Probing Nanoelectroporation and Resealing of the Cell Membrane by the Entry of Ca2+ and Ba2+ Ions

International Journal of Molecular Sciences

Vitalij Novickij¹  Wenfei Bo²  Uma Mangalanathan²  Olga N. Pakhomova²  Shu Xiao²  Iurii Semenov²  Maura Casciola²  Andrei G. Pakhomov²  Mantas Silkunas² 

[1] Faculty of Electronics, Vilnius Gediminas Technical University, 03227 Vilnius, Lithuania;Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508, USA;

关键词: electroporation;    electropermeabilization;    membrane repair;    membrane integrity;    nsPEF;    nsEP;   

DOI  :  10.3390/ijms21093386

来源: DOAJ

【 摘 要 】

The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca²⁺, Cd²⁺, Zn²⁺, and Ba²⁺ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca²⁺ and Ba²⁺ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within < 1 ms. The threshold for one 300-ns pulse was at 1.5-2 kV/cm, much lower than > 7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba²⁺ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca²⁺ entry could elicit calcium-induced calcium release (CICR) followed by Ca²⁺ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca²⁺ and Ba²⁺ proved as sensitive nanoporation markers, with Ba²⁺ being more reliable for monitoring membrane damage and resealing.

【 授权许可】

Unknown