Inhibition of voltage-gated Na+ current by nanosecond pulsed electric field (nsPEF) is not mediated by Na+ influx or Ca2+ signaling
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. Bioelectromagnetics
(Impact Factor: 1.71).
09/2012; 33(6):443-51. DOI: 10.1002/bem.21703
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.
Available from: Daniel Remondini
- "Specifically, the present work was devoted to testing the possibility that MF-tuned ion parametric resonance conditions (according to Lednev   and Blanchard and Blackman ) may affect the TEA-sensitive voltage-dependent outward K þ currents measured by the patch-clamp technique in whole-cell configuration before, during, and after exposure. The patch clamp is the best experimental method to investigate the biophysical properties and functions of ion channels, and test, with high accuracy, ion conductance changes following application of chemical or physical agents including pulsed electric fields [Nesin and Pakhomov, 2012], low-frequency EMFs [Tonini et al., 2001; Obo et al., 2002; Grassi et al., 2004; Marchionni et al., 2006] and static MFs [Rosen, 1996, 2003]. Moreover, the peculiar property of voltage-gated channels of coupling membrane potential to a contextual measurable cellular response (ion flux) allows an intrinsic positive control of the sensitivity of the recording system in each measurement, in terms of ion conductance changes in response to voltage variations. "
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ABSTRACT: Despite the experimental evidence of significant biological effects of extremely low frequency (ELF) magnetic fields (MFs), the underlying mechanisms are still unclear. Among the few mechanisms proposed, of particular interest is the so called "ion parametric resonance (IPR)" hypothesis, frequently referred to as theoretical support for medical applications. We studied the effect of different combinations of static (DC) and alternating (AC) ELF MFs tuned on resonance conditions for potassium (K(+) ) on TEA-sensitive voltage-dependent outward K(+) currents in the human neuroblastoma BE(2)C cell line. Currents through the cell membrane were measured by whole-cell patch clamp before, during, and after exposure to MF. No significant changes in K(+) current density were found. This study does not confirm the IPR hypothesis at the level of TEA-sensitive voltage-dependent outward K(+) currents in our experimental conditions. However, this is not a direct disprove of the hypothesis, which should be investigated on other ion channels and at single channel levels also. Bioelectromagnetics. © 2013 Wiley Periodicals, Inc.
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- "In other words, the cells were left ''untouched'' until immediately before the measurements, so any discussions about the shift of ''voltage dependency of I Na inactivation'' due to the prolonged holding of cells under patch clamp conditions are not applicable. Notably, Verkerk et al. omitted any discussion of important Figure 7 [Nesin et al., 2012], which shows that I Na may decrease with I leak as low as 50 pA, or may increase despite I leak as high as 1,500 pA. Poor correlation between I leak and the inhibition of I Na does not fit with the hypothesis of Verkerk et al. "
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ABSTRACT: In two recent publications in Bioelectromagnetics it has been demonstrated that the voltage-gated sodium current (I(Na) ) is inhibited in response to a nanosecond pulsed electric field (nsPEF). At the same time, there was an increase in a non-inactivating "leak" current (I(leak) ), which was attributed to the formation of nanoelectropores or larger pores in the plasma membrane. We demonstrate that the increase in I(leak) , in combination with a residual series resistance, leads to an error in the holding potential in the patch clamp experiments and an unanticipated inactivation of the sodium channels. We conclude that the observed inhibition of I(Na) may be largely, if not fully, artifactual. Bioelectromagnetics. © 2012 Wiley Periodicals, Inc.
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