Nanosecond pulsed electric field induced dose dependent phosphatidylinositol--bisphosphate signaling and intracellular electro-sensitization.

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol--bisphosphate (PIP) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose-dependent PIP hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP hydrolysis: PLCδ-PH-EGFP and GFP-C1-PKCγ-C. The 50% maximum response occurs with a single 600ns pulse achieving an effective dose (ED) of EF~8kV/cm within our model cell system. At 16.2kV/cm, the ED for the pulse width was 484ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60ns 16.2kV/cm pulses produced an effect similar to a single 600ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions.