Kinetics of pore size during irreversible electrical breakdown of lipid bilayer membranes.

The kinetics of pore formation followed by mechanical rupture of lipid bilayer membranes were investigated in detail by using the charge-pulse method. Membranes of various compositions were charged to a sufficiently high voltage to induce mechanical breakdown. The subsequent decrease of membrane voltage was used to calculate the conductance. During mechanical breakdown, which was probably caused by the widening of one single pore, the membrane conductance was a linear and not exponential function of time after the initial starting process. In a large number of experiments using various lipids and electrolytes, the characteristic opening process of the pore turned out to be independent of the actual membrane potential and electrolyte concentration. Our theoretical analysis of the pore formation suggested that the voltage-induced irreversible breakdown is due to a decrease in edge energy when the pore had formed. After initiation of the pore, the electrical contribution to surface tension is negligible. The time course of the increase of pore size shows that our model of the irreversible breakdown is in good agreement with mechanical properties of membranes reported elsewhere.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Pulse-length dependence of the electrical breakdown in lipid bilayer membranes

Charge-pulse experiments were performed on artificial lipid bilayer membranes with charging times in the range between 10 ns and 10 μs. If the membranes are charged to voltages in the order of 100 mV, the membrane voltage at the end of the charge pulse is a linear function of the injected charge. However, if the membranes are charged to voltages in the range of 1 V, this relationship no longer holds and a reversible high conductance state occurs. This state is defined as an electrical breakdown and it does not allow the membranes to charge to higher voltages than the breakdown voltage, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$. Between charging times of 300 ns and 5 μs at 25°C and between 100 ns and 2 μs at 40°C, $\text{V}{}_{\mathrm{<mtext>c</mtext>}}$ showed a strong dependence on the charging time of the membrane and decreased from 1.2 to 0.5 V (25°C) and from 1 to 0.4 V (40°C). For other charging times below and above these ranges, the breakdown voltage seemed to be constant. The results indicate that the breakdown phenomenon occurs in less than 10 ns.The pulse-length dependence of the breakdown voltage is consistent with the interpretation of the electrical breakdown mechanism in terms of the electromechanical model. However, it seems possible that below a charging time of the membrane of 300 ns (25°C) and 100 ns (40°C) other processes (such as the Born energy) become possible.     

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.     

Reversible electrical breakdown of squid giant axon membrane

Charge pulse relaxation experiments were performed on squid giant axon. In the low voltage range, the initial voltage across squid axon membrane was a linear function of the injected charge. For voltages of the order of 1 V this relationship between injected charge and voltage across the membrane changes abruptly. Because of a high conductance state caused by these large electric fields the voltage across the membrane cannot be made large enough to exceed a critical value, Vc, defined as the breakdown voltage, Vc has for squid axon membrane a value of 1.1 V at 12 degrees C. During breakdown the specific membrane conductance exceeds 1 S. cm-2. Electrical breakdown produced by charge pulses of few microseconds duration have no influence on the excitability of the squid axon membrane. The resealing process of the membrane is so fast that a depolarizing breakdown is followed by the falling phase of a normal action potential. Thus, membrane voltages close to Vc open the sodium channels in few microseconds, but do not produce a decrease of the time constant of potassium activation large enough to cause the opening of a significant percentage of channels in a time of about 10 mus. It is probable that the reversible electrical breakdown is mainly caused by mechanical instability produced by electrostriction of the membrane (electrochemical model), but the decrease in the Born energy for ion injection into the membrane, accompanying the decrease in membrane thickness, may play also an important role. Because of the high conductance of the membrane during breakdown it seems very likely that this results in pore formation.     

High electric field effects on the cell membranes of Halicystis parvula

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage V_c. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 s and 100 s. Below 10 s the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 s the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvula. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains).     

Biphasic voltage relaxation pattern observed in cells of Eremosphaera viridis after injection of charge-pulses of short duration: detection of tip clogging of intracellular microelectrodes by charge-pulse technique

Charge pulse experiments performed on the peat-bog alga Eremosphaera viridis revealed an unusual voltage relaxation behaviour. Injection of charge pulses of 1 microseconds duration resulted in an immediate charging of the membranes (time constant of the order of 40 ns). Nevertheless, the potential-measuring microelectrode recorded an exponential increase in membrane voltage with a time constant of about 1.3 ms. The maximum voltage value was recorded after about 3 ms, followed by an exponential decay with a time constant of about 9.6 ms. This biphasic time course was independent of the amplitude of the injected charge and of the location of the impaled microelectrodes in the vacuole. Centrifuged cells in which the chloroplasts and the other organelles were pelleted in one part of the cells showed the same electrical response. Electrical breakdown of the cell membranes resulted in the disappearance of the biphasic voltage response. In this case only the decaying relaxation process could be recorded with a time constant of 3 ms. After resealing of the membranes the original biphasic relaxation response was restored. Increasing concentrations of KCl in the bathing medium reduced both time constants almost correspondingly. The experimental findings were evaluated with an electrical equivalent circuit. Theoretical analysis with reference to the experimental data suggested that the delayed voltage response of the potential-recording electrode resulted from a membrane seal across the tip of this electrode. The resistance of this seal was calculated to be about 400 M omega. The specific resistances and capacitances of tonoplast and plasmalemma membranes were calculated from the decaying part of the biphasic relaxation curves. The average values were found to be 2.58 omega.m2 and 5 mF.m-2. The investigations reported here suggest that charge pulse experiments can be generally used for the detection of membrane and cytoplasmic material clogging of the tip of intracellular microelectrodes, a problem with which most electrophysiologists are faced when interpreting data obtained from impaled microelectrodes.     

An investigation of electrical breakdown of bimolecular lipid membranes

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

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

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

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.     

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation.     

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

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

Dielectric breakdowm in the membranes of itValonia utricularis. The role of energy dissipation

The electrical properties of the membranes of Valoniautricularis were investigated using intracellular electrodes. Using short (0.5–1.0 ms) current pulses it was found that at a critical membrane potential difference of 0.85 V there was a large and discontinuous decrease in the membrane impedance and the slope resistance beyond this potential was virtually zero.The electrical breakdown of the membranes did not lead to global damage of the cells and after a resealing time of approx. 5 s could be repeated with identical results.Experiments with long current pulses and long bursts of pulses repeated at 1 kHz are described which show that the electrical breakdown is not due to thermal damage arising from localized heating in the membrane. Thus a dissipation of some 10³–10⁵ times the energy normally dissipated during the onset of breakdown did not lead to breakdown itself unless the critical membrane potential was exceeded.The results also show that punch-through and avalanche ionization are not likely to be important in the breakdown mechanism. The results are consitent, however, with there being a critical instability in the electro-mechanical stresses set up in the membrane at large electric field strengths.     

Electric field-mediated cell fusion

The use of electrical fields to guide, hold and fuse cells is described. The electrical fusion process consists of two steps: the cells are collected to form pearl-chains between Pt electrodes by the action of dielectrophoresis, then a brief DC pulse is applied, such that the breakdown voltage of the membranes is briefly exceeded and cell-to-cell juncture of the membranes occurs around the pore formed by the pulse. Giant fused cells (diameter up to 100 m) can be formed by the electrically mediated fusion of mouse 3T3 fibroblast cells, provided that pronase is added just before field application.     

Evidence for the presence of mobile charges in the cell membrane of Valonia utricularis.

Charge-pulse relaxation studies were performed on cells of the giant marine alga Valonia utricularis with microelectrodes inserted into the vacuole. If the cell was charged by short pulses of 200 ns duration, the decay of the initial membrane voltage could be described by two relaxation processes at normal pH (8.2). The fast exponential relaxation had a time constant of approximately 100 microseconds whereas the the time constant of the slow relaxation ranged between 2 and 15 ms. The ratio of the two amplitudes varied between 10 and 20 and was found to be independent of the initial voltage, up to 400 mV. In contrast to the time constants, the amplitude ratio was a function of the duration of the charge pulse. As the pulse length was increased to 10 ms, the fast relaxation disappeared. A change in pH of the natural sea water from 8.2 to 4 resulted in the disappearance of both exponential processes and the appearance of one single exponential with a 1-ms time constant over the whole pulse-length range. The analysis of the data in terms of a two-membrane model leads to unusual values and a pH-dependence of the specific capacitances (0.6 and 6 microF cm-2) of the two membranes, which can be treated as two serial circuits of a capacitor and a resistor in parallel. The charge-pulse and the current-clamp data are consistent with the assumption that the cell membrane of V. utricularis contains mobile charges with a total surface concentration of approximately 4 pmol cm-2. These charges cross the membrane barrier with a translocation rate constant around 500 s-1 and become neutralized at low pH. From our experimental results it cannot be completely excluded that the tonoplast has also a high specific resistance. But in this case it has to be assumed that the tonoplast and plasmalemma have very similar electrical properties and contain both mobile charges, so that the two membranes appear as a single membrane. Experiments on artificial lipid bilayer membranes in the presence of the lipophilic ion dipicrylamine, support our mobile charge concept for the cell membrane of V. utricularis.     

Life Cycle of an Electropore: Field-Dependent and Field-Independent Steps in Pore Creation and Annihilation

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.     

Voltage-dependent lipid flip-flop induced by alamethicin.

Alamethicin appears to allow voltage-dependent lipid exchange ("flip-flop") between leaflets of a planar bilayer. In membranes with one leaflet of phosphatidyl serine and one of phosphatidyl ethanolamine, the shape of the nonactin current-voltage curve accurately reports the difference in surface potential between the two sides of the membrane. The surface potential is itself a good measure of membrane asymmetry. Alamethicin added to the bathing solutions of an asymmetric membrane does not per se reduce the membrane asymmetry, but turning on the alamethicin conductance by application of a voltage pulse does. Immediately after application of a voltage pulse, large enough to turn on the alamethicin conductance, the asymmetry of the nonactin-K+ current voltage curve decreases, in some cases, nearly to zero. During the pulse, the alamethicin conductance activates if a decrease in surface potential favors turn-on of the alamethicin conductance or inactivates if a decrease in surface potential favors turn-off of the alamethicin conductance. After the pulse, the nonactin-K+ asymmetry returns to its original value if the alamethicin conductance is not turned on. The time-course of this return allows an estimate of the diffusion constant of lipid in the planar bilayer. The value obtained is 5.1 x 10(-8) cm2/s.     

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

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

Numerical study of lipid translocation driven by nanoporation due to multiple high-intensity,ultrashort electrical pulses

The dynamical translocation of lipids from one leaflet to another due to membrane permeabilization driven by nanosecond, high-intensity (> 100 kV/cm) electrical pulses has been probed. Our simulations show that lipid molecules can translocate by diffusion through water-filled nanopores which form following high voltage application. Our focus is on multiple pulsing, and such simulations are relevant to gauge the time duration over which nanopores might remain open, and facilitate continued lipid translocations and membrane transport. Our results are indicative of a N^½ scaling with pulse number for the pore radius. These results bode well for the use of pulse trains in biomedical applications, not only due to cumulative behaviors and in reducing electric intensities and pulsing hardware, but also due to the possibility of long-lived thermo-electric physics near the membrane, and the possibility for pore coalescence.