane permeabilization, we conducted extensive MD simulations of water bridge formation, stabilization, and collapse in palmitoyloleoylphosphatidylcholine bilayers and in water–vacuum–water systems, in which two groups of water molecules are separated by a 2.8 nm vacuum gap, a simple analog of a phospholipid bilayer. Certain features, such as the exponential decrease in water bridge initiation time with increased external electric field, are similar in both systems. Other features, such as the relationship between water bridge lifetime and the diameter of the water bridge, are quite different between the two systems. Data such as these contribute to a better and more quantitative understanding of the relative roles of water and lipid in membrane electropore creation and annihilation, facilitating a mechanism-driven development of electroporation protocols. These methods can be extended to more complex, heterogeneous systems that include membrane proteins and intracellular and extracellular membrane attachments, leading to more accurate models of living cells in electric fields. 相似文献
11.
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. 相似文献
12.
In vivo electroporation (EP) is gaining momentum for drug and gene delivery. In particular, DNA transfer by EP to muscle tissue can lead to highly efficient long-term gene expression. We characterized a vascular effect of in vivo EP and its consequences for drug and gene delivery. Pulses of 10-20,000 micros and 0.1-1.6 kV/cm were applied over hind- and forelimb of mice and perfusion was examined by dye injection. The role of a sympathetically mediated vasoconstrictory reflex was investigated by pretreatment with reserpine. Expression of a transferred gene (luciferase), permeabilization (determined using (51)Cr-EDTA), membrane resealing and effects on perfusion were compared to assess the significance of the vascular effects. Above the permeabilization threshold, a sympathetically mediated Raynaud-like phenomenon with perfusion delays of 1-2 min was observed. Resolution of this phase followed kinetics of membrane resealing. Above a second threshold, irreversible permeabilization led to long perfusion delays. These vascular reactions (1) affect kinetics of drug delivery, (2) predict efficient DNA transfer, which is optimal during short perfusion delays, and (3) might explain electrocardiographic ST segment depressions after defibrillation as being caused by vascular effects of EP of cardiac muscle. 相似文献
13.
P. Luitel D. F. Schroeter J. W. Powell 《Journal of biomolecular structure & dynamics》2013,31(5):495-503
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. 相似文献
14.
Tarek M 《Biophysical journal》2005,88(6):4045-4053
We present results of molecular dynamics simulations of lipid bilayers under a high transverse electrical field aimed at investigating their electroporation. Several systems are studied, namely 1), a bare bilayer, 2), a bilayer containing a peptide nanotube channel, and 3), a system with a peripheral DNA double strand. In all systems, the applied transmembrane electric fields (0.5 V.nm(-1) and 1.0 V.nm(-1)) induce an electroporation of the lipid bilayer manifested by the formation of water wires and water channels across the membrane. The internal structures of the peptide nanotube assembly and that of the DNA strand are hardly modified under field. For system 2, no perturbation of the membrane is witnessed at the vicinity of the channel, which indicates that the interactions of the peptide with the nearby lipids stabilize the bilayer. For system 3, the DNA strand migrates to the interior of the membrane only after electroporation. Interestingly enough, switching of the external transmembrane potential in cases 1 and 2 for few nanoseconds is enough to allow for complete resealing and reconstitution of the bilayer. We provide evidence that the electric field induces a significant lateral stress on the bilayer, manifested by surface tensions of magnitudes in the order of 1 mN.m(-1). This study is believed to capture the essence of several dynamical phenomena observed experimentally and provides a framework for further developments and for new applications. 相似文献
15.
Modeling electroporation in a single cell. I. Effects Of field strength and rest potential.
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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. 相似文献
16.
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. 相似文献
17.
Externally applied voltages can create transient, non-selective pores in a cell’s membrane, a phenomenon known as electroporation. Electroporation has reduced toxicity, is easy to perform, and does not induce the immune system. Therefore, the technique has a wide range of biological and medical applications. Previous experiments show that a two-pulse protocol, which consists of a fast, large-magnitude pulse and a slow, small-magnitude pulse, can increase the efficiency of drug delivery such as gene electrotransfer. In this work, we investigate the dynamics and control of the two-pulse protocol using a macroscopic model of electroporation. Numerical simulations show that there exists a range of pore radii that cannot be sustained using the conventional, open-loop, two-pulse protocol. As a result, one may need to use pores that are significantly larger than the sizes of the targeted molecules. Moreover, it is not possible to know the rate of delivery a priori. To ensure accurate drug delivery and avoid potential damage to the cell’s membrane, we explore feedback mechanisms to eliminate the gap in sustainable pore radii and thus to precisely control the electroporation process. Numerical simulations show that a straightforward feedback algorithm can achieve robust control effects. Moreover, the control algorithm is effective without knowledge of the model and thus has the potential to be implemented in experiments. 相似文献
18.
Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15°C or higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 μM for Ca2+ and 200 μM for Mg2+. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca2+, loss of spectrin does not occur. These results suggest that an intact spectrin network is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described. 相似文献
19.
A theory of osmotic lysis of lipid vesicles 总被引:3,自引:0,他引:3
Osmotic lysis of vesicles is shown to begin when the membrane expansion due to osmotic pressure exceeds its critical value, delta S, at which a membrane ruptures to form a pore. The dependence of delta S on the vesicle radius and respective osmotic pressures are obtained. It is found that osmotic pressure necessary for small (100 A) vesicles to rupture should exceed 30 atm, for large (10 000 A) vesicles it being as small as 10(-3) atm. In the case of large (greater than or approximately 1000 A) vesicles the value of relative expansion of the membrane at which its rupture occurs in a reasonable time only depends slightly on the vesicle radius. For instance, for 10 000 A vesicles it amounts to 3%. The tension of membrane rupture is about 8 dyn/cm for large vesicles. Membrane tension, although it decreases considerably as a result of rupture and pore formation, does not vanish completely. It supports the residual intravesicular pressure causing the efflux of vesicle (cell) contents. Simultaneously, osmotic influx of water through the membrane occurs that results in either complete rupture of the membrane with the efflux of the whole of the contents, or its gradual washout in either of two, quasi-steady or pulse-wise regimes. In the first case a pore is steadily open, whereas in the second case it alternately opens and closes, ejecting about 5% of internal solution each time. Lysis kinetics is analyzed. Pulse-wise regime of lysis is shown to be the most likely one. 相似文献
20.
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. 相似文献