title: Inhibition of voltage-gated Na(+)  current by nanosecond pulsed electric field (nsPEF) is not mediated by Na(+)  influx or Ca(2+)  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.
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors.forename: Vasyl
authors.initials: V
authors.lastname: Nesin
authors.forename: Andrei G
authors.initials: AG
authors.lastname: Pakhomov
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004553:Electric Conductivity; D051379:Mice; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D013997:Time Factors
chemical_list: D002614:Chelating Agents; D004533:Egtazic Acid; D012964:Sodium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid