Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study...

Area of Science:

• Cell Biology
• Biophysics
• Biotechnology

Background:

• Microsecond pulsed electric fields (μsPEF) are established tools for plasma membrane (PM) permeabilization.
• Nanosecond pulsed electric fields (nsPEF) are used for internal membrane permeabilization but are complex.
• Modulating intracellular calcium is crucial for understanding cell physiology.

Purpose of the Study:

• To investigate if μsPEF can permeabilize internal cell membranes, specifically the endoplasmic reticulum (ER).
• To compare the effectiveness and complexity of μsPEF versus nsPEF for internal membrane permeabilization.
• To explore the potential of μsPEF in controlling cytosolic calcium concentration.

Main Methods:

• Applied a single 100 µs pulse to two cell types.
• Used calcium (Ca2+) as a marker for permeabilization, monitoring cytosolic Ca2+ peaks.
• Utilized thapsigargin and specific inhibitors (IP3R, RyR) to confirm calcium source and mechanism.
• Compared permeabilization thresholds between cell types.

Main Results:

• A single 100 µs pulse successfully permeabilized the ER membrane in both cell types without affecting cell viability.
• Observed cytosolic Ca2+ peaks, dependent on ER calcium release, confirming ER permeabilization.
• Ruled out ER Ca2+ channel activation as the cause of Ca2+ release.
• Identified cell and ER size as factors influencing PM and ER permeabilization thresholds.

Conclusions:

• μsPEF can effectively permeabilize internal membranes like the ER, offering a simpler alternative to nsPEF.
• μsPEF provides a controllable method to modulate cytosolic calcium levels, aiding research into calcium's role in cell physiology.
• This technique has potential applications in research, medicine, and biotechnology.