Abstract
Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.
Highlights
Microsecond pulsed electric fields permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology
In DMEM, cells displaying a pulse-induced Ca2+ peak could be detected at a field E0PM as low as 120 V/cm (Fig. 1A). 50% of cells presented a calcium peak when pulse amplitude E50PM was about 210 V/cm and plateau was reached at E100PM at about 320 V/cm
The present study shows, for the first time, the electropermeabilization of the endoplasmic reticulum (ER) membrane by μsPEF, experimentally, and with cell viability assessments
Summary
Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. Using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 μs pulse in a medium without Ca2+. Cell electroporation involves the application of electric pulses in order to increase the plasma membrane (PM) permeability. This concept emerged in the mid-60s with the work of Sale and Hamilton (1968)[1]. Electric pulses of a typical duration of 100 microseconds and an electric field amplitude of the order of 1000 V/ cm (μsPEF), have been widely used in many biotechnological or medical applications, notably for antitumor electrochemotherapy[4,5,6], tumor ablation[7,8], cell transfection in vitro[3,9] and even gene transfer in vivo. Electric pulses of a typical duration of 100 microseconds and an electric field amplitude of the order of 1000 V/ cm (μsPEF), have been widely used in many biotechnological or medical applications, notably for antitumor electrochemotherapy[4,5,6], tumor ablation[7,8], cell transfection in vitro[3,9] and even gene transfer in vivo10,11. μsPEF have been used to permeabilize many cell types and allow internalization of non-permeant molecules such as www.nature.com/scientificreports/
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