Optical imaging of cell membrane potential changes induced by applied electric fields.

We report the first imaging of the spatial distributions of transmembrane potential changes induced in nonexcitable cells by applied external electric fields. These changes are indicated by the fluorescence intensity of a charge-shift potentiometric dye incorporated in the cell plasma membrane and measured by digital intensified video microscopy.     

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.     

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.     

Distinction between changes in membrane potential and surface charge upon chemotactic stimulation of Escherichia coli.

Galactose and other chemotactic attractants have been shown to trigger an apparent hyperpolarization in Escherichia coli (Eisenbach, M., 1982, Biochemistry, 21:6818-6825). The probe used to measure membrane potential in that study, tetraphenylphosphonium (TPP+), may respond also to surface-charge changes in the membrane. The distinction between true changes in membrane potential and changes in the surface charge of the membrane is crucial for the study of this phenomenon in bacterial chemotaxis. To distinguish between these parameters, we compared the response to galactose with different techniques: K+ distribution in the presence of valinomycin (measured with a K+-selective electrode), TPP+ distribution (measured with a TPP+-selective electrode) at different ionic strengths, absorbance changes of bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethineoxonol (oxonol V), and fluorescence changes of three probes with different mechanisms of response. All the techniques revealed stimulation by galactose of transient hyperpolarization, of comparable magnitude. This indicates the involvement of ion currents rather than alterations of local surface properties.     

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.     

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.     

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.     

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.     

Changes in the membrane surface charge density and/or membrane potential of the porcine intestinal brush-border membrane vesicles induced by treatment with neuraminidase

The effects of neuraminidase treatment on the membrane surface charge density and/or membrane potential of the porcine intestinal brush-border membrane vesicles were studied by using three fluorescent dyes, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-anilino-8-naphthalene sulfonate (ANS), and 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (DiS-C3(5]. The results of quenching studies of DPH-labeled membranes using cationic (T1+) and anionic (I-) quenchers suggested an increase of negative charge on the membrane surface by desialylation upon neuraminidase treatment. This interpretation was further supported by a decrease of ANS-binding affinity of the membranes after treatment with the enzyme. In addition, the degree of valinomycin-induced fluorescence change of DiS-C3(5)-probed membranes in the presence of various concentrations of KCl was reduced by treatment of the membranes with neuraminidase. This suggests that penetration of the dye molecules into the vesicle interior is facilitated by the treatment. The membrane potentials estimated from the null point of valinomycin-induced changes in the DiS-C3(5) fluorescence of the control and neuraminidase-treated membranes were -25 to -29.7 and -40 to -48.8 mV, respectively. From these results, it is suggested that the membrane surface charge density and/or membrane potential of the intestinal brush-border membranes are susceptible to modification of carbohydrate moieties on the membrane surface by neuraminidase treatment.     

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.     

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+].     

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.     

Intrinsic electric fields and membrane bending

The fragmentation of human erythrocytes heated in a range of ionic environments has been examined by video microscopy, , the average number of surface wave crests growing on the cell rim during fragmentation by membrane externalization, andI, the percentage of cells internalizing membrane, were scored.The membrane diffusion potential was altered experimentally on decreasing the extracellular chloride concentration by substituting either membrane-impermeant sorbitol or Na gluconate for some NaCl. The external-membrane-face surface potential was altered either by surface charge depletion or by ionic strength changes. The dependence of morphological change on diffusion potential at constant cell volume and surface potentials was established over a 34-mV change in diffusion potential. The rate constants for morphological change with charge depletion at different diffusion potentials are largely independent of the diffusion potential. A l.O-mV increase in diffusion potential has an effect on morphological change of comparable magnitude to that of a 1.0-mV decrease in the modulus of the negative surface potential. When the diffusion potential increased on decreasing both the extracellular diffusible ion concentration and extracellular ionic strength, the effect on cell morphology of increasing the modulus of the surface potential was overcome by the effects of the diffusion potential change.     

Electric fields and surface charges induced by ELF magnetic fields

A method is described for evaluating electric fields induced by ELF magnetic fields into electrically inhomogeneous, low-conductivity (less than 5 S/m) structures. It is applied to cylinders and spheres, and numerical results are given for electrical properties that are representative of some tissues, or of cells embedded either in saline solution or a tissue matrix. Surface currents on spherical cell boundaries are estimated and compared with thermal noise due to ion motion.     

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.     

Schwan equation and transmembrane potential induced by alternating electric field.

The transmembrane potential generated by an alternating electric field (ac) depends strongly on the frequency of the field and can be calculated using the Schwan Equation. We have measured the critical electric breakdown potential, delta psi crit, of the plasma membrane of murine myeloma cell line (Tib9) using ac fields, by monitoring the entry of a fluorescence probe, propidium iodide, into the cells. This dye is weakly fluorescent in solution but becomes strongly fluorescent when it binds to DNA. Experiments were done under a microscope by direct visual examination of single cells or by examining photographic prints. When an ac field reached the intensity, Ecrit, that generated a maximal membrane potential delta psi max, equal to or greater than the delta psi crit, the membrane was perforated at the two loci facing the electrodes. The dye diffused into the cell, giving rise to two bright, narrow bands, which expanded to the whole cell in 1-3 min. delta psi crit's were measured in three media of different resistivities, rho ext, (52,600, 7,050, and 2,380 omega cm), over the range of 0.1-300 kHz, with the field duration of 200 ms. Regression analysis based on the Schwan Equation showed that in a medium of given resistivity, the delta psi crit was constant over the frequency range studied. When the capacitance of the membrane, Cmembr, was taken to be 0.90 microF cm-2, the resistivity of the cytoplasmic medium, rho int, was determined to be 910-1,100 omega cm. The delta psi crit were 0.33, 0.48, and 0.53 V, respectively, for the three media in decreasing resistivities.(ABSTRACT TRUNCATED AT 250 WORDS)