Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, ΔΨ m) and, for sufficiently strong fields, in a transient increase of ...membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of ΔΨ m and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between ΔΨ m and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of ΔΨ m are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.
The paper presents an approach that reduces several difficulties related to the determination of induced transmembrane voltage (ITV) on irregularly shaped cells. We first describe a method for ...constructing realistic models of irregularly shaped cells based on microscopic imaging. This provides a possibility to determine the ITV on the same cells on which an experiment is carried out, and can be of considerable importance in understanding and interpretation of the data. We also show how the finite-thickness, nonzero-conductivity membrane can be replaced by a boundary condition in which a specific surface conductivity is assigned to the interface between the cell interior (the cytoplasm) and the exterior. We verify the results obtained using this method by a comparison with the analytical solution for an isolated spherical cell and a tilted oblate spheroidal cell, obtaining a very good agreement in both cases. In addition, we compare the ITV computed for a model of two irregularly shaped CHO cells with the ITV measured on the same two cells by means of a potentiometric fluorescent dye, and also with the ITV computed for a simplified model of these two cells.
The development of a high performance protein probe for the measurement of membrane potential will allow elucidation of spatiotemporal regulation of electrical signals within a network of excitable ...cells. Engineering such a probe requires a functional screen of many candidates. Although the glass-microelectrode technique generally provides an accurate measure of a given test probe, throughputs are limited. In this study, we focused on an approach that uses the membrane potential changes induced by an external electric field in a geometrically simple mammalian cell. For quantitative evaluation of membrane voltage probes that rely on the structural transition of the S1–S4 voltage sensor domain and hence have non-linear voltage dependencies, it was crucial to introduce exogenous inwardly rectifying potassium conductance to reduce cell-to-cell variability in resting membrane potentials. Importantly, the addition of the exogenous conductance drastically altered the profile of the field-induced potential. Following a site-directed random mutagenesis and the rapid screen, we identified a mutant of a voltage probe Mermaid, exhibiting positively shifted voltage sensitivity. Due to its simplicity, the current approach will be applicable under a microfluidic configuration to carry out an efficient screen. Additionally, we demonstrate another interesting aspect of the field-induced optical signals, ability to visualize electrical couplings between cells.
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•External electric-fields can be used to characterize membrane potential probes.•Incorporation of Kir2.1 channels to the host cells was crucial for quantitativity.•Cells expressing Kir2.1 channels show altered field-induced potential changes.•We identified a Mermaid variant with positively-shifted voltage sensitivity.•Field-induced optical response also visualizes a cellular electrical coupling.
Knowledge of the parameters which influence the efficiency of gene electrotransfer has importance for practical implementation of electrotransfection for gene therapy as well as for better ...understanding of the underlying mechanism. The focus of this study was to analyze the differences in gene electrotransfer and membrane electropermeabilization between plated cells and cells in a suspension in two different cell lines (CHO and B16F1). Furthermore, we determined the viability and critical induced transmembrane voltage (ITVc) for both cell lines. In plated cells we obtained relatively little difference in electropermeabilization and gene electrotransfection between CHO and B16F1 cells. However, significant differences between the two cell lines were observed in a suspension. CHO cells exhibited a much higher gene electrotransfection rate compared to B16F1 cells, whereas B16F1 cells reached maximum electropermeabilization at lower electric fields than CHO cells. Both in a suspension and on plated cells, CHO cells had a slightly better survival rate at higher electric fields than B16F1 cells. Calculation of ITVc in a suspension showed that, for both electropermeabilization and gene electrotransfection, CHO cells have lower ITVc than B16F1 cells. In all cases, ITVc for electropermeabilization was lower than ITVc for gene electrotransfer, which is in agreement with other studies. Our results show that there is a marked difference in the efficiency of gene electrotransfer between suspended and plated cells.
Electropermeabilization and cell death caused by the exposure to high voltage electric pulses depends on the parameters of pulses, as well as the composition of the extracellular medium. We studied ...the influence of extracellular conductivity on electropermeabilization and survival of cells in vitro. For this purpose, we used a physiological medium with a conductivity of 1.6 S/m and three artificial media with conductivities of 0.14, 0.005, and 0.001 S/m. Measurements of pH, osmolarity, and cell diameter were made to estimate possible side effects of the media on the cells. Our study shows that the percentage of surviving cells increases with the decreasing medium conductivity, while the percentage of electropermeabilized cells remains unaffected. Our results show that cell survival in experiments involving electropermeabilization can be improved by decreasing the medium conductivity. To provide an interpretation of experimental results, we have theoretically estimated the resting transmembrane voltage, the induced transmembrane voltage, the time constant of the voltage inducement, and heating of the cell suspension for each of the media used. These calculations imply that for accurate interpretation of experimental results, both the induced and the resting transmembrane voltage must be considered, taking into account the conductivity and the ionic composition of the extracellular medium.
Electropermeabilization of biological membranes is applied more and more in medicine and in biotechnologies as a useful technique to facilitate the transfer of various substances between the inner ...and outer sides of a cell. It has considerably improved in the last decades, under intensive study, through experiments and simulations. A numerical study aiming to provide data for setting the main experimental parameters is presented in the paper, for simplified models of cells in suspension exposed to time-harmonic electric field.
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