Inhibition of voltage-gated Na+ current by nanosecond pulsed electric field (nsPEF) is not mediated by Na+ influx or Ca2+ signaling
ABSTRACT In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na(+) current (I(Na)) resulted from (i) reduction of the transmembrane Na(+) gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca(2+)-dependent mechanism. We found that a single 300 ns electric pulse at 1.6-5.3 kV/cm triggered sustained Na(+) influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a Sodium Green Dye. In the whole-cell patch clamp configuration, this influx was efficiently buffered by the pipette solution so that the increase in the intracellular concentration of Na(+) ([Na](i)) did not exceed 2-3 mM. [Na](i) increased uniformly over the cell volume and showed no additional peaks immediately below the plasma membrane. Concurrently, nsPEF reduced VG I(Na) by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na(+)] (by 5 mM) did not attenuate VG I(Na), thereby indicating that the nsPEF-induced Na(+) influx was not the cause of VG I(Na) inhibition. Similarly, adding 20 mM of a fast Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the pipette solution did not prevent or attenuate the inhibition of the VG I(Na) by nsPEF. These findings point to possible Ca(2+)-independent downregulation of the VG Na(+) channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel.
SourceAvailable from: Iurii Semenov[Show abstract] [Hide abstract]
ABSTRACT: Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the nsPEF-induced swelling, the bath buffer was isoosmotically supplemented with an electropore-impermeable solute (sucrose). A similar addition of a smaller, electropore-permeable solute (adonitol) served as a control. We demonstrated that sucrose efficiently blocked disassembly of actin features by nsPEF, whereas adonitol did not. Sucrose also attenuated bleaching of mApple-tagged actin in nsPEF-treated cells (as integrated over the cell volume), although did not fully prevent it. We conclude that disintegration of the actin cytoskeleton was a result of cell swelling, which, in turn, was caused by cell permeabilization by nsPEF and transmembrane diffusion of solutes which led to the osmotic imbalance.Bioelectrochemistry (Amsterdam, Netherlands) 12/2014; DOI:10.1016/j.bioelechem.2014.01.004 · 3.87 Impact Factor
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ABSTRACT: Physiological electric field (EF) is a potent guidance cue for many physiological development and pathological conditions. The EF induced cellular responses such as migration and proliferation, are considered to be regulated by multiple signaling pathways in a coordinated way. Unlike the signaling transduction regulating the cellular responses toward chemical gradients, the signaling network involved in electric stimulation shows a unique manner, combining the regulation of ion channels, membrane receptors and associated intracellular signaling pathways. This review shall discuss the cellular responses in EF, and summarize the primary signaling network activated during the EF-induced cellular response.The International Journal of Biochemistry & Cell Biology 09/2014; 55. DOI:10.1016/j.biocel.2014.09.014 · 4.24 Impact Factor
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ABSTRACT: Nanosecond electric pulses (nsEP) are defined as very short high intensity electric pulses which present great potential for the destabilization of intracellular structures. Their theoretical descriptions first suggested specific effects on organelles that have been confirmed by various observations both in vitro and in vivo. However, due to their concomitant effects on the plasma membrane, nsEP can also affect cell functions. In this mini-review, nsEP effects on cells are described following three topics: effects at the plasma membrane level, intracellular effects, and the impact on cell survival. Eventually, a short description of the major results obtained in vivo will be presented. This study shows that the use of nsEP has evolved during the last decade to focus on low voltage for practical applications.Bioelectrochemistry 08/2014; 103. DOI:10.1016/j.bioelechem.2014.07.008 · 3.87 Impact Factor