Bioelectromagnetics Inhibition of Voltage-Gated Na þ Current by Nanosecond Pulsed Electric Field (nsPEF) Is Not Mediated by Na þ Influx or Ca 2þ Signaling Vasyl Nesin and Andrei G. Pakhomov* Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk,Virginia In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompa- nied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na þ current (I Na ) resulted from (i) reduction of the trans- membrane Na þ gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca 2þ -dependent mechanism. We found that a single 300 ns electric pulse at 1.6–5.3 kV/cm triggered sustained Na þ influx in exposed NG108 cells and in primary chromaf- fin 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 þ ([Na] i ) did not exceed 2–3 mM. [Na] i increased uniform- ly over the cell volume and showed no additional peaks immediately below the plasma mem- brane. 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 þ ] (by 5 mM) did not attenuate VG I Na , thereby indicating that the nsPEF-induced Na þ influx was not the cause of VG I Na inhibition. Similarly, adding 20 mM of a fast Ca 2þ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetra- acetic 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 2þ -independent downregulation of the VG Na þ channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel. Bioelectromagnetics ß 2012 Wiley Periodicals, Inc. Key words: electropermeabilization; ion influx; cell membrane; sodium channels; patch-clamp INTRODUCTION Our previous studies found that permeabi- lization of the cell plasma membrane by intense nsPEF may be accompanied by prolonged inhibition of voltage-gated (VG) currents through Na þ , Ca 2þ , and K þ channels [Pakhomov et al., 2007a,b; Bow- man et al., 2008; Nesin et al., 2011]. This previously unknown effect adds to the understanding of nsPEF interactions with living matter and may lead to appli- cations in experimental biology and medicine. How- ever, mechanisms underlying the inhibition of VG currents have not been identified. At the first glance, the most likely and well- expected mechanism is the ion leak current (I leak ) through electropores in the plasma membrane. This leak can reduce the transmembrane ion gradient, thereby reducing the driving force for ion flow when VG channels open. This mechanism is purely electro- chemical and does not imply any damage or alter- ation of VG channels or any regulatory biological response. Arguments in favor of this mechanism are its simplicity and the fact that nsPEF did not cause inhibition of VG currents without a concurrent or preceding increase in I leak (although sometimes the I leak increase was very small and brief). On the other hand, the intracellular ion composition in ‘‘patched’’ cells is strongly buffered by a practically unlimited ion supply from the recording pipette, and it is not clear if I leak can efficiently compete with it to affect any intracellular ion concentration. The central role of the ion gradient reduction is also questioned by the poor correlation between the I leak amplitude and Grant sponsors: National Cancer Institute (R01CA125482); National Institute of General Medical Sciences (R01GM088303); Air Force Office of Scientific Research (LRIR 09RH09COR). *Correspondence to: Andrei G. Pakhomov, 4211 Monarch Way, Suite 300, Norfolk, VA 23508. E-mail: andrei@pakhomov.net, apakhomo@odu.edu Received for review 20 August 2011; Accepted 12 December 2011 DOI 10.1002/bem.21703 Published online in Wiley Online Library (wileyonlinelibrary.com). ß 2012 Wiley Periodicals,Inc.