Electromobile surface charge alters membrane potential changes induced by applied electric fields.

The relation between extracellular electric fields and changes in membrane potential that such fields directly induce has previously been described both theoretically and experimentally. It is clearly established that extracellular electric-field-induced membrane potential changes are well described by Poisson's equation of electrostatics. A modification of this simple theory to include effects of the electric-field-induced redistribution of charged cell surface components is introduced and is shown to produce major alterations in calculated membrane potential changes over times of the order of minutes to hours. Implications for biological systems which respond to extracellular electric fields are discussed.     

Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.     

Threshold effects observed in conformation changes induced by electric fields

Single-stranded polynucleotides are used as model systems for the investigation of conformational changes induced by electric fields. It is demonstrated that the single-strand helix–coil transition in poly(A), poly(dA), and poly(C) can be induced by application of high electric fields. The transition is measured by UV absorbance using polarized light at an angle of 54.8° with respect to the vector of the electric field and by electrodichroism. A linear increase of the absorbance, reflecting the helix-to-coil transition, is observed at increasing field strength. When ions are added to the polymer, electric fields do not induce conformation changes, unless a threshold value of the electric field strength E₀ is exceeded. At field strengths above this threshold, the degree of transition is a linear function of the increase in field strength. The threshold values E₀ show a linear increase with the logarithm of the ion concentration. Bivalent ions cause thresholds at much lower ion concentrations than mo-novalent ions. The shielding efficiency of ions is correlated to the binding affinity of these ions to the polymer. The conformation changes induced by the field and the existence of thresholds can be explained on the basis of dissociation field effects. Similar threshold effects may be expected for other macromolecules as well as for membrane structures and may be important in the regulation of bioelectricity.     

Control of electric field induced cell membrane permeabilization by membrane order

Cells can be made temporarily permeable if pulsed by high-intensity short-duration electric fields. The molecular mechanisms underlying this electropermeabilization are still unknown. The kinetic events may be described by four successive steps: induction, expansion, stabilization, and resealing. On one hand, cell electropermeabilization is detected only under more stringent conditions when cells have been treated by ethanol. On the other hand, lysolecithin is observed to facilitate cell electropermeabilization. More precisely, these molecules that modify membrane order, when used in concentrations compatible with cell viability, are shown to affect only the expansion and resealing steps. Electropermeabilization is inducing a transition in the membrane organization. Membrane order is modulating the energy barrier needed to evoke this membrane transition which occurs when cells are submitted to a field larger than a characteristic threshold (expansion step). Less order would increase the magnitude of this energy barrier; more order would decrease it.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0. We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0 We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Membrane potential perturbations induced in tissue cells by pulsed electric fields

Pulsed electric fields directly influence the electrophysiology of tissue cells by transiently perturbing their transmembrane potential. To determine the magnitude and time course of this interaction, electrotonic cable theory was used to calculate the membrane potential perturbations induced in tissue cells by a spatially uniform, pulsed electric field. Analytic solutions were obtained that predict shifts in membrane potential along the length of cells as a function of time in response to an electrical pulse. For elongated tissue cells, or groups of tissue cells that are coupled electrotonically by gap junctions, significant hyperpolarizations and depolarizations can result from millisecond applications of electric fields with strengths on the order of 10–100 mV/cm. The results illustrate the importance of considering cellular cable parameters in assessing the effects of transient electric fields on biological systems, as well as in predicting the efficacy of pulsed electric fields in medical treatments. © 1995 Wiley-Liss, Inc.     

Theoretical evaluation of cell membrane ion channel activation by applied magnetic fields

This letter re-examines a recently published calculation of the forces exerted on a membrane ion channel by a cation passing through in the presence of an externally applied magnetic field. We show here, in contradiction to the originally published calculation, that the forces generated due to the Lorentz force of the magnetic field on the cation are negligible compared with the forces required to activate an ion channel protein conformation change associated with the gating of the channel. Received: 11 August 1998 / Revised version: 25 October 1998 / Accepted: 11 November 1998     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Study of conductance changes of bilayer lipid membrane induced by electric field

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.     

Alterations of the apparent area expansivity modulus of red blood cell membrane by electric fields.

Red blood cell membrane exhibits a large resistance to changes in surface area. This resistance is characterized by the area expansivity modulus K, which relates the isotropic membrane force resultant, T, to the fractional change in membrane surface area delta A/Ao. The experimental technique commonly used to determine K is micropipette aspiration. Using this method, E. A. Evans and R. Waugh (1977, Biophys. J. 20:307-313) obtained a value of 450 dyn/cm for the modulus. In the present report, it is shown that the value of K, as determined using this method, is affected by electric potential differences applied across the tip of the pipette. Using Ag-AgCl electrodes and current clamping electronics, we obtained values for K ranging from 150 dyn/cm with -1.0 V applied, to 1,500 dyn/cm with 1.0 V applied. At 0.0 V the modulus obtained was approximately 500 dyn/cm. A reversible, voltage- and pressure-dependent change in the cell volume probably accounts for the effect of the voltage on the calculated value of the modulus. The use of lanthanum chloride or increasing the extra- and intracellular solute concentrations reduced the voltage dependence of the measurements. It was also found that when dissimilar metals were used to "ground" the pipette to the chamber to prevent lysis of cells by static charge, values for K ranged from 121 to 608 dyn/cm. Based on measurements made at zero applied volts, in the presence of 0.4 mM lanthanum and at high solute concentration, we conclude that the true value of the modulus is approximately 500 dyn/cm.     

Skin surface electric potential induced by ion-flux through epidermal cell layers

The skin surface electric potential has been widely used for psychological studies because it is sensitive to emotional conditions. We measured the electric potential on the surface of hairless mice skin in organ culture with several physiological factors. Disruption of mitochondrial function and inhibition of ATPase reduced the skin surface potential 50-70%. Calcium, potassium, and sodium channel blockers also reduced the potential. A calcium-specific and potassium ionophore reduced the potential, but the calcium and magnesium ionophore increased it. EDTA decreased the potential but EGTA had no effect. Skin surface barrier disruption reduced the potential and calcium and potassium channel blockers partially prevented the decrease. Substance P and corticotropin-releasing factor decreased the potential, and antagonists blocked the decreases. These results suggest that the ion flux in the nucleated layer of the epidermis induce the skin surface potential and it is influenced by environmental and neuroendocrinological factors.     

A model for the polarization of neurons by extrinsically applied electric fields.

A model is presented for the subthreshold polarization of a neuron by an applied electric field. It gives insight into how morphological features of a neuron affect its polarizability. The neuronal model consists of one or more extensively branched dendritic trees, a lumped somatic impedance, and a myelinated axon with nodes of Ranvier. The dendritic trees branch according to the 3/2-power rule of Rall, so that each tree has an equivalent cylinder representation. Equations for the membrane potential at the soma and at the nodes of Ranvier, given an arbitrary specified external potential, are derived. The solutions determine the contributions made by the dendritic tree and the axon to the net polarization at the soma. In the case of a spatially constant electric field, both the magnitude and sign of the polarization depend on simple combinations of parameters describing the neuron. One important combination is given by the ratio of internal resistances for longitudinal current spread along the dendritic tree trunk and along the axon. A second is given by the ratio between the DC space constant for the dendritic tree trunk and the distance between nodes of Ranvier in the axon. A third is given by the product of the electric field and the space constant for the trunk of the dendritic tree. When a neuron with a straight axon is subjected to a constant field, the membrane potential decays exponentially with distance from the soma. Thus, the soma seems to be a likely site for action potential initiation when the field is strong enough to elicit suprathreshold polarization. In a simple example, the way in which orientation of the various parts of the neuron affects its polarization is examined. When an axon with a bend is subjected to a spatially constant field, polarization is focused at the bend, and this is another likely site for action potential initiation.     

Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A.

The effect of the mitogenic lectin concanavalin A on the membrane potential of murine lymphocytes was investigated by observing the fluorescence of cells stained with carbocyanine and oxonol dyes. We describe a rapid and reliable method for detecting lectin-induced membrane potential changes in individual cells by flow cytometric analysis of oxonol fluorescence. By 10 min after addition of lectin to suspensions of isolated cells from lymph node, 7-15% of the cells have responded by releasing oxonol dye, indicating a membrane hyperpolarization. The dose onset of this response is similar to that for mitogenesis, which was assessed by measuring [3H]thymidine incorporation. The effect is abolished by alpha-methyl mannoside (100mM), which prevents concanavalin A from binding to the cells, but not by fucose (100mM). When cells are treated with lectin in medium from which Ca2+ has been omitted or to which quinine (0.5mM) has been added, a membrane depolarization is observed. Since these are conditions under which activation of plasma membrane Ca2+-dependent K+ channels is prevented, these findings support the view that the early hyperpolarization of these cells is brought about by an increase in intracellular free [Ca2+].     

Fluorescence imaging of local membrane electric fields during the excitation of single neurons in culture.

The spatial distribution of depolarized patches of membrane during the excitation of single neurons in culture has been recorded with a high spatial resolution (1 micron2/pixel) imaging system based on a liquid-nitrogen-cooled astronomical camera mounted on an inverted microscope. Images were captured from rat nodose neurons stained with the voltage-sensitive dye RH237. Conventional intracellular microelectrode recordings were made in synchrony with the images. During an action potential the fluorescence changes occurred in localized, unevenly distributed membrane areas, which formed clusters of depolarized sites of different sizes and intensities. When fast conductances were blocked by the addition of tetrodotoxin, a reduction in the number and the intensities of the depolarized sites was observed. The blockade by tetrodotoxin of voltage-clamped neurons also reduced the number of depolarized sites, although the same depolarizing voltage step was applied. Similarly, when a voltage-clamped neuron was depolarized by a constant-amplitude voltage step, the number of depolarized sites varied according to the degree of activation of the voltage-sensitive channels, which was modified by changing the holding potential. These results suggest that the spatial patterns of depolarization observed during excitation are related to the operations of ionic channels in the membrane.     

Optical imaging of plastic changes induced by fear conditioning in the auditory cortex

The plastic changes in the auditory cortex induced by a fear conditioning, through pairing a sound (CS) with an electric foot-shock (US), were investigated using an optical recording method with voltage sensitive dye, RH795. In order to investigate the effects of association learning, optical signals in the auditory cortex in response to CS (12 kHz pure tone) and non-CS (4, 8, 16 kHz pure tone) were recorded before and after normal and sham conditioning. As a result, the response area to CS enlarged only in the conditioning group after the conditioning. Additionally, the rise time constant of the auditory response to CS significantly decreased and the relative peak value and the decay time constant of the auditory response to CS significantly increased after the conditioning. This study introduces an optical approach to the investigation of fear conditioning, representational plasticity, and the cholinergic system. The findings are synthesized in a model of the synaptic mechanisms that underlie cortical plasticity.     

Cellular membrane potentials induced by alternating fields

Membrane potentials induced by external alternating fields are usually derived assuming that the membrane is insulating, that the cell has no surface conductance, and that the potentials are everywhere solutions of the Laplace equation. This traditional approach is reexamined taking into account membrane conductance, surface admittance, and space charge effects. We find that whenever the conductivity of the medium outside the cell is low, large corrections are needed. Thus, in most of the cases where cells are manipulated by external fields (pore formation, cell fusion, cell rotation, dielectrophoresis) the field applied to the cell membrane is significantly reduced, sometimes practically abolished. This could have a strong bearing on present theories of pore formation, and of the influence of weak electric fields on membranes.     

Morphological transitions of vesicles induced by alternating electric fields

When subjected to alternating electric fields in the frequency range 10²-10⁸ Hz, giant lipid vesicles attain oblate, prolate, and spherical shapes and undergo morphological transitions between these shapes as one varies the field frequency and/or the conductivities λ_in and λ_ex of the aqueous solution inside and outside the vesicles. Four different transitions are observed with characteristic frequencies that depend primarily on the conductivity ratio λ_in/λ_ex. The theoretical models that have been described in the literature are not able to describe all of these morphological transitions.