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 (INa) 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 Ca2+ -dependent mechanism. We found that a single 300?ns electric pulse at 1.65.3?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 INa by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na+] (by 5mM) did not attenuate VG INa, thereby indicating that the nsPEF-induced Na+ influx was not the cause of VG INa inhibition. Similarly, adding 20 mM of a fast Ca2+ 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 INa by nsPEF. These findings point to possible Ca2+ -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.
Original Publication Citation
Nesin, V., & Pakhomov, A. G. (2012). Inhibition of voltage-gated Na+ current by nanosecond pulsed electric field (nsPEF) is not mediated by Na+ influx or Ca2+ signaling. Bioelectromagnetics, 33(6), 443-451. doi:10.1002/bem.21703
Nesin, Vasyl and Pakhomov, Andrei G., "Inhibition of Voltage-Gated Na+ Current by Nanosecond Pulsed Electric Field (nsPEF) is Not Mediated by NA+ Influx or Ca²+ Signaling" (2012). Bioelectrics Publications. 179.