Mechanism of acetylcholine receptor cluster formation induced by DC electric field

Background

The formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.

Methodology/Principal Findings

To understand its molecular mechanism, quantum dots (QDs) were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering.

Conclusions

These results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.     

Anisotropic electric conductivity of bacterial suspensions induced by external electric field

The anisotropy of electrical conductivity of suspensions of such bacteria, as E. coli, Serratia marcescens, Pseudomonas fluorescens induced by a sinusoidal external electric field and relaxation of the anisotropy after switching off the field were investigated. On the basis of the experimental relationships the anisotropy of electrical polarizability and coefficient of rotational diffusion of the cells were evaluated. The anisotropy of electrical polarizability and coefficient of the rotational diffusion obtained are in a good agreement with the available data of other methods.     

Synchronization of Na/K pump molecules by an oscillating electric field

Synchronization of the Na/K pump molecules in a cell membrane was studied in frog skeletal muscle fibers using double Vaseline-gap voltage-clamp techniques. We found that the pumping rate of naturally random-paced pump molecules can be artificially synchronized by a pulsed, symmetric, oscillating membrane potential with a frequency comparable to the physiological turnover rate. The synchronized pump currents show separated outward and inward components, where the magnitude of the outward component is about three times the randomly-paced pump currents, and the magnitude-ratio of the outward to inward pump currents is close to 3:2, which reflects the stoichiometric ratio of the pump molecules. Once synchronized, the pumping rate is restricted to the field frequency, and the pump currents are mainly dependent on the field frequency, but not the field strength. In contrast to previous work, which by restraining the pumps at a presteady state succeeded in triggering the steps of the pump cycle only individually and between interruptions, here we synchronize the pumps running continuously and in a normal running mode.     

外加电场刺激对菲降解菌的影响

以菲为主要C源,吐温80作为增溶剂,研究细菌Enterobacter dissolvens在直流电和交流电条件下的生长和代谢过程。实验结果表明:当施加10 mA直流电时,通电8 h后,菌液中细胞的菲降解率较对照增长1.6倍,细胞生长亦有所加快;而施加交流电时,细菌生长和菲降解率均低于直流电刺激。     

Phase transition of dimyristoylphosphatidylglycerol bilayers induced by electric field pulses

The phase transition of dimyristoylphosphatidylglycerol (DMPG) bilayers has been studied by measurements of light scattering under high electric field pulses. Midpoints of phase transitions have been identified by a clear discontinuity of field induced relaxation amplitudes. We show that the phase transition of DMPG suspensions in monovalent salt is virtually independent of the electric field strength up to approx. 35 kV/cm. A shift of the lipid phase by electric field pulses has been observed, however, for DMPG suspensions in the presence of Ca2+ ions. DMPG suspensions exhibit a jump of the phase transition temperature from 17 degrees C at Ca/DMPG molar ratios r less than 1/7 to 32 degrees C at r greater than 1/7. Field pulses of 60 to 100 microseconds applied to DMPG suspensions with Ca2+ at r greater than 1/7 induce discontinuities of relaxation amplitudes in the temperature range 15 to 22 degrees C in addition to the 'standard' one at 32 degrees C, when the electric field strength is above 15 kV/cm. These results indicate that electric field pulses induce a transition from the phase formed at 'high' Ca(2+)- to the one formed at 'low' Ca(2+)-ion concentrations. Our results are consistent with a dissociation field effect on Ca(2+)-lipid complexes which drives the phase transition.     

Conductance transition induced by an electric field in lipid bilayers

A cooperative phenomenon showing a structural change in the organization of bilayer lipid membranes at a critical value of the applied electric field is presented. The transition is characterized by a sharp increase in conductance. The phenomenon can be observed under current-clamp conditions (rather than the usual voltage-clamp conditions) to avoid rupturing the membrane. At a critical potential value the conductance increases and therefore the potential decreases to keep the current constant. Results refer to membranes made of egg phosphatidylcholine (PC), diphytanoylphosphatidylcholine and cholesterol/egg PC. It is found that the critical potential at which the transition occurs depends dramatically on pH and ionic concentration, indicating that the electrical properties of the external surface determine the major characteristics of such a transition.     

Roles of electric field and fiber structure in cardiac electric stimulation.

This study investigated roles of the variation of extracellular voltage gradient (VG) over space and cardiac fibers in production of transmembrane voltage changes (DeltaV(m)) during shocks. Eleven isolated rabbit hearts were arterially perfused with solution containing V(m)-sensitive fluorescent dye (di-4-ANEPPS). The epicardium received shocks from symmetrical or asymmetrical electrodes to produce nominally uniform or nonuniform VGs. Extracellular electric field and DeltaV(m) produced by shocks in the absolute refractory period were measured with electrodes and a laser scanner and were simulated with a bidomain computer model that incorporated the anterior left ventricular epicardial fiber field. Measurements and simulations showed that fibers distorted extracellular voltages and influenced the DeltaV(m). For both uniform and nonuniform shocks, DeltaV(m) depended primarily on second spatial derivatives of extracellular voltages, whereas the VGs played a smaller role. Thus, 1) fiber structure influences the extracellular electric field and the distribution of DeltaV(m); 2) the DeltaV(m) depend on second spatial derivatives of extracellular voltage.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Aspects of plant plasmalemma charging induced by external electric field pulses

The charging of the plasmalemma is a necessary condition for permeabilization of the plasma membrane (electroporation) in response to external electric field exposure. Common theories explain this permeabilization by formation of pores in the lipid bilayer. Using pulsed laser fluorescence microscopy, we measured the charging process of the membrane during the application of an external electric field with a temporal resolution of 5 ns. Visualization of the charging process of protoplasts plasma membrane (Nicotiana tabacum Bright Yellow 2) was achieved by staining of the plasma membrane with the voltage-sensitive fluorescent dye ANNINE-6. Measurements on membranes exhibiting negligible membrane permeabilization confirm the sine-shaped azimuthal distribution of the membrane voltage predicted by the relation of Cole. At higher membrane voltages, enhanced pore formation allows for the exchange of charge carriers, leading to deviations from the sine-shaped curve progression, i.e., a saturation of the membrane voltage at membrane segments facing the electrodes. Additionally, measurements on protoplasts exposed to multiple successive pulses indicate that the recovery of the membrane seems to be a fast process, occurring within seconds after termination of the external electric field pulse.     

Exploring how extracellular electric field modulates neuron activity through dynamical analysis of a two-compartment neuron model

To investigate how extracellular electric field modulates neuron activity, a reduced two-compartment neuron model in the presence of electric field is introduced in this study. Depending on neuronal geometric and internal coupling parameters, the behaviors of the model have been studied extensively. The neuron model can exist in quiescent state or repetitive spiking state in response to electric field stimulus. Negative electric field mainly acts as inhibitory stimulus to the neuron, positive weak electric field could modulate spiking frequency and spike timing when the neuron is already active, and positive electric fields with sufficient intensity could directly trigger neuronal spiking in the absence of other stimulations. By bifurcation analysis, it is observed that there is saddle-node on invariant circle bifurcation, supercritical Hopf bifurcation and subcritical Hopf bifurcation appearing in the obtained two parameter bifurcation diagrams. The bifurcation structures and electric field thresholds for triggering neuron firing are determined by neuronal geometric and coupling parameters. The model predicts that the neurons with a nonsymmetric morphology between soma and dendrite, are more sensitive to electric field stimulus than those with the spherical structure. These findings suggest that neuronal geometric features play a crucial role in electric field effects on the polarization of neuronal compartments. Moreover, by determining the electric field threshold of our biophysical model, we could accurately distinguish between suprathreshold and subthreshold electric fields. Our study highlights the effects of extracellular electric field on neuronal activity from the biophysical modeling point of view. These insights into the dynamical mechanism of electric field may contribute to the investigation and development of electromagnetic therapies, and the model in our study could be further extended to a neuronal network in which the effects of electric fields on network activity may be investigated.     

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.     

Actin polymerization induced by pulsed electric stimulation of bone cells in vitro

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.     

Breakdown of highly excited oxygen in a DC electric field

The breakdown of oxygen in a dc electric field is studied. A high concentration of oxygen molecules in the a ¹Δ_g excited state is obtained in a purely chemical reactor. A decrease in the breakdown voltage at degrees of excitation exceeding 50% is observed. The theoretical decrement in the breakdown voltage obtained by solving the Boltzmann equation is in good agreement with the experimental data.     

Model development and numerical simulation of electric-stimulus-responsive hydrogels subject to an externally applied electric field

Based on a multi-phasic mixture theory with consideration of ionic diffusion and convection, a multi-physic model, called the multi-effect-coupling electric-stimulus (MECe) model, is developed for simulation of responsive behavior of the electric-sensitive hydrogels when they are immersed into a bathing solution subject to an externally applied electric field. In the developed model, with chemo-electro-mechanical coupling effects, the convection-diffusion equations for concentration distribution of diffusive ions incorporate the influence of electric potential. The electroneutrality condition is replaced by the Poisson equation for distribution of electric potential. The steady and transient analyses of hydrogel deformation are easily carried out by the continuity and momentum equations of the mixture phase. Further, the computational domain of the present model covers both the hydrogel and the surrounding solution. In order to solve the present mathematical model consisting of multi-field coupled nonlinear partial differential governing equations, a hierarchical iteration technique is proposed and a meshless Hermite-Cloud method (HCM) is employed. The steady-state simulation of the electric-stimulus responsive hydrogel is numerically conducted when it is subjected to an externally applied electric field. The hydrogel deformation and the ionic concentrations as well as electric potentials of both the hydrogel and external solution are investigated. The parameter influences on the swelling behaviors of the hydrogel are also discussed in detail. The simulating results are in good agreement with the experimental data and they validate the presently developed model.     

The magnetic and electric fields induced by superparamagnetic magnetite in honeybees

Hairs on the abdomen of honeybees contain dendrites and a rod and ring structure composed of black particles, presumed to be superparamagnetic (SPM) magnetite. The rod and ring were divided into compartments and each compartment approximated by a dipole. The magnetic fields were calculated at a point P at various locations for a change of the external geomagnetic field from zero to 0.5 G in 0.1 s. The magnetite amplifies the external field at the rod/ring-dendrite interface. The induced electric field and potential difference for a small circular area are in the order of 10^–7 V/m and 10^–13 V respectively. Mechanisms are proposed for amplifying the electric fields in the dendrite and in an integrating nerve fibre. A hypothesis is developed for associative learning of visual and magnetic stimuli. If magnetic and visual inputs are associated in the ganglion and in the brain, very small changes of either magnetic or visual inputs could be perceived. A bee could sequentially follow the images associated with magnetic gradients on a cloudy day and find the food source.This paper is dedicated to the memory of the late Prof. Dr. W. Reichardt, an outstanding scientist and personality, who will be greatly missed by all who knew him and his work     

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)     

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.