Membrane permeabilization and cell damage by ultrashort electric field shocks

Mammalian cells exposed to electric field pulses of nanosecond duration (nsPEF; 60-ns, 12 kV/cm) experienced a profound and long-lasting increase in passive electrical conductance (G_m) of the cell membrane, probably caused by opening of stable conductance pores (CPs). The CPs were permeable to Cl⁻ and alkali metal cations, but not to larger molecules such as propidium iodide (PI). CPs gradually resealed; the process took minutes and could be observed even in dialyzed cells and in ATP- and glucose-free solutions. Cells subjected to long nsPEF trains (up to 200 pulses) underwent severe and immediate necrotic transformation (cell swelling, blebbing, cytoplasm granulation), but remained impermeable to PI for at least 30-60 min after the exposure. Both G_m increase after short nsPEF trains and necrotic changes after long nsPEF trains were cell type-dependent: they were much weaker in HeLa than in GH3 cells. La³⁺ and Gd³⁺ ions significantly inhibited the nsPEF-induced G_m increase (probably by blocking the CPs), and effectively protected intensely exposed cells from developing necrosis. We conclude that plasma membrane permeabilization is the principal cause of necrotic transformation in nsPEF-exposed cells and probably contributes to other known nsPEF bioeffects.     

Ultrashort electric pulse induced changes in cellular dielectric properties

The interaction of nanosecond duration pulsed electric fields (nsPEFs) with biological cells, and the models describing this behavior, depend critically on the electrical properties of the cells being pulsed. Here, we used time domain dielectric spectroscopy to measure the dielectric properties of Jurkat cells, a malignant human T-cell line, before and after exposure to five 10ns, 150kV/cm electrical pulses. The cytoplasm and nucleoplasm conductivities decreased dramatically following pulsing, corresponding to previously observed rises in cell suspension conductivity. This suggests that electropermeabilization occurred, resulting in ion transport from the cell's interior to the exterior. A delayed decrease in cell membrane conductivity after the nsPEFs possibly suggests long-term ion channel damage or use dependence due to repeated membrane charging and discharging. This data could be used in models describing the phenomena at work.     

Nanosecond Electroporation: Another Look

As the medical field moves from treatment of diseases with drugs to treatment with genes, safe and efficient gene delivery systems are needed to make this transition. One such safe, non-viral, and efficient gene delivery system is electroporation (electrogenetherapy). Exciting discoveries using electroporation could make this technique applicable to drug and vaccine delivery in addition to gene delivery. Typically milli and microsecond pulses have been used for electroporation. Recently, the use of nanosecond electrical pulses (10-300 ns) at very high magnitudes (10-300 kV/cm) has been studied for direct DNA transfer to the nucleus in vitro. This article reviews the work done using high-intensity nanosecond pulses, termed as nanosecond electroporation (nsEP), in electroporation gene delivery systems.     

What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues

Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what are the actual limits of the DNA delivery into cells. Jean-Michel Escoffre and Thomas Portet have contributed equally to this work.     

Nanosecond pulse electric field (nanopulse): a novel non-ligand agonist for platelet activation

Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca²⁺) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca²⁺ ion concentration ([Ca²⁺]_i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300 ns pulse with an electric field of 30 kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7 kV/cm with maximum effects seen with approximately 30 kV/cm. The increases in intracellular Ca²⁺ release and Ca²⁺ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30 kV/cm. The increases in [Ca²⁺]_i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca²⁺ to leak out of intracellular Ca²⁺ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca²⁺ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca²⁺]_i, however by a different mechanism.     

Modeling the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations

Calcium (Ca2+) oscillations play fundamental roles in various cell signaling processes and have been the subject of numerous modeling studies. Here we have implemented a general mathematical model to simulate the impact of store-operated Ca2+ entry on intracellular Ca2+ oscillations. In addition, we have compared two different models of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and their influences on intracellular Ca2+ oscillations. Store-operated Ca2+ entry following Ca2+ depletion of endoplasmic reticulum (ER) is an important component of Ca2+ signaling. We have developed a phenomenological model of store-operated Ca2+ entry via store-operated Ca2+ (SOC) channels, which are activated upon ER Ca2+ depletion. The depletion evokes a bi-phasic Ca2+ signal, which is also produced in our mathematical model. The IP3R is an important regulator of intracellular Ca2+ signals. This IP3 sensitive Ca2+ channel is also regulated by Ca2+. We apply two IP3R models, the Mak-McBride-Foskett model and the De Young and Keizer model, with significantly different channel characteristics. Our results show that the two separate IP3R models evoke intracellular Ca2+ oscillations with different frequencies and amplitudes. Store-operated Ca2+ entry affects the oscillatory behavior of these intracellular Ca2+ oscillations. The IP3 threshold is altered when store-operated Ca2+ entry is excluded from the model. Frequencies and amplitudes of intracellular Ca2+ oscillations are also altered without store-operated Ca2+ entry. Under certain conditions, when intracellular Ca2+ oscillations are absent, excluding store-operated Ca2+ entry induces an oscillatory response. These findings increase knowledge concerning store-operated Ca2+ entry and its impact on intracellular Ca2+ oscillations.     

Ion channel clustering enhances weak electric field detection by neutrophils: apparent roles of SKF96365-sensitive cation channels and myeloperoxidase trafficking in cellular responses

We have tested Galvanovskis and Sandblom’s prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K_v1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca²⁺ channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K⁺ and Ca²⁺ channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K⁺ channel blockers act by reducing the neutrophil’s membrane potential. Mibefradil and SKF93635, which block T-type Ca²⁺ channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca²⁺ signaling. Electric fields enhanced Ca²⁺ spike amplitude and triggered formation of a second traveling Ca²⁺ wave. Mibefradil blocked Ca²⁺ spikes and waves. Although 10 μM SKF96365 mimicked mibefradil, 7 μM SKF96365 specifically inhibited electric field-induced Ca²⁺ signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-β-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.     

Orientation of membrane fragments by electric field

Purple membrane fragments from Halobacterium halobium were oriented by a static electric field in a water suspension. It was found that an electric field of approx. 20 V/cm is sufficient to achieve practically complete orientation; the purple membranes have a permanent electric dipole moment of (6 ±1)· 10^?23 C · m, the orientation of the retinal transition moment relative to the direction of the electric dipole moment, θ, is (59 ± 1)⁰, and the purple membrane rotational diffusion constant D_rot = 0.65 s^?1. It was found that because of the electrophoretic movement of the particles a hydrodynamic velocity gradient builds up which also orients the purple membranes.     

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.     

Increase of intracellular Ca(2+) during ischemia/reperfusion injury of heart is mediated by cyclic ADP-ribose

While the molecular mechanisms by which oxidants cause cytotoxicity are still poorly understood, disruption of Ca(2+) homeostasis appears to be one of the critical alterations during the oxidant-induced cytotoxic process. Here, we examined the possibility that oxidative stress may alter the metabolism of cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing second messenger in the heart. Isolated heart perfused by Langendorff technique was subjected to ischemia/reperfusion injury and endogenous cADPR level was determined using a specific radioimmunoassay. Following ischemia/reperfusion injury, a significant increase in intracellular cADPR level was observed. The elevation of cADPR content was closely correlated with the increase in ADP-ribosyl cyclase activity. Inclusion of oxygen free radical scavengers, 2,2,6,6-tetramethyl-1-piperidinyloxy and mannitol, in the reperfusate prevented the ischemia/reperfusion-induced increases in cADPR level and the ADP-ribosyl cyclase activity. Exposure of isolated cardiomyocytes to t-butyl hydroperoxide increased the ADP-ribosyl cyclase activity, cADPR level, and intracellular Ca(2+) concentration ([Ca(2+)](i)) and consequently resulting in cell lethal damage. The oxidant-induced elevation of [Ca(2+)](i) as well as cell lethal damage was blocked by a cADPR antagonist, 8-bromo-cADPR. These results provide evidence for involvement of cADPR and its producing enzyme in alteration of Ca(2+) homeostasis during the ischemia/reperfusion injury of the heart.     

Electropermeabilization, a physical method for the delivery of therapeutic molecules into cells

Electropermeabilization designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. A position-dependent reversible local membrane permeabilization is induced leading to an exchange of hydrophilic molecules across the membrane. This permeabilized state can be used to load cells with therapeutic molecules. In the case of small molecules, such as anticancer drugs, transfer occurs through simple diffusion. In the case of DNA, transfer occurs through a multi-step mechanism, a process that involves the electrophoretically driven association of the DNA molecule with the destabilised membrane and then its passage.     

Ontogeny of the electric organ discharge and the electric organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus (Hypopomidae, Gymnotiformes)

I recorded the electric organ discharges (EODs) of 331 immature Brachyhypopomus pinnicaudatus 6–88 mm long. Larvae produced head-positive pulses 1.3 ms long at 7 mm (6 days) and added a second, small head-negative phase at 12 mm. Both phases shortened duration and increased amplitude during growth. Relative to the whole EOD, the negative phase increased duration until 22 mm and amplitude until 37 mm. Fish above 37 mm produced a “symmetric” EOD like that of adult females. I stained cleared fish with Sudan black, or fluorescently labeled serial sections with anti-desmin (electric organ) or anti-myosin (muscle). From day 6 onward, a single electric organ was found at the ventral margin of the hypaxial muscle. Electrocytes were initially cylindrical, overlapping, and stalk-less, but later shortened along the rostrocaudal axis, separated into rows, and formed caudal stalks. This differentiation started in the posterior electric organ in 12-mm fish and was complete in the anterior region of fish with “symmetric” EODs. The lack of a distinct “larval” electric organ in this pulse-type species weakens the hypothesis that all gymnotiforms develop both a temporary (larval) and a permanent (adult) electric organ. Accepted: 1 March 1997     

TPCs: Endolysosomal channels for Ca mobilization from acidic organelles triggered by NAADP

Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca²⁺ and Na⁺ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca²⁺ release channels that respond to activation by the Ca²⁺ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled.     

Differences in the electric birefringence of spectrin dimers and tetramers as shown by the fast reversing electric pulse method

The electric birefringence of purified Spectrin has been examined in medium of low ionic strength ai 20°C and for electric fields smaller than 4 × 10⁴ V m^?1. using the reversing electric pulse method. This technique allows study of the permanent and induced dipole electric moment of macromolecules more easily than in measurements using only rectangular pulses. We show that spectrin heterodimers and heterotetramers have different electro-optical properties. The relaxation time of the tetramer (7 μs) is significantly longer than that of the dimer (4.5 μs). Tetramers and dimers have also different polarizability parameters.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.     

Lack of correlation between the amplitudes of TRP channel-mediated responses to weak and strong stimuli in intracellular Ca imaging experiments

It is often observed in intracellular Ca²⁺ imaging experiments that the amplitudes of the Ca²⁺ signals elicited by newly characterized TRP agonists do not correlate with the amplitudes of the responses evoked subsequently by a specific potent agonist. We investigated this rather controversial phenomenon by first testing whether it is inherent to the comparison of the effects of weak and strong stimuli. Using five well-characterized TRP channel agonists in commonly used heterologous expression systems we found that the correlation between the amplitudes of the Ca²⁺ signals triggered by two sequentially applied stimuli is only high when both stimuli are strong. Using mathematical simulations of intracellular Ca²⁺ dynamics we illustrate that the innate heterogeneity in expression and functional properties of Ca²⁺ extrusion (e.g. plasma membrane Ca²⁺ ATPase) and influx (TRP channels) pathways across a cellular population is a sufficient condition for low correlation between the amplitude of Ca²⁺ signals elicited by weak and strong stimuli. Taken together, our data demonstrate that this phenomenon is an expected outcome of intracellular Ca²⁺ imaging experiments that cannot be taken as evidence for lack of specificity of low-efficacy stimuli, or as an indicator of the need of other cellular components for channel stimulation.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.