Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1860, Issue 11, Pages 2175-2183Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2018.08.017
Keywords
Voltage-gated Ca2+ channels; Nanosecond pulsed electric field; HEK293 cells; Electroporation; Nanopores
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Funding
- AFOSR MURI [FA9550-15-1-0517]
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We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca2+. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (> 80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC + cells) was about twofold greater than in cells which did not (VGCC cells). Among VGCC + cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca2+ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC + cells. However, it remains to be established whether larger transients reflected additional Ca2+ entry through VGCC, or were a result of more severe electropermeabilization of VGCC + cells.
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