Gap junctions increase the sensitivity of tissue cells to exogenous electric fields

Cells coupled by gap junctions will react as a single unit to an applied electric field. In a given field, the hyperpolarization and depolarization of cell membranes at the ends of an electrotonically coupled tissue are increases by a factor of 10-100 over single, uncoupled cells. Gap junctional coupling therefore increases the likelihood that cells are influenced by the weak electric fields which are commonly found in developing and regenerating tissues.     

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.     

Cell culture dosimetry for low-frequency magnetic fields

Calculations of the current density and electric field distributions induced in cell cultures by an applied low-frequency magnetic field have assumed that the medium is uniform. This paper calculates these distributions for a more realistic, inhomogeneous, anisotropic model in which the cells are regarded as conducting squares surrounded by insulating membranes. Separate parameters are used to specify the resistivities of the cell interior, the cell membrane parallel to its surface, the cell membrane perpendicular to its surface, and the intercellular junction parallel to the membrane. The presence of gap junctions connecting the interiors of adjacent cells is also considered. For vertical applied magnetic fields, the induced currents and field distributions may deviate considerably from the homogeneous medium model if there is sufficiently tight binding of the cells to each other. The presence of gap junctions can produce relatively large transmembrane electric fields or intracellular current densities. These considerations are generally less important for horizontal applied fields. A simple microscopic model of the cell surface is also discussed. © 1996 Wiley-Liss, Inc.     

Steady-state current flow through gap junctions. Effects on intracellular ion concentrations and fluid movement.

Double voltage clamp studies were performed on gap junctions contained in septal membranes of the earthworm median giant axon. The gap junctions exhibited no conductance changes in response to voltages imposed across either the septal membrane or the plasma membrane. However, the trans-septal current displayed a slow (10 s) relaxation in response to transjunctional voltage steps. The experimental evidence suggests that this relaxation is a polarization of the septum due to local accumulation/depletion of permeant ions. A theoretical analysis of this observation suggests that the applied electric field causes accumulation of impermeant anions on one side of the junction and depletion on the other, which leads to a change in concentration of permeant ions to maintain macroscopic electroneutrality. The change in concentration of permeant ions generates a transjunctional equilibrium potential that opposes junctional current flow. These results indicate that currents flowing through gap junctions can have an influence on the distribution of intracellular ions. Moreover, the theoretical analysis suggests that such currents will be accompanied by significant intracellular and intercellular water flow.     

Dynamic gap junctional communication: a delimiting model for tissue responses.

Gap junctions are aqueous intercellular channels formed by a diverse class of membrane-spanning proteins, known as connexins. These aqueous pores provide partial cytoplasmic continuity between cells in most tissues, and are freely permeable to a host of physiologically relevant second messenger molecules/ionic species (e.g., Ca2+, IP3, cAMP, cGMP). Despite the fact that these second messenger molecules/ionic species have been shown to alter junctional patency, there is no clear basis for understanding how dynamic and transient changes in the intracellular concentration of second messenger molecules might modulate the extent of intercellular communication among coupled cells. Thus, we have modified the tissue monolayer model of Ramanan and Brink (1990) to account for both the up-regulatory and down-regulatory effects on junctions by second messenger molecules that diffuse through gap junctions. We have chosen the vascular wall as our morphological correlate because of its anisotropy and large investment of gap junctions. The model allows us to illustrate the putative behavior of gap junctions under a variety of physiologically relevant conditions. The modeling studies demonstrated that transient alterations in intracellular second messenger concentrations are capable of producing 50-125% changes in the number of cells recruited into a functional syncytial unit, after activation of a single cell. Moreover, the model conditions required to demonstrate such physiologically relevant changes in intercellular diffusion among coupled cells are commonly observed in intact tissues and cultured cells.     

Electrical properties of developing oocytes of the migratory locust, Locusta migratoria.

The electrical properties of developing nonfertilized oocytes of Locusta migratoria were studied, using intracellular microelectrodes. The inseries potential of the combined oomembrane and of the follicular cells was about 20 mV in the youngest oocytes. It increased as the oocytes developed and it reached a plateau of about 50 mV before full maturation, generally four to seven oocytes away from the fully-developed terminal oocyte. Current-voltage relations were always linear for hyperpolarizing currents. Most oocytes exhibited, however, rectification to outward current. Input resistance values varied with oocyte size from about 5 X 10(6) ohm for young oocytes to about 0.2 X 10(6) ohm for the more developed ones. Some oocytes displayed a transient depolarization on turning off a hyperpolarizing step of current. This depolarization was not correlated with the size of the oocyte or with any observed morphological feature. Any two adjacent oocytes were electrotonically coupled. A single ovariole thus represented a longitudinal chain of developing oocytes which were connected electrically. This was supported by electron microscope observations which revealed junctions partially impermeable to lanthanum and gap junctions between the follicular cells themselves and between follicular cells and oocytes. The coupling coefficient was dependent on the direction of current flow. The attenuation of voltage along an ovariole was always greater at the distal than at the proximal side.     

Gap junction blockage limits intercellular spreading of astrocytic apoptosis induced by metabolic depression

Astrocytes are highly coupled by gap junction channels, which allow transfer of intracellular signalling molecules and metabolites between connected cells. Astrocytic gap junctions remain open during ischemic conditions as previously demonstrated in vitro and in situ. In this study, we investigated the effect of gap junction blockage on iodoacetate-induced ATP depression and cell death progression in astrocytes in primary rat hippocampal cultures. We demonstrated that blockage of gap junctions during iodoacetate-induced inhibition of the glycolysis induced an earlier onset of the ATP depression. Moreover, initiation of apoptotic processes, demonstrated by binding of Annexin V, was critically dependent on the ATP levels. The apoptotic event was also shown to spread and involve neighbouring cells, a process that was inhibited by blockage of gap junction communication. Chelating intracellular calcium using BAPTA-AM decelerated the iodoacetate-induced ATP depression. The chelation also decelerated the spreading of apoptotic processes. Inhibition of caspases did not alter the expansion of cell groups being Annexin V positive. However, the proportion of Annexin V positive cells also being propidium iodide positive was increased after caspase inhibition. The results show that inhibition of gap junctions during cellular metabolic depression interferes with the metabolic status and cell death progression in astrocytes.     

Does direction of induced electric field or current provide a test of mechanism involved in nerve regeneration?

We suggest an experimental comparison of two directions for applying the time-varying magnetic fields which have been found to speed spontaneous regeneration of injured peripheral nerves and in attempts to repair spinal cord injuries. Time-varying magnetic fields induce currents in a plane perpendicular to the magnetic field direction. The lower conductivity of the spinal cord's sheath (dura matter) or of the myelin sheath of peripheral nerves would seem to confine the induced electric fields and currents to the spinal cord or nerve itself. The proposed comparison could allow choosing between two possible modes of action of the fields: (1) Magnetically-induced electric fields or currents may be encouraging ion flow or otherwise helping enzyme, channel or other interactions at the cell membrane, as is thought to be the case in field stimulation of healing in bone. This mechanism should be independent of field direction. (2) Work in developing organisms and with fields applied to nerve cells in vitro has shown that neurite growth is guided parallel to both endogenous and external electric fields. This mechanism would be effective when induced electric fields are parallel, but not when they are perpendicular to the nerve. Any experimental test should seek to produce as close as possible to the same induced current intensity with both field directions. Possible confounding factors, as well as breakdowns in the assumptions of the simple model presented here, would have to be considered. This proposal was motivated by a recent report in which the authors listed a changed field direction as one of several possible reasons for an unsuccessful experiment.     

Dye and electrical coupling of endothelial cells in situ

Electron microscopic studies show that endothelial cells of pig coronary arteries are linked by gap junctions. We investigated the dye and electrical coupling of these junctions in a strip of pig coronary artery in vitro. The membrane potential of two neighbouring (about 0.2 mm) endothelial cells were simultaneously recorded with two microelectrodes. The fluorescent dye lucifer yellow was microiontophoretically injected through one of the microelectrodes. The endothelial cells in situ were dye and electrically coupled. The dye coupling extended parallel to the longitudinal axis of the arteries. We conclude that an electrical message like the bradykinin and substance P hyperpolarizations of the endothelial cells can be conveyed electrotonically by the endothelium along the longitudinal axis of arteries.     

Voltage-dependent blockade of connexin40 gap junctions by spermine

The effects of spermine and spermidine, endogenous polyamines that block many forms of ion channels, were investigated in homotypic connexin (Cx)-40 gap junctions expressed in N2A cells. Spermine blocked up to 95% of I(j) through homotypic Cx40 gap junctions in a concentration- and transjunctional voltage (V(j))-dependent manner. V(j) was varied from 5 to 50 mV in 5-mV steps and the dissociation constants (K(m)) were determined from spermine concentrations ranging from 10 micro M to 2 mM. The K(m) values ranged from 4.9 mM to 107 micro M for 8.6 < or = V(j) < or = 37.7 mV, within the physiological range of intracellular spermine for V(j) > or = 20 mV. The K(m) values for spermidine were > or = 5 mM. Estimates of the electrical distance (delta) for spermine (z = +4) and spermidine (z = +3) were 0.96 and 0.76 respectively. Cx40 single channel conductance was 129 pS in the presence of 2-mM spermine and channel open probability was significantly reduced in a V(j)-dependent manner. Similar concentrations of spermine did not block I(j) through homotypic Cx43 gap junctions, indicating that spermine selectively blocks Cx40 gap junctions. This is contrary to our previous findings that large tetraalkylammonium ions, also known to block several forms of ion channels, block junctional currents (I(j)) through homotypic connexin Cx40 and Cx43 gap junctions.     

Dye and electrical coupling of endothelial cells in situ

Electron microscopic studies show that endothelial cells of pig coronary arteries are linked by gap junctions. We investigated the dye and electrical coupling of these junctions in a strip of pig coronary artery in vitro. The membrane potential of two neighbouring (about 0.2 mm) endothelial cells were simultaneously recorded with two microelectrodes. The fluorescent dye lucifer yellow was microiontophoretically injected through one of the microelectrodes. The endothelial cells in situ were dye and electrically coupled. The dye coupling extended parallel to the longitudinal axis of the arteries. We conclude that an electrical message like the bradykinin and substance P hyperpolarizations of the endothelial cells can be conveyed electrotonically by the endothelium along the longitudinal axis of arteries.     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

Numerical simulation of gel electrophoresis of DNA knots in weak and strong electric fields

Gel electrophoresis allows one to separate knotted DNA (nicked circular) of equal length according to the knot type. At low electric fields, complex knots, being more compact, drift faster than simpler knots. Recent experiments have shown that the drift velocity dependence on the knot type is inverted when changing from low to high electric fields. We present a computer simulation on a lattice of a closed, knotted, charged DNA chain drifting in an external electric field in a topologically restricted medium. Using a Monte Carlo algorithm, the dependence of the electrophoretic migration of the DNA molecules on the knot type and on the electric field intensity is investigated. The results are in qualitative and quantitative agreement with electrophoretic experiments done under conditions of low and high electric fields.     

Calmodulin transit via gap junctions is reduced in the absence of an electric field

Gap junctional transport of Calmodulin (CaM) from epithelial cells to insect oocytes is enhanced by alignment of the molecules via an electric field. It has recently been shown that CaM is needed for uptake of vitellogenins, is produced in the epithelial cells and reaches oocytes via gap junctions. For CaM to transit the gap junctions something must align these elongated molecules with the lumina of the gap junctions. This might be accomplished by the electric field that exists at the membrane of any cell with an Em of >0 mV. Fluorescently labeled CaM was injected into oocytes. At t=0, the epithelial cell/oocyte "fluorescence" ratio showed epithelial cells to be 24%+/-1.5% as bright as the injected oocyte. In follicles which maintained an electric field for one hour the epithelial cell/oocyte fluorescence ratio had risen to 79%+/-1.4%, while for follicles in which the field was cancelled by holding Em at 0 mV the ratio was only 45%+/-1.7%. After termination of the holding current follicles regained their original Em and their original electric field. At the end of a second hour of incubation the ratio had risen to 76%+/-1.2%, very close to what was observed in the untreated control follicles.     

Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.

Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved.     

N3ICD with the transmembrane domain can effectively inhibit EMT by correcting the position of tight/adherens junctions

EMT allows a polarized epithelium to lose epithelial integrity and acquire mesenchymal characteristics. Previously, we found that overexpression of the intracellular domain of Notch3 (N3ICD) can inhibit EMT in breast cancer cells. In this study, we aimed to elucidate the influence of N3ICD or N3ICD combined with the transmembrane domain (TD+N3ICD) on the expression and distribution of TJs/AJs and polar molecules. We found that although N3ICD can upregulate the expression levels of the above-mentioned molecules, TD+N3ICD can inhibit EMT more effectively than N3ICD alone. TD+N3ICD overexpression upregulated the expression of endogenous full-length Notch3 and contributed to correcting the position of TJs/AJs molecules and better acinar structures formation. Co-immunoprecipitation results showed that the upregulated endogenous full-length Notch3 could physically interact with E-ca in MDA-MB-231/pCMV-(TD+N3ICD) cells. Collectively, our data indicate that overexpression of TD+N3ICD can effectively inhibit EMT, resulting in better positioning of TJs/AJs molecules and cell-cell adhesion in breast cancer cells.

Abbreviations: EMT: Epithelial-mesenchymal transition; TJs: Tight junctions; AJs: Adherens junctions; aPKC: Atypical protein kinase C; Crb: Crumbs; Lgl: Lethal (2) giant larvae; LLGL2: lethal giant larvae homolog 2; PAR: Partitioning defective; PATJ: Pals1-associated TJ protein     

The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.     

Synchronization in a model of two inferior olive cells with variable electrotonic coupling

Synchronization was assessed in a model describing the dynamics of two inferior olive cells coupled electrotonically via gap junctions and surrounded with inhibitory synaptic terminals (modeled from first-order kinetics) that can block this coupling. Depending on the parameters, the system gives rise to various synchronization regimes: 1:1, 1:2, spike “time binding” etc. Even small changes of coupling parameters (coupling strength and decoupling delay) can quite significantly affect the regimes of synchronization between interacting neurons. In some cases, because of collective dynamics the activity of one cell is suppressed while the other cell remains active.     

Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers--in cells and in silico

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.     

On the electromotor neurons of both electric organs of Pollimyrus isidori (Mormyridae, Teleostei)

The electromotor neurons (EMNs) of the adult electric organ of Pollimyrus isidori and the "giant neurons" (GNs) found in the more rostral part of the spinal cord were investigated both with light and electron microscopical methods. The two kinds of neuron are more or less similar in size, about 30 microns, and are found in the more dorsal part of the spinal cord. The EMNs show somatosomatic gap junctions, as do the GNs. In addition, chemical synapses could be found between nerve fibres and both types of neurons. Histochemical investigations show that the EMNs and the GNs are both AChE positive. On the basis of the arguments presented in the discussion, we believe that the "giant neurons" are the electromotor neurons of the larval electric organ of Pollimyrus isidori.