Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response.

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP) depletion resulting in physiological responses similar to those observed following stimulation of G-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP uptake compared to Gd. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP and Ca cannot be used solely as markers of nsEP-induced nanoporation.