Elementary [Ca²⁺]_isignals generated by electroporation functionally mimic those evoked by hormonal stimulation

Abstract

The generation of oscillations and global Ca2+ waves relies on the spatio-temporal recruitment of elementary Ca2+ signals, such as 'Ca2+ puffs'. Each elementary signal contributes a small amount of Ca2+ into the cytoplasm, progressively promoting neighboring Ca2+ release sites into an excitable state. Previous studies have indicated that increases in frequency or amplitude of such hormone-evoked elementary Ca2+ signals are necessary to initiate Ca2+ wave propagation. In the present study, an electroporation device was used to rapidly and reversibly permeabilize the plasma membrane of HeLa cells and to allow a limited influx of Ca2+. With low field intensities (100-500 V/cm), brief (50-100 micros) electroporation triggered localized Ca2+ signals that resembled hormone-evoked Ca2+ puffs, but not global signals. With such low intensity electroporative pulses, the Ca2+ influx component was usually undetectable, confirming that the electroporation-induced local signals represented Ca2+ puffs arising from the opening of intracellular Ca2+ release channels. Increasing either the frequency at which low-intensity electroporative pulses were applied, or the intensity of a single electroporative pulse (>500 V/cm), resulted in caffeine-sensitive regenerative Ca2+ waves. We suggest that Ca2+ puffs caused by electroporation functionally mimic hormone-evoked elementary events and can activate global Ca2+ signals if they provide a sufficient trigger.

Stimulation of Capacitative Calcium Entry in HL-60 Cells by Nanosecond Pulsed Electric Fields

“…This response was observed in the absence of classical plasma membrane electroporation. Previous studies in HeLa cells showed that classical electroporation pulses (100 s, 500 V/cm) also stimulated calcium responses similar to those seen with hormones (30). The results presented here further support the hypothesis that when nsPEFs that are below the threshold for plasma membrane electroporation and apoptosis induction are applied, intracellular signal transduction cascades can be triggered, resulting in signaling events that are common with natural ligands.…”

Upper threshold of extracellular neural stimulation

“…It also cannot be explained by the reversible electroporation (formation of pores in cell membrane), since such pores close within tens of seconds to tens of minutes (Bobanović et al 1999;Saulis 1997), whereas spike generation in our measurements resumed immediately after reduction of the stimulus below the upper threshold, within one period of the stimulation waveform (1 s). Electroporation is also likely to change the cell resting potential, which has not been observed at the upper threshold.…”

Electroporation of cells and tissues

“…Essentially, any size molecule can be introduced into the cell cytoplasm or transported across a vesicle membrane. For example, Ca ions can be gently introduced to mimic exposure to a hormone [32], antibody molecules (∼150 000 gmol ) can be introduced to block specific biochemical pathways, and even micrometer-size particles can be introduced [33], [34]. In a very different application, biochemical reactions involving surfaceimmobilized vesicles can be rapidly initiated using microelectrodes [35].…”