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.

Fig. 1

Timeline of the experiment and typical effects of nsPEF in a chromaffin cell. Top: Cell images were taken and quantified repeatedly before and after the delivery of one 300 ns pulse at 4 kV/cm at 0 s (dashed line). Center: Whole-cell currents in response to voltage steps from −100 mV to +30 mV, in 10-mV increments, as measured 20 s before exposure and 20 and 30 s after (arrows). The membrane potential was stepped from the holding value of −80 mV to voltages from −100 mV to +30 mV, in 10-mV increments. Bottom: Respective current-voltage (I-V) curves for the fast-inactivating current (VG I_Na) and non-inactivating current (I_leak). This is a representative experiment out of 12; mean data are shown in Figures 2 and 3.

Fig. 2

Increase in Sodium Green fluorescence and respective change in [Na]_i triggered by nsPEF exposure in NG108 cells and adrenal chromaffin cells. Fluorescence was quantified as an average value within a contour of the cell body. Cells were exposed at 0 s (vertical dashed lines) to one 300 ns pulse at the indicated E-field amplitude. Mean values ± s.e. for 5–12 experiments per group; for clarity, error bars are drawn in one direction only. See Figure 3A and B for concurrent measurements of membrane currents in these cells.

Fig. 3

Effect of nsPEF and increasing Na⁺ concentration in the pipette buffer on membrane currents in NG108 cells (A and C) and adrenal chromaffin cells (B). A and B: Currents were measured 20 s prior to one 300 ns pulse at the indicated E-field amplitude and 20 s after. Note the E-field-dependent enhancement of I_leak and inhibition of the VG I_Na in both cell lines. C: VG I_Na was measured 1–2 min after forming the whole-cell patch clamp recording configuration, without any nsPEF exposure.

Fig. 4

Lack of localized increase in [Na]_i in the vicinity of the cell plasma membrane following nsPEF exposure. Inset shows a fluorescent image of the cell loaded with 1 µm of Sodium Green. The emission intensity was measured along the dashed line, which connects the tips of nsPEF-delivering electrodes (not shown). A: The emission along the line was measured repeatedly before and after exposure to one 300 ns pulse at 5.3 kV/cm (arrow). Increased emission following exposure reflects the Na⁺ influx through the membrane. B: Individual line scans of the emission intensity immediately prior to exposure and 20 s after. The emission gain due to exposure is the arithmetic difference between these two curves. Note maximum emission gain above the center of the cell body and lack of any additional peaks close to the plasma membrane. The calibration of dye emission against [Na]_i that was used in Figure 2 does not apply to line scan measurements presented here.

Fig. 5

High intracellular concentration of a fast Ca²⁺ chelator, BAPTA, does not prevent inhibition of VG I_Na by nsPEF. Membrane currents were measured in individual NG108 cells 20 s prior to nsPEF exposure and 30 s after; the former was subtracted from the latter using a “control subtraction” function of the pClamp software. This difference reflected the change in current caused by nsPEF (one 300 ns pulse at 4 kV/cm) or sham exposure. Note that sham-treated cells showed a minor reduction in VG I_Na (presumably due to cell rundown) and no change in I_leak. The nsPEF-treated cells displayed significantly greater reduction of VG I_Na and profound I_leak. Adding 20 mM BAPTA instead of 5 mM EGTA into the pipette buffer did not attenuate the nsPEF effects.