Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

Introduction

The advent of systems to deliver nanosecond duration electric pulses (NEPs) to biological cells has sparked a great deal of interest in exploring uses of these pulses for biomedical applications, such as neurostimulation. An example of the potential use of NEPs for neurostimulation is the report by Jiang and Cooper [1] that a single 12 ns pulse was capable of activating skin nociceptors. Also, a recent study by Casciola et al. [2] demonstrated that repeated stimulation of isolated peripheral nerves of Xenopus leavis by 12 ns pulses continuously elicited action potentials without damaging the nerve fibers. Previous studies from our group additionally have shown that a 5 ns pulse can stimulate catecholamine release in neuroendocrine adrenal chromaffin cells by causing Ca2+ influx via voltage-gated calcium channels (VGCCs) [3].

Efforts aimed at elucidating the mechanisms by which NEPs stimulate neural cells and tissues have revealed some interesting differences with respect to the involvement of voltage-gated Na+ channels. In peripheral nerve, 12 ns pulse exposure triggers Na+ influx via voltage-gated Na+ channels, which is responsible for the generation of action potentials [2]. In bovine chromaffin cells, in contrast, Na+ influx via voltage-gated Na+ channels is not responsible for the membrane depolarization that evokes VGCC activation in cells exposed to a 5 ns pulse [4]. Instead, membrane depolarization appears to be the result of Na+ influx via putative nanopores [4, 5]. Moreover, a 5 ns pulse actually causes an inhibition of voltage-gated Na+ channels in these cells [6].

Nesin et al. [7, 8] were the first to report that voltage-gated Na+ channels can be inhibited by NEPs. In their studies, a single 300 ns pulse was found to cause a prolonged inhibition of Na+ conductance in several excitable cell types that included NG108 cells, GH3 cells, as well as adrenal chromaffin cells. The inhibitory effect was enhanced when the E-field magnitude of the applied pulse was increased. Of interest to us was whether the much shorter duration 5 ns pulses that we had been studying also caused inhibitory effects on voltage-gated Na+ channels in chromaffin cells and if so, how the inhibition was affected by E-field intensity. To this end we recently evaluated the effects of a single 5 ns pulse on fast voltage-dependent Na+ currents (INa) recorded in whole-cell voltage clamped bovine chromaffin cells. The gating properties of the net current were consistent with those of tetrodotoxin-sensitive Na+ channels, and of NaV1.7, the pore-forming Na+ channel subunit thought to be the main voltage-gated Na+ channel expressed in bovine chromaffin cells [9-12]. Our results showed that a single 5 ns pulse applied at 5 MV/m, an E-field magnitude that is just above the threshold for evoking responses in these cells [13] produced a rapid inhibition (~ 4%) of INa at a test potential of +10 mV [6], which was in agreement with the inhibition of voltage-gated Na+ channels previously reported in these cells [8]. The decrease in INa was associated with a decrease in maximal conductance without a change in its voltage-dependence of steady-state activation and inactivation [6]. Our results also showed that a single 5 ns pulse at the higher E-field of 8 MV/m and 10 MV/m produced a rapid inhibitory effect that was ~ twofold greater (from ~ 4% to ~ 9%). The purpose of this study was to determine whether the application of a second pulse of identical characteristics leads to additive effects on INa.

Our data indicate that the application of a second NEP produced as anticipated an additive inhibitory effect on INa but surprisingly, only when the interval between the pulses exceeded 100 ms. In addition, the second pulse in a pair caused a paradoxical time-dependent recovery of the Na+ current after inhibition, a phenomenon that varied with E-field magnitude and the time interval between the pulses, and that appeared to involve lipid rafts. Our study strengthens the notion that voltage-dependent ionic currents can be altered by NEPs and offers a new perspective on how the application of multiple pulses could be tailored to augment or attenuate inhibitory effects on voltage-gated Na+ channel activity in excitable cells.

Materials and methods

Chromaffin cell culture

As described previously [14], chromaffin cells were isolated by collagenase digestion of the medulla from fresh bovine adrenal glands obtained from a local abattoir (Wolf Pack Meats, University of Nevada, Reno). After isolation, the cells were maintained in suspension culture in Petri dishes at 36.5°C under a humidified atmosphere of 5% CO2 in Ham’s F-12 medium supplemented with 10% bovine calf serum, 1% antibiotic (100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone) and 6 μg/ml cytosine arabinoside. Cells were used up until two weeks in culture. For experiments, single, isolated cells were obtained by treatment of cell aggregates with the protease dispase [15] and attached to fibronectin-coated glass coverslips.

Whole-cell patch clamp experiments

Coverslips containing the attached cells were placed in a perfusion chamber positioned on the stage of a Nikon Eclipse TS100 inverted microscope. The cells were continuously perfused at a rate of 0.5 ml/min with a balanced salt solution (BSS) consisting of 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM Na2HPO4, 1.3 mM MgCl2, 10 mM glucose and 15 mM Hepes, pH 7.4 (adjusted with NaOH). The bath solution was connected to the ground via an Ag/AgCl pellet. The patch pipette internal solution contained 10 mM NaCl, 30 mM KCl, 110 mM K-gluconate, 1 mM MgCl2, 10 mM EGTA, 3 mM Mg-ATP, and 10 mM Hepes, pH 7.2 (adjusted with KOH). Whole-cell currents were recorded in voltage clamp mode using an Axopatch 200B amplifier and Digidata 1322A (Axon Instruments, Sunnyvale, CA) or Digidata 1550B data acquisition system (Molecular Devices, Sunnyvale, CA), and PClamp software (version 8.2 or 11, Molecular Devices, Sunnyvale, CA) at a sampling rate of 20 kHz and low-pass filtering at 1 kHz. The series resistance (Rs), which was compensated to 90%, varied between 7 and 32 MΩ. The seal resistance (Rseal) varied between 1.1 and 3.0 GΩ and was measured by applying a +10 mV test pulse from a holding potential of ‒70 mV. Cell capacitance (Cm) ranged between 4 and 12 pF. All recordings were performed at ambient room temperature. Fire-polished patch micropipettes having a tip size of 0.8–1.2 μm with a typical resistance of ~ 3 MΩ were fabricated from borosilicate glass (#BF150-110-7.5, Sutter Instruments, Novato, CA) using a P-97 pipette puller (Sutter Instruments, Novato, CA) and fire-polished with a MF-830 microforge (Narishige, Tokyo, Japan). The cell being recorded was viewed with a 40X objective and a bright field image of the cell was captured with a CoolSnap HQ DIFF CCD camera (Photometrics, Tucson, AZ) and SimplePCI software (version 6.6.0.0, Hamamatsu Corporation, Hamamatsu City, Japan) at the beginning and end of each experiment. For recordings obtained in the absence of extracellular Na+, an equimolar concentration of N-methyl-D-glucamine (NMDG+) was used to replace Na+ in the external bath solution. For recordings conducted under K+-free conditions, the patch pipette internal solution contained 10 mM NaCl, 20 mM tetraethylammonium chloride (TEA-Cl), 10 mM CsCl, 110 mM aspartic acid, 1 mM MgCl2, 10 mM EGTA, 3 mM Mg-ATP, and 10 mM Hepes, pH 7.2 (adjusted with CsOH) at room temperature. The external bath solution consisted of 145 mM NaCl, 10 mM TEA-Cl, 1.2 mM Na2HPO4, 2 mM CaCl2, 1.3 mM MgCl2, 5.5 mM glucose and 10 mM Hepes, pH 7.4 (adjusted with CsOH). For the Ca2+-free experiments, an equimolar concentration of MgCl2 was used to replace CaCl2 in the external bath solution.

The effects of NEPs on peak inward currents were recorded by applying a total of 200 voltage clamp steps, with a 3 s interval between each step as described in our previous work [6]. The voltage clamp protocol consisted of applying a 100 ms voltage step to +10 mV from a holding potential of ‒70 mV. A single pulse or a pulse pair was applied 1.5 s after the 21st voltage clamp step. The detailed protocols are described in the supplemental information (S1 Fig). Currents of the 8 voltage steps prior to NEP exposure were averaged and used as baseline.

The NEP-induced leak current (Ileak) was recorded during a 2 s voltage ramp protocol ranging from ‒70 to +80 mV from a holding potential of ‒70 mV. The ramp protocol was applied 5 times prior to the application of a pair of NEPs to determine control baseline membrane current, and 15 times following the application of twin pulses. The interval between each ramp was 6 s. Ileak was measured in K+-free pipette internal solution and in K+-free external bath solution containing 20 μM nitrendipine. Ileak was determined by subtracting the ramp current measured after the pair of NEPs from the baseline ramp current.

Current-voltage (I-V) relationships for inward currents were generated using a voltage step protocol consisting of 50 ms steps ranging from –70 mV to +80 mV applied in 10 mV increments every 2 s. Steady-state activation curves for Na+ currents were constructed by measuring the peak Na+ conductance (GNa) calculated from the equation: where INa is the peak Na+ current during the test depolarization (V), and Erev is the reversal potential of the inward current determined as the voltage producing null current on the I-V relationship. Data were normalized to maximum peak conductance (Gmax) and fitted to a Boltzmann function: from which the slope factor (k) and half-maximal activation voltage (V0.5) were derived.

The steady-state inactivation curve of INa was generated by holding the cell at –70 mV and applying a series of 1 s conditioning voltage clamp steps ranging from –100 to 0 mV in 10 mV increments, with each voltage step followed by a constant 100 ms test pulse to +10 mV to record INa. INa was normalized to its respective maximal value and plotted as a function of the conditioning potential. Normalized peak inward currents were plotted as a function of the conditioning potentials and the data fitted to an appropriate Boltzmann function: from which the slope factor (k) and half-maximal inactivation voltage (V0.5) were derived.

NEP exposure

The 5 ns duration pulses were delivered by a nanosecond pulse generator, designed and fabricated by Transient Plasma Systems, Inc. (Torrance, CA), to chromaffin cells using a pair of cylindrical, gold-plated tungsten rod electrodes (127 μm diameter) with a gap of 100 μm between the electrode tips as detailed in previous publications [6, 13]. After achieving the whole-cell recording mode by rupturing the cell plasma membrane, the NEP-delivering electrodes were positioned to a predefined “working” position 40 μm from the bottom of the coverslip by a motorized MP-225 micromanipulator (Sutter Instruments, Novato, CA), with the patched cell situated midway between the electrode tips. A single or pair of 5 ns pulses was delivered to the cells at E-field amplitudes ranging from 5 to 10 MV/m. The E-field distribution at the location of the target cell was computed by the Finite-Difference Time-Domain (FDTD) method using the software package SEMCAD X (version 14.8.5, SPEAG, Zurich, Switzerland) as previously described [5, 6, 13]. In all experiments, two sham (control) exposures preceded the delivery of a single or twin NEP(s) controlled by a program written in LabVIEW [5, 6, 13].

Reagents

Ham’s F-12 medium, dispase II and the antibiotics-antimycotics were obtained from Gibco Laboratories (Grand Island, NY, USA) and bovine calf serum was purchased from Gemini Bio-Products (West Sacramento, CA, USA). Collagenase B and methyl-β-cyclodextrin were purchased from MilliporeSigma (St. Louis, MO, USA). Tetrodotoxin was purchased from Alomone Labs (Jerusalem, Israel). All other chemicals were reagent grade and purchased from standard commercial sources.

Data analysis

For all experiments, the results were obtained using cells from different days in culture and different cell preparations and presented as the mean ± standard error (SE). The data were analyzed with SPSS or Origin 2020 (v. 9.7, OriginLab, Northampton, MA) software using either an unpaired two-tailed t test when the means of two groups were compared, or a One-Way or Two-Way ANOVA test for repeated measures followed by Tukey post hoc multiple range tests in multiple group comparisons. P < 0.05 was considered statistically significant.

Results

Voltage-gated Na+ channels are responsible for the early inward current

An initial series of experiments performed in bovine chromaffin cells exposed to normal K+-based external (BSS) and internal solutions was carried out to determine the ionic nature of the early inward current elicited by depolarizing voltage clamp steps from a holding of –70 mV. Three families of membrane currents elicited by the voltage clamp protocol shown in Fig 1A are presented in Fig 1B. The mean peak inward current measured for each was plotted as a function of step potential as shown in Fig 1C. For cells in BSS, the early inward current activated near –30 mV, peaked around +10 mV and reversed at ~ +50 mV (Fig 1B and 1C). Total replacement of external Na+ with the non-permeant NMDG+ abolished the inward current (Fig 1B and 1C), which confirms the results of a recent study by our group performed under similar conditions [6] and demonstrates that Na+ was the charge carrier responsible for this voltage-dependent inward current. Finally, the specific voltage-gated Na+ channel inhibitor tetrodotoxin (TTX; 5 μM) also eliminated the inward current (Fig 1B and 1C). Taken together, these results support the idea that even when recorded in normal K+-based salt solutions, the early inward current was predominantly carried by Na+ and reflects the activity of TTX-sensitive voltage-dependent Na+ channels and will thus be referred to as INa.

Fig 1

Voltage-dependent properties of the early inward current recorded in whole-cell voltage clamped bovine chromaffin cells.

(A) The voltage clamp step protocol used to determine the voltage-dependence of peak inward current consisted of 50 ms steps ranging from –70 to +80 mV, applied in 10 mV increments every 2 s from a holding potential of –70 mV. (B) Representative families of membrane current traces elicited by the voltage step protocol shown in (A) recorded in different experimental conditions. BSS: normal K+-based external bath solution; NMDG+: an equimolar concentration of NMDG+ was used to replace Na+ in the external bath solution; TTX: 5 μM TTX was added to the BSS to block voltage-dependent Na+ channels. (C) I-V relationship for the peak inward current (arrow in B). Each data point represents the mean value ± SE (BSS, n = 4; NMDG+, n = 6; TTX, n = 3).

Comparison of the effects of single and twin NEPs on INa

A constant voltage clamp step protocol consisting of repetitive voltage steps to +10 mV (near the peak of the I-V relationship; see Fig 1C) applied every 3 s from a holding potential of ‒70 mV was performed to monitor the time-dependent changes in INa before and after cell exposure to 5 ns pulses. INa was recorded for 1 min before NEP exposure to establish a baseline and then for 9 min afterwards to monitor the response.

The results presented in Fig 2A show that in cells unexposed to a NEP, INa ran down spontaneously ~ 6% over the course of 9 min, a phenomenon most likely attributable to a negative shift of the voltage-dependence of voltage-gated Na+ channels when recorded using the whole-cell variant of the patch clamp technique [16]. A single 5 ns pulse applied at an E-field of 5 MV/m resulted in a rapid decrease of ~ 4% in peak INa that then declined to a level that was ~ 89% of the initial level after 9 min (Fig 2A). These results are in agreement with those previously reported [6]. The application of a second 5 ns pulse 30 s after the first pulse amplified the inhibitory effect of a single pulse on INa from ~ 4% to 7% (Fig 2B). Representative traces of INa are shown in S2 Fig. Interestingly and in contrast to the effects of a single NEP, the second pulse in the pair led to a progressive recovery of the current that stabilized near the level of INa observed in control cells unexposed to NEPs (Fig 2B).

Fig 2

Effects on INa of a single or twin NEP applied at an E-field of 5 MV/m.

(A) Time course of changes in INa for unexposed cells (control, black circles) compared to cells exposed to a single NEP (1 pulse, blue circles). (B) Time course of changes of INa in cells exposed to a twin pulse applied with a 30 s interpulse interval (red circles) compared to that recorded in unexposed cells (control, black circles). For both panels, normalized current represents the magnitude of peak inward current evoked by the constant voltage step protocol described in the Methods normalized to the mean peak inward current recorded during the 8 voltage clamp steps to +10 mV (holding potential = ‒70 mV) that preceded the NEP(s). Each data point is a mean ± SE (control, n = 20; 1 pulse, n = 12; twin pulse, n = 10).

Decreasing the time interval between the twin pulses from 30 to 5 s (Fig 3A) again produced a similar inhibitory effect (~ 7%) on INa. However, the recovery of INa was accelerated and enhanced relative to intervals between pulses that are longer. Further shortening the time interval between the two pulses to 1 s or 100 ms progressively attenuated the inhibitory effect (~ 6% and ~ 5%, respectively) on INa (Fig 3B and 3C). Additionally, when the time interval between the two pulses was decreased to 10 ms (Fig 3D), the inhibition of INa was slightly less (~ 3%) than the effect of a single pulse on peak INa. Again, shorter intervals between pulses accelerated and amplified the recovery of INa. In fact, when the interval was only 10 ms, the current leveled off near that recorded at the beginning of the experiment, that is prior to the application of NEPs (Fig 3D). Representative traces of INa are shown in S2 Fig. One-Way ANOVA test revealed a significant difference between the cells exposed to the twin pulses and single pulse (P< 0.001).

Fig 3

Effects on INa of the time interval between twin NEPs applied at an E-field of 5 MV/m.

Time course of changes in INa for cells exposed to a single pulse (blue circles, data reproduced from Fig 2A) compared to cells exposed to twin NEPs (red circles) separated by a 5 s (A), 1 s (B), 100 ms (C) or 10 ms time interval (D), respectively. Each data point is a mean ± SE (1 pulse, n = 12; 2 pulses with a 5 s interval, n = 9; 2 pulses with a 1 s interval, n = 9; 2 pulses with a 100 ms interval, n = 9; 2 pulses with a 10 ms interval, n = 9).

At the higher E-field amplitude of 8 MV/m, INa rapidly decreased by ~ 9% following the application of a single pulse (Fig 4A) as reported previously [6]. Exposure to a second 5 ns pulse 30 s after the first pulse increased the inhibitory effect produced by a single NEP from ~ 9% to 14% (Fig 4B). Representative traces of INa are shown in S2 Fig. Decreasing the time interval between the twin pulses from 30 s to 5 s or 1 s led to a comparable inhibitory effect on INa (~ 14%). For both intervals, INa recovered to a level that was similar to that achieved by a single NEP. Decreasing the interval between the two pulses from 1 s to 100 ms (Fig 4C) led to a reduction of the inhibition from ~ 14% to 11%. Furthermore, when the time interval between the two pulses was decreased to 10 ms (Fig 4D and S2 Fig), the inhibition of INa was slightly less (~ 10%) than that caused by the 100 ms time interval, which was still larger than the effect of a single pulse on peak INa. Similar to the effects of twin NEPs at 5 MV/m, intervals shorter than 1 s between pulses accelerated and increased the magnitude of the recovery of INa, which stabilized above that produced by a single NEP at 8 MV/m (Fig 4C and 4D). One-Way ANOVA test showed a significant difference between the cells exposed to the twin pulses and single pulse (P< 0.001).

Fig 4

Effects on INa of the time interval between twin NEPs applied at an E-field of 8 MV/m.

(A) Time course of changes in INa for unexposed cells (control, black circles) compared to cells exposed to a single pulse (blue circles). Time course of changes of INa in cells exposed to a single pulse (blue circles) compared to cells exposed to twin pulses (red circles) with a 30 s (B), 100 ms (C) or 10 ms time interval (D), respectively. Each data point is a mean ± SE (control, n = 20; 1 pulse, n = 15; 2 pulses with a 30 s interval, n = 5; 2 pulses with a 100 ms interval, n = 10; 2 pulses with a 10 ms interval, n = 10). The control data set in panel A (black circles) are the same as those shown in Fig 2.

A single NEP at 10 MV/m produced a 9% decrease in INa, which is comparable to that seen with a single pulse at an E-field of 8 MV/m (Fig 5A) as previously reported [6]. Once inhibited the current remained stable for ~ 9 min post-NEP. These results indicate that the effect of a single NEP on INa saturated at the higher E-field amplitudes of 8 and 10 MV/m [6].

Fig 5

Effect of a single pulse and twin NEPs applied at different time intervals on INa at an E-field of 10 MV/m.

(A) Time course of changes in INa for unexposed cells (control, black circles) compared to cells exposed to a single pulse (blue circles). Time course of changes in INa for cells exposed to a single pulse (blue circles) compared to cells exposed to twin pulses (red circles) with a 30 s (B) or 10 ms time interval (C), respectively. Each data point is a mean ± SE (control, n = 20; 1 pulse, n = 15; 2 pulses with a 30 s interval, n = 8; 2 pulses with a 10 ms interval, n = 7). The control data set in panel A (black circles) are the same as those shown in Fig 2.

The time course of changes in INa resulting from twin NEP exposures are shown in Fig 5. Increasing the E-field amplitude to 10 MV/m increased the inhibitory effect of twin NEPs on peak INa (~ 15%) regardless of the interval between twin NEPs. At this E-field, the second pulse in the pair applied 10 ms to 30 s after the first pulse consistently increased the inhibitory effect of a single NEP from ~ 9% to ~ 15%. Representative traces of INa are shown in S2 Fig. This contrasts with the effects of twin pulses at lower E-fields (≤ 8 MV/m), which produced less inhibitory effects when the interval was progressively shortened (Figs 3 and 4). The data in Fig 5 also reveal that although the inhibitory effect of twin NEPs on INa was still apparent at this E-field, current recovery was severely impaired, never reaching the level produced by a single NEP. Taken together, these results indicate that the effects of twin NEPs on INa are highly dependent on the interval between the pulses and E-field magnitude. One-Way ANOVA test revealed a significant difference between the cells exposed to the pulse pair and single pulse (P< 0.001).

Fig 6 summarizes the effects of E-field strength and interpulse interval on the inhibition and recovery of INa in response to twin NEPs. As shown in Fig 6A and 6B, twin pulses applied at the E-fields of 5 and 8 MV/m, respectively, produced an additive inhibitory effect on INa compared to that elicited by a single pulse when the interval between the pulses was longer than 100 ms. The additional inhibition was progressively reduced when the interval between pulses was 100 ms and 10 ms. At the E-field of 10 MV/m (Fig 6C), a twin pulse caused a consistently larger inhibitory effect relative to a single pulse, the magnitude of which was independent of pulse interval in the range of 10 ms to 30 s.

Fig 6

Time interval and E-field strength dependence of the inhibitory and recovery effect of twin NEPs on INa.

For the left panel (inhibitory), each semi-logarithmic plot reports the mean ± SE % inhibition of peak inward Na+ current elicited in cells exposed to a single pulse (blue circles), or cells exposed to twin NEPs (red circles) as a function of the interval between the pulse pair at an E-field of 5 MV/m (A), 8 MV/m (B) or 10 MV/m (C), respectively. For the right panel (recovery), each plot reports the mean ± SE normalized peak inward Na+ current registered in unexposed cells (control, black circles), cells exposed to a single pulse (blue circles), or cells exposed to twin NEPs (red circles) as a function of the interval between the pulse pair at an E-field of 5 MV/m (D), 8 MV/m (E) or 10 MV/m (F), respectively. In panels D, E and F, all measurements were performed 9 min after the application of the twin pulses. In panels A and D, the solid line is a logistic fit to the data points. The solid lines in panels B and E are a sigmoidal fit to the data points. In panels C and F, the solid line is a linear fit. Results were obtained from the same cells as those shown in Figs 2, 3, 4 and 5.

With respect to the level of recovery of INa determined 9 min after NEP exposure, a single pulse applied at an E-field magnitude of 5 MV/m resulted in a decline in INa to a level that was ~ 89% of the initial level compared with control cells in which INa was ~ 94% of the initial level (Fig 6D). Paradoxically, the application of twin pulses at 5 MV/m (Fig 6D) or 8 MV/m (Fig 6E) led to a recovery of INa after the inhibition that was inversely proportional to the time interval between the twin NEPs. In fact, at intervals of 10 or 100 ms, INa magnitude was similar to or slightly greater than the level reached in cells unexposed to NEPs. Interestingly, the application of a twin pulse at an E-field of 10 MV/m (Fig 6F) did not lead to recovery of INa, which stabilized below the level reached with a single pulse regardless of the interval between the twin pulses.

The application of twin NEPs led to similar effects on INa when blocking K+ and Ca2+ currents

The experiments described above examining the effects of NEPs on INa recorded at +10 mV were performed under physiological conditions with normal transmembrane gradients of Na+, K+ and Ca2+. To isolate the effect of the NEPs on Na+ channels, additional experiments were carried out by suppressing K+ and Ca2+ currents. First, K+ currents were inhibited by replacing K+ with Cs+ and TEA both in the internal and external solutions, and by replacing K-gluconate with Cs-aspartate in the internal solution. An equimolar concentration of Mg2+ was used to replace Ca2+ in the external bath solution to suppress Ca2+ currents instead of using the dihydropyridine antagonist nitrendipine because this compound also reduced INa as previously reported by others [17] at the high concentration (20 μM) that is necessary to fully inhibit Ca2+ currents in chromaffin cells [18]. As shown in Fig 7A, the activation threshold potential and magnitude of the inward current recorded between –30 to +40 mV were not affected by using K+- and Ca2+-free solutions; however, a small positive shift of the reversal potential (~ +5 mV) was noted, which can be attributed to a small but significant contribution from outward K+ currents due to the reduced driving force for Na+ at higher voltages (> +40 mV). Application of twin pulses with a 10 ms interpulse interval applied at an E-field of 8 MV/m produced a similar inhibitory effect on INa shortly after pulse application, which recovered following a nearly superimposable time course to that recorded in regular BSS (unpaired t test; P = 0.7; Fig 7B), confirming the results obtained with normal K+-based external and internal solutions (e.g. Fig 4D) and that the paradoxical effects of twin pulses were targeted to INa.

Fig 7

Effect of twin pulses on INa recorded in K+- and Ca2+-free BSS at an E-field of 8 MV/m.

(A) Effect of eliminating K+ and Ca2+ currents on the I-V relationship of the early peak inward current. I-V relationships were obtained either in regular BSS (black circles) or in K+- and Ca2+-free BSS (Cs+ + TEA + Mg2+; red circles). (B) Mean time course of changes of peak INa recorded with normal (BSS) or Cs+-TEA-Mg2+-based solutions in cells exposed to twin pulses (8 MV/m) with a 10 ms interpulse interval. Data are expressed as the mean ± SE (BSS, n = 5; Cs+ + TEA + Mg2+, n = 6).

Twin NEPs reduced the maximal conductance but not the voltage-dependence of INa

The inhibitory and reversal effects of twin 5 ns pulses on INa could be caused by a shift of the steady-state inactivation or activation curve, or both. In order to test for possible changes in steady-state inactivation, a typical double-pulse protocol was used whereby the cells were held over a range of potentials for 1 s and then depolarized to a test pulse (+10 mV) to evaluate the availability of Na+ channels. As for Fig 7, all these experiments were carried out under conditions isolating INa. Fig 8A shows representative traces of INa recorded just prior (left), immediately after (middle) and 9 min following (right) the application of twin NEPs. Fig 8B shows the effects of twin pulses with a 1 s interpulse interval at E-field strength of 8 MV/m on the voltage-dependence of inactivation. This interval and E-field were chosen because we started to observe the largest inhibition of INa with these pulse parameters (Fig 6E). Half-maximal inactivation voltages V0.5 were –50.4 ± 1.2 mV (k = 8.3 ± 0.6), –51.3 ± 0.7 mV (k = 7.1 ± 0.4) and –54.4 ± 0.6 mV (k = 8.1 ± 0.4) before the pulses, immediately after and 9 min after the pulses, respectively. These data show that there was no significant effect of the twin pulses on the steady-state inactivation of INa (One-Way ANOVA, P > 0.05).

Fig 8

Effects of twin NEPs applied with a 1 s time interval on the voltage-dependence of steady-state inactivation, activation and conductance of INa at an E-field of 8 MV/m.

(A) Three families of INa recorded in the same cell from a typical experiment; as depicted, these families of traces were recorded before (Control; left traces), immediately after (After pulses; middle traces) and 9 min after (right traces) twin pulse exposure. For each family of traces, step potentials spanned from –40 to +60 mV. (B) Steady-state inactivation curves of INa recorded before (control, black circles), right after (red circles) and 9 min after (blue circles) twin pulse exposure. INa in each case was normalized to its respective maximal value, plotted as a function of the conditioning potential and fitted to a Boltzmann function (solid lines). The data represent the mean value ± SE, n = 5. The V0.5 and slope factor values derived from all Boltzmann fits can be found in the text. Overall One-Way ANOVA yielded P values for V0.5 (P = 0.24373) and k (P = 0.8831) that revealed no significant differences between the means of the three groups. (C) I-V relationships recorded before (control, black circles), immediately after (red circles), and 9 min after (blue circles) twin pulse exposure; the I-V relationship labeled with green circles (“After pulses with correction”) was obtained by subtraction of the I-V of the NEP-induced leak current (grey circles; Ileak) from that measured after delivery of the NEPs (red circles). Ileak was measured in separate experiments using a voltage ramp protocol and K+-free external solution containing 20 μM nitrendipine as described in the Methods. All data points are expressed as the mean ± SE (I-V, n = 5; Ileak, n = 4). (D) Steady-state activation curves of INa recorded before (control, black circles), immediately after with correction (green circles) and 9 min following (blue circles) twin pulse exposure. For each data set, INa in each case was normalized to its respective maximal conductance (calculated as indicated in the Methods), plotted as a function of the test potential and fitted to a Boltzmann function (solid lines). All data points are expressed as the mean ± SE (n = 5). The V0.5 and slope factor values derived from all Boltzmann fits can be found in the text. Overall One-Way ANOVA yielded P values for V0.5 (P = 0.18424) and k (P = 0.46385) that revealed no significant differences between the means of the three groups. (E) Relative conductance, calculated by normalizing the conductance estimated before and after the pulses to the maximum conductance before the pulses, was plotted against test potential. Peak INa was converted to conductance as described in the Methods. The data were fitted to a Boltzmann function (solid lines). All data points are expressed as the mean ± SE (n = 5). An overall Two-Way ANOVA test revealed significant differences between Pulse treatment (P < 0.001), Voltage (P < 0.001) and the Interaction between Pulse treatment and Voltage (P = 0.00679). Overall Tukey post hoc tests revealed a significant difference between “Control” and “After pulses with Ileak correction” (P < 0.001) and between “Control” and “9 min after pulses” (P = 0.00616), but no significant difference between “After pulses with Ileak correction” and “9 min after pulses” (P = 0.84134). Tukey Post hoc tests only showed significant differences between “Control” and “After pulses with Ileak correction” at +10 (P = 0.04208) and +20 mV (P < 0.001), and between “Control” and “9 min after pulses” at +20 mV (P = 0.01159).

The three families of INa shown in Fig 8A, which were recorded in the same cell just prior, immediately after and 9 min following the application of twin NEPs with an interval of 1 s at 8 MV/m, show the inhibitory effect of twin pulses on INa and partial recovery of the current over a 9 min post-pulse period. Fig 8C shows the I-V relationships of INa recorded for these same time periods. As previously reported by our group for a single NEP [6], INa was significantly reduced by twin NEPs over a wide range of membrane potentials. Twin pulses also produced a significant negative shift (~ –6 mV) of the reversal potential relative to the control I-V obtained prior to pulse delivery. We hypothesized that this shift might be attributable to a relatively small leak current (Ileak; Fig 8A, middle family of traces) elicited by twin NEPs an observation consistent with the concept of membrane electronanoporation [5, 6]. To determine the impact of this basal conductance on peak INa measurements, Ileak was estimated in separate experiments using K+-free pipette and bath solutions containing Cs+ and TEA, which were used to suppress voltage-gated K+ channels, and 20 μM nitrendipine applied to the bath to inhibit voltage-gated Ca2+ channels [18]. A 2 s voltage ramp protocol spanning from ‒70 to +80 mV was used to generate I-V relationships before and after the application of twin pulses with a 1 s interval at E-field of 8 MV/m. Voltage-gated Na+ channels were voltage-inactivated by this slow voltage ramp protocol. Fig 8C depicts the small NEP-sensitive I-V for the leak current (Ileak; grey circles; obtained by digital subtraction of the control current recorded prior to the application of twin NEPs), which displayed mild inward rectification and a reversal potential near 0 mV. Leak current measurements were performed by averaging the current during the voltage ramp for a range spanning ± 0.5 mV at each target potential (used for peak INa measurements). Subtraction of the I-V of Ileak from the I-V of INa recorded immediately after delivery of twin NEPs yielded a “corrected” I-V for INa (green circles) that displayed a very similar amplitude over the entire range of membrane potentials and a reversal potential (~ +62 mV) that coincided with the control I-V for INa recorded prior the application of twin NEPs. This correction procedure was not necessary for INa measured 9 min after the NEPs were applied because Ileak slowly disappeared over this time span, which is similar to our previous report with a single NEP [5]. Taken together, our data demonstrate that peak INa measured at +10 mV, which is the test potential mostly used in our study, was nearly superimposable to the uncorrected INa, indicating that the changes in peak Na+ current mediated by NEPs were minimally contaminated by the presence of Ileak.

We next examined whether twin NEPs affect the steady-state activation curves of INa using the data presented in Fig 8C. These curves were constructed by estimating the normalized Na+ conductance (GNa/Gmax) from measurements of corrected peak inward Na+ current evoked by the depolarizing steps used to build the I-V curve and the driving force (V–Erev) as described in the Methods. As for the steady-state inactivation curve, our results indicate that twin pulses with a 1 s interval at an E-field of 8 MV/m produced no significant effect on the voltage-dependence of activation of INa (Fig 8D). Half-maximal activation voltages V0.5 were –8.2 ± 0.5 mV (k = 6.3 ± 0.2), –10.7 ± 0.5 mV (k = 6.6 ± 0.2) and –10.5 ± 0.5 mV (k = 6.3 ± 0.3) before, immediately after with correction and 9 min after pulse delivery, respectively. One-Way ANOVA tests revealed no significant differences between the three groups for both parameters (P > 0.05). The Erev (derived from the I-Vs in Fig 8C) were 59.6 ± 2.6 mV, 62.1 ± 2 mV (corrected) and 57.6 ± 1.6 mV before, immediately after and 9 min after the pulses, respectively, which were also not significantly different from each other (P > 0.05).

Another possibility that could account for the effects of twin NEPs on peak INa is a change in maximal conductance of the Na+ channel, as previously reported for a single pulse [6]. To assess this possibility, we determined the absolute Na+ conductance before and after twin pulses with a 1 s interpulse interval at an E-field of 8 MV/m. For this determination, relative Na+ channel conductance was calculated by normalizing the conductance of the channel after the pulses to the conductance of the channel measured before the pulses. As shown in Fig 8E, the maximal conductance of INa estimated immediately after the twin pulses was significantly reduced at +10 and +20 mV (P < 0.05). For example, at +20 mV, the decrease in maximal Na+ conductance was ~ 26% after the pulses. However, 9 min after the pulses the maximal Na+ conductance did not significantly recover, an observation consistent with the lack of recovery of INa after the twin pulse-induced inhibition with longer intervals as described above in Fig 6D and 6E.

Disruption of lipid rafts alters the effects of twin NEPs on INa

It is well known that lipid rafts and one of its subset structures called caveolae are specialized membrane assemblies involved in compartmentalized signaling and protein trafficking [19]. In this regard, TTX-resistant NaV1.5 and NaV1.8 channels were respectively shown to be clustered in caveolae in breast cancer cells and sensory dorsal root ganglion neurons [20, 21]. Although it has not yet been established whether TTX-sensitive NaV1.7 channels are similarly clustered in lipid rafts, we nevertheless hypothesized that twin NEPs may be altering the function of voltage-gated Na+ channels in chromaffin cells by indirectly perturbing the composition and/or structure of lipid rafts regulating their activity and recycling. Methyl-β-cyclodextrin (MβCD), an agent known to deplete membrane cholesterol and impair lipid rafts and caveolae [19, 22], was used to investigate whether the disruption of lipid rafts altered the response of INa to twin NEPs. Cells were pre-incubated for a minimum of 30 min at a concentration of 3 mg/ml MβCD. We first tested the effects of MβCD on INa in cells unexposed to NEPs. As shown in Fig 9A, MβCD produced a 42% inhibition of the inward current at the test potential of +10 mV and caused a small negative shift of the reversal potential (~ 5 mV). This small shift in reversal potential is probably attributable to a relatively greater contribution from outward voltage-dependent K+ currents triggered by the MβCD-induced inhibition of peak Na+ current measured at voltages where the driving force for Na+ is diminished. These results imply that Na+ channels in chromaffin cells may too be regulated by lipid rafts in a manner suggestive of a permissive role.

Fig 9

Disruption of lipid rafts reduces INa and alters the impact of twin NEPs on the inward current.

(A) The voltage step protocol to construct the I-V relationship of peak INa was described in the Methods. The mean I-V relationships were obtained either in the presence of normal BSS (black circles) or in BSS containing MβCD (red circles). (B) Time course of changes in INa for cells exposed to a single pulse at an E-field of 8 MV/m recorded in the absence (black circles) or presence (red circles) of MβCD. (C) Time course of changes in INa for cells exposed to twin NEPs at an E-field of 5 MV/m with a 10 ms time interval between the pulses recorded in the absence (black circles) or presence (red circles) of MβCD. (D) Time course of changes in INa for cells exposed to twin pulses at an E-field of 8 MV/m with a 10 ms time interval between the pulses recorded in the absence (black circles) or presence (red circles) MβCD. Cells exposed to MβCD were pre-incubated with the drug at a concentration of 3 mg/ml for a minimum of 30 min. Data are expressed as the mean ± SE. (A): BSS, n = 16; MβCD, n = 14; (B): BSS, n = 15; MβCD, n = 6; (C): BSS, n = 9; MβCD, n = 5; (D): BSS, n = 10; MβCD, n = 8).

We next examined how depleting membrane cholesterol affected the inhibition of INa in cells exposed to a single 5 ns pulse at the E-field of 8 MV/m that caused a ~ 9% drop in Na+ conductance, followed by a stabilization of the current (Fig 4A). As shown in Fig 9B, pretreating cells with MβCD minimally altered the response to the pulse, reducing slightly the inhibitory effect of the pulse on INa, and causing a minor increase in the level of recovery of the current that was just at the limit of statistical significance (unpaired t test P = 0.07). However, MβCD pretreatment of the cells impacted the effect of twin NEPs on the inward current in a different manner. In cells pretreated with MβCD, a pair of NEPs at an E-field of 5 MV/m with a 10 ms interval between pulses increased the inhibitory effect on INa from ~ 3% to ~ 5% (Fig 9C). Interestingly, the recovery of INa in MβCD-treated cells was reduced but not significantly relative to that registered from cells unexposed to MβCD (unpaired t test P = 0.19). The effects noted at an E-field of 5 MV/m were similar but much more prominent at an E-field of 8 MV/m (Fig 9D). The inhibition caused by twin NEPs was enhanced from ~ 14% to ~ 22% in cells pretreated with MβCD compared to untreated cells. Moreover, the recovery of INa was nearly abolished by cholesterol depletion (unpaired t test P < 0.001).

Discussion

Stimulation of adrenal chromaffin cells with 5 ns pulses is effective for evoking neurosecretion in the absence of overt deleterious effects [3, 4, 23, 24]. As reported in this study, we found that application of 5 ns pulses as a pulse pair (twin pulse) can modulate voltage-gated Na+ channels in a unique manner, which points to the potential for NEPs to influence overall cell excitability in novel ways.

The early rapid inward current is mediated by voltage-gated Na+ channels

In this study, we investigated in whole-cell voltage clamped bovine chromaffin cells the effects of twin nanosecond pulses on the activity of the early and rapid inward current recorded in physiological gradients of Na+, K+ and Ca2+ across the membrane. Our data showed that this current was largely carried by Na+ influx across voltage-gated Na+ channels because: 1) the inward current was eliminated by external Na+ substitution with the non-permeant NMDG+, as similarly reported by our group [6], or 2) by the specific NaV inhibitor tetrodotoxin, 3) the voltage-dependence was similar to that of the main Na+ channel pore-forming subunit expressed in chromaffin cells, NaV1.7 [9-12], 4) the properties of the inward current were nearly identical to those of the inward current recorded in K+-free solutions containing Cs+ and TEA to inhibit K+ channels, and nitrendipine applied externally to inhibit Ca2+ channels, with the exception of the current measured near the reversal potential, and 5) the effects of twin NEPs on the inward current were very virtually superimposable whether the current was recorded using physiological solutions or solutions designed to isolate INa. A small exception was the contamination of the inward current recorded in normal solutions around the reversal potential, with some contamination from outward K+ currents that shifted Erev by a few millivolts in the negative direction. This had no effect on our findings since the effects of twin NEPs were investigated at +10 mV. Moreover, a more detailed analysis of the effects of twin NEPs on INa was performed under conditions that isolated INa. These arguments give credence to the notion that the fast inward current modulated by NEPs in this study was INa.

Inhibitory effects of twin NEPs on INa

A previous study from our group reported that a single 5 ns pulse was sufficient to produce a small but consistent inhibition of INa in bovine chromaffin cells [6]. This was reminiscent of studies showing that much longer nanosecond pulses (300 ns) also reduced INa in chromaffin cells [8]. Based on these findings, we tested in this study the hypothesis that the application of a second NEP of identical duration and E-field strength would produce an amplified inhibitory effect on INa. We showed that irrespective of the E-field tested (5 to 10 MV/m), the inhibition of INa approximately doubled relative to that produced by a single pulse when the interval between the two pulses was long (≥ 5 s). One puzzling observation was that for twin pulses at E-field strengths of 5 and 8 MV/m but not 10 MV/m, reducing the interval between the pulses decreased the additive inhibitory activity on INa. In fact, for 5 MV/m, the magnitude of the inhibition produced by twin pulses with a 10 ms interpulse gap was slightly less than that evoked by a single NEP. These data suggest that the process initiated by the first pulse was somehow interrupted or reversed by the second pulse in the pair. This is analogous to the phenomenon of bipolar cancellation whereby the application of a second nanosecond pulse of opposite polarity can attenuate or abolish a cellular response (Ca2+ transient, electronanoporation, fluorescent dye uptake, etc.) triggered by the corresponding unipolar pulse [25-33].

As for a single NEP [6], the inhibitory effects of twin NEPs on INa were not accompanied by a change in the voltage-dependence of steady-state activation and inactivation, indicating that single or twin NEPs did not appear to interfere with the voltage sensor. After correction for the presence of a small inwardly-rectifying basal conductance induced by a pulse pair, which was previously reported by our group in chromaffin cells for a single NEP [5], the reversal potential of the inward current was also unchanged by twin NEPs, suggesting that the ion selectivity and perhaps the ion conduction pathway remained intact. The only parameter that was altered by twin NEPs was a reduction in the maximal Na+ conductance G and this observation was similar to that evoked by a single NEP in this and a previous study from our group [6].

For a macroscopic ionic current, G is defined by the following equation: where g is the single-channel conductance (in pS), P is the maximal open probability and N the number of active channels in the membrane. It is likely that based on the lack of effect of twin NEPs on the reversal potential that the single-channel conductance, an indicator of ion flux across a single open ion channel, was minimally altered by twin pulses. On the basis of unperturbed voltage sensing by the channels, it is possible that P may also have been unaffected by twin NEPs although this will require further testing.

One plausible hypothesis is that twin NEPs produced a transient suppression of the number of active channels through perturbations of the lipid bilayer closely interacting with the channel protein. This suggestion is based on a number of indirect observations from our group and others: 1) theoretical molecular dynamics studies [34-36] demonstrated that high intensity NEPs are capable of triggering the formation of hydrophilic nanopores in the phospholipid bilayer that lower the energy barrier for ion conduction across the membrane; 2) single or multiple NEPs of various durations are known to cause important perturbations of the lipid bilayer, a process called electronanoporation, that allows the membrane to become permeable not only to ions [37, 38] but in some cell types also to fluorescent organic molecules such as propidium iodide and YO-PRO-1 [25, 26, 29, 33, 38–42]. Even though the application of up to ten 5 ns pulses does not lead to YO-PRO-1 uptake in chromaffin cells [23], a non-selective membrane conductance (Ileak) permeable to Na+ could be elicited by a single 5 ns pulse in chromaffin cells clamped near the resting membrane potential (‒70 mV) in these cells [5], which has also been detected for longer NEPs in GH3 and NG108 cells [7, 8] and also in bovine chromaffin cells [8]. A similar inwardly rectifying conductance was also detected in response to twin NEPs in this study. Interestingly, the time course of onset (ms) and decay (min) of Ileak elicited by a single 5 ns pulse [5], which may be the product of ion flux through lipid nanopores and/or voltage-independent ion channel proteins, correlated well with that of the rapid inhibition (ms to s) and delayed recovery (min) of INa by twin 5 ns pulses observed in this study.

Because many types of ion channels are localized and regulated in highly organized lipid microenvironments such as lipid rafts and caveolae [43-45], another way that twin NEPs could produce a transient suppression of the number of active channels is by influencing endocytosis and membrane protein recycling, resulting in the transient disappearance of active Na+ channels (discussed in the section below). Unequivocal evidence in support of this hypothesis will require further experiments in which the movement of fluorescently tagged voltage-gated Na+ channels expressed in a mammalian cell line is monitored over time before, during and after the application of a single or twin NEPs by total internal reflection fluorescence or super-resolution fluorescence microscopy.

Another viable hypothesis to explain the inhibitory effects of NEPs on INa is that NEPs may directly influence the Na+ channel protein. Recent molecular dynamics simulations by Rems et al. [46] showed that pulsed electric fields could create alternate conductive pores near the voltage-sensitive domains (VSDs) of voltage-gated ion channels including Na+ channels. These alternate pathways were shown to spontaneously evolve into more complex pores stabilized by lipid head groups and were accompanied by unfolding of the VSD from the channel. Unfolded VSDs were unable to regulate the opening/closure of the channel pore leading to a dysfunctional ion channel that could no longer respond to changes in transmembrane potential, which is in agreement with our electrophysiological measurements showing a decrease in maximal Na+ channel conductance. These complex alternate conduction pathways, and possibly the ion channel pore itself (e.g. if the channel was locked into a subconductance state), could also possibly contribute to the induction of the so-called “leak” current, which would be interpreted as current flowing through these alternate ion channel pathways, lipid nanopores or other permeable defects caused by the pulsed electric field. However, such profound defects would have to be reversible to account for the fact that the maximal conductance of Na+ channels fully recovered 9 min after the second NEP at 8 MV/m, especially at short twin pulse intervals.

Paradoxical recovery of INa after twin NEP-mediated inhibition

A very interesting observation of the present study was the paradoxical finding that shortening the interval between twin pulses led to a progressive recovery of INa, which after 9 min exceeded the level reached following a single NEP, and in some cases the level seen in unexposed cells (e.g. for 10 ms interval at 5 and 8 MV/m). Even for twin NEPs at 10 MV/m, there were clear signs of a partial time-dependent recovery over a 9 min period of recording after delivery of the pulse pair, and minimal additive effects relative to a single NEP. As mentioned in the previous section, similar effects were observed whether INa was recorded using physiological salt solutions, or K+- and Ca2+-free solutions containing Cs+ and TEA on both sides of the membrane, indicating that twin NEPs targeted INa and that the recorded effects did not include contamination by voltage-dependent Ca2+ and K+ currents. The progressive attenuation of the inhibition of INa by twin NEPs by shortening the interval between pulses correlates well with the interval-dependent enhanced recovery.

In the last few decades, our understanding of the structure of membrane proteins has expanded considerably with the rapid development of new microscopy techniques with unprecedented spatial and temporal resolution, improvement in crystallization methods for X-ray diffraction imaging, and the development of new computer algorithms to reconstruct images [47, 48]. Despite these developments, our understanding of the properties of transmembrane proteins, and in particular ion channels in their native environment, still remains limited because the vast majority of protein structures that have been uncovered were determined without the stabilizing effects of a surrounding lipid bilayer. Many recent studies have highlighted important roles for the lipid bilayer in dynamically regulating ion channel activity [6, 49–54]. Cholesterol is an integral lipid component that conveys many structural properties to the cell membrane as well as regulating the activity of ion channels in the lipid bilayer. This natural steroid is thought to influence ion channels by several mechanisms that include: 1) a direct interaction with the channel protein, 2) changes in the fluidity of the bilayer, which indirectly affect ion channel gating and conformational changes, and 3) compartmentalization of ion channels into spatially restricted lipid rafts [55-60]. Many classes of voltage-gated ion channels have been reported to be influenced by cholesterol, including voltage-dependent Ca2+ and K+ channels [56]. Also, many ion channels are found in caveolae, a subset of lipid rafts [60-63], which are organized for optimal compartmentalized signal transduction and turnover of membrane proteins. Importantly, cholesterol may also regulate phosphatidylinositol 4,5-bisphosphate (PIP2) levels, which is a key second messenger molecule regulating multiple types of ion channels [64, 65]. Depletion of membrane cholesterol affected the cellular response to 600 nanosecond electric pulses [66]; the same pulses were also reported to initiate hydrolysis or depletion of PIP2 in the plasma membrane [67, 68], which could, at least in part, explain the NEP-induced inhibition of voltage-gated channels [6-8]. Therefore, transient disruption of the phospholipid bilayer by a NEP could be a possible step leading to the subsequent inhibition of voltage-gated Na+ channels and/or the slow recovery of INa following the application of a pulse pair.

In this study, the effect of disrupting the structure of the phospholipid bilayer on INa was first investigated in chromaffin cells unexposed to NEPs. Cell exposure to MβCD, an agent used in our experiments as a cholesterol depleting agent [69], produced a partial inhibition of the inward Na+ current (Fig 9). Whether this effect was the result of a direct acute blocking effect of MβCD on Na+ channels, cholesterol depletion per se and/or lipid raft disruption is unclear as time-dependent effects of the agent on INa were not investigated. However, distinguishing between these alternative possibilities would be difficult to interpret because in one study, a 5 min preincubation was sufficient to deplete membrane cholesterol, disrupt lipid rafts and increase Ca2+-activated Cl- currents in vascular smooth muscle cells [63]. In most studies, a preincubation with this agent for 15 min or more (30 min in our study), is a standard procedure to deplete membrane cholesterol and eliminate caveolae in cells in which these membrane structures are found [19, 56, 69–72]. Interestingly, in the presence of MβCD, a pulse pair with a 10 ms interval still inhibited INa but its recovery was nearly abolished. In fact, the inhibition was accentuated and was similar to the near doubling of the inhibition produced by the pulse pair at longer intervals (e.g. ≥ 1 s). These results suggest that the inhibition and recovery of INa by twin pulses reflect different processes as they were completely dissociated by MβCD. At longer intervals, twin pulses led to cumulative functional disruption of Na+ channels, perhaps by inflicting damage to the channel protein as suggested by the molecular dynamics study by Rems et al. [46]. At shorter intervals, we propose that the rapid timing of delivery of the second pulse stimulated the trafficking of newly synthesized and readily assembled NaV subunits from the Golgi and/or endoplasmic reticulum systems toward the membrane. This is supported by the observation that twin pulses applied at intervals shorter than 1 s led to a recovery of INa that partially masked its inhibition by twin pulses, and exceeded the amplitude of INa in control cells unexposed to NEPs, suggesting the recruitment of additional NaV subunits leading to a larger number of functional Na+ channels in the membrane. Consistent with this hypothesis was the fact that the enhancement of INa by twin NEPs was not associated with a change in the voltage-dependence of activation and inactivation. Clearly, additional experiments will be required to investigate this mechanism in more detail.

Conclusion

The results of this study have shown that INa in excitable adrenal chromaffin cells can be differentially modulated by a single versus a pair of 5 ns pulses, with the differential effect of the pulse pair depending on both the time interval between the two pulses and the E-field amplitude. Of particular significance is that a pulse pair at 5 MV/m having a very short interpulse interval (10 ms) can not only attenuate the inhibitory effect of a single pulse on INa but also lead to a paradoxical progressive recovery of the inward current. The enhanced recovery of INa disappeared at longer interpulse intervals, an effect accentuated at a higher E-field. While the biophysical mechanisms underlying these complex effects on INa are unknown and remain to be explored, our results nevertheless point to the potential of utilizing NEPs to modulate NaV1.7 channel activity and hence cell excitability for therapeutic applications. As an example, the hyperactivity of NaV1.7 channels in neurons that has been associated with producing pain could be transiently reduced by applying twin NEPs [73-75]. It is also possible that other classes of NaV subunits might be similarly or differentially influenced by these pulses, which could affect the excitability of other types of neurons in both the central and peripheral nervous systems. To address this possibility, future experiments will be to investigate the effects of NEPs on INa in mouse adrenal chromaffin cells that is mediated by both NaV1.3 and NaV1.7 channels [76].

Sample traces illustrating the timing of application of a single or pulse pair at E-field of 8 MV/m and recording of the inward Na current evoked by a constant voltage step protocol.

Traces in all panels were from different experiments. The constant voltage step protocol consisted of applying a 100 ms voltage clamp step to +10 mV from a holding potential as shown at the top of panel A. Traces of inward currents were continuously recorded by applying a total of 200 voltage clamp steps, with a 3 s interval between each step. The inset in panel A shows an expanded view of the peak inward current. Representative traces of inward current following exposure of a cell to a single 5 ns pulse (A) or a pulse pair with time interval of 10 ms (B), 100 ms (C), 1 s (D), 5 s (E) or 30 s (F). For panels A thru D, the single pulse or pulse pair (red line) was applied just prior to the 21st voltage clamp step in the sequence to record to current, with an interval of 1.5 s for the single pulse (P) or the first pulse (1st P) of a pulse pair. For the 10 ms (B) 100 ms (C) and 1 s data set (D), the second pulse (2nd P) of a pulse pair interval was applied with an interval of ~ 1.49, ~ 1.4 and ~ 0.5 s, respectively, prior to the application of the voltage clamp step. For panels E and F, because the interval was longer than the membrane recording sweep duration, the membrane current traces from different sweeps were superimposed and displayed by different colors. For the 5 s data set (E), the second pulse (black line) was delivered after the 22nd voltage clamp step with an ~ 2.5 s interval between the second pulse and recording the sodium current recording during the 23rd voltage clamp step (not shown). For the 30 s data set (F), the second pulse interval (black line) was ~ 1.5 s prior to recording the inward current during the 31st voltage clamp step.

(TIF)

Click here for additional data file.

Typical effects on INa of twin NEPs applied at different time intervals and E-fields.

Traces in all panels were from different experiments. INa traces were elicited by voltage clamp steps to +10 mV from a holding potential of -70 mV as described in S1 Fig. Representative traces of INa recorded before, and after the first (1st P) and second pulses (2nd P) at an E-field of 5 MV/m (A), 8 MV/m (C) or 10 MV/m (E), respectively. Representative traces of INa recorded before, immediately after the twin pulses with an interval of 10 ms (After pulses) and 9 min after the application of the twin pulses (9 min after pulses) at an E-field of 5 MV/m (B), 8 MV/m (D) or 10 MV/m (F), respectively.

(TIF)

Click here for additional data file.

20 Nov 2019

PONE-D-19-29052

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses

PLOS ONE

Dear Dr. Leblanc,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-19-29052

This manuscript presents results from experiments to examine the effect of two closely spaced high intensity nanosecond electric pulses on voltage-dependent sodium (NaV) current in bovine chromaffin cells. The twin pulses are varied by intensity and interval and clear differences are observed as these parameters are altered. While the effects on the inward current are interesting, there are no definitive experiments that point to a clear mechanism. In addition, there is one result that appears to suggest that NaV channels are not directly affected by these pulses, which is against one of the main conclusions of this work.

One of the first observations presented is that the NaV current ‘runs down’ with time. The authors speculate that this results from a slow leftward drift in the voltage-dependent gating parameters, which has been observed previously. However, the data presented in their earlier paper (on a single high intensity pulse) argues against such a shift. Figure 2a of that paper shows that peak inward current remains at +10 mV for the entire 15 min recording period. On the other hand, outward currents show a large reduction over the same time period at +50 mV. The slow loss of the ‘N’ shape of the outward current IV suggests that the current actually running down is voltage-dependent calcium (CaV) current, which is well known to express this phenomenon. Since the NaV current has not been isolated in these experiments, it seems that CaV current rundown is a likely explanation for the slow loss of peak inward current. If this is true, one wonders if these experiments are focused on the wrong current.

One of the most difficult aspects of this work to understand is why isolated NaV current was not studied. This was also the case in the previous publication. The authors argue that peak inward current is primarily comprised of NaV current, but at +10 mV it is clear that CaV, KV and Ca-activated K currents also contribute. If the effect of the nanopulses were larger, the author’s argument for a NaV channel effect would be strengthened. However, the reduction varies between 5-14%, which supports the idea that any of the above-mentioned currents could be mediating these effects. The experiments on isolated NaV currents should be done.

While the majority of data shown are time courses of inward current measured at +10 mV, Figure 6 shows both normalized and relative conductance vs. voltage (GV) curves. Figure 6C shows the relative GV curve, which appears to discount the hypothesis that this is an NaV channel effect. The relative conductance is only reduced at voltages > 0 mV by the twin nanopulses. If the nanopulse effect were due to a reduction in the number of active NaV channels (as proposed by the authors), one would observe a 14% reduction over the entire GV curve, not only at voltages > 0 mV. One possible explanation is that KV current is enhanced by the nanopulse. This idea is supported by the voltage-dependent activation of KV current shown in the previous publication (activation > -10 mV). If this were true, a shift in the reversal potential would likely be observed. Unfortunately, IV curves are not shown, so it not possible to see if such a shift exists. Another problem is that none of the discussed mechanisms can explain the preferential reduction of inward current at voltages > 0 mV. The relative GV curve shown in the previous publication shows a similar preferential effect at depolarized voltages.

In the Discussion section, the authors describe the possibility that the nanopulses produce new pathways for ions to permeate the cells as leak currents (not voltage-dependent). However, do data are presented on the holding current. If such ‘leak’ pathways were generated, there should be an increase in holding current if those pathways were Na+ or Ca2+ selective, or if they were non-selective for cations. If such leak pathways were K+ selective, holding current may not be significantly altered at -70 mV (the holding potential). Such an increase in K+ leak could be observed by making brief, strong hyperpolarizing steps (-120 mV perhaps) to look for increased holding current at a voltage with a driving force strong enough to observe K+ flux.

One thing missing from the figures is the actual ionic currents. The currents at +10 mV should be shown with the time courses for the three times used for the GV curves, namely control, peak reduction of inward current and at 9 min. It is also important that a biophysical analysis be done on the currents. At least the rise time of the inward current and information on the outward current(s) needs to be added. Otherwise the reader is not able to independently assess the author’s conclusions.

The authors state that the reduction of current due to the nanopulses is instantaneous, but does not appear to be true. There is a clear time course to the current reduction, which is easily observed for the larger nanopulse effects. Such a time course should be investigated as it is possible that clues to the mechanism could be gleaned from such an analysis.

The methyl-β-cyclodextrin experiment is interesting, but impossible to interpret without more information. The currents need to be shown so that the reader can see if outward currents are also affected. Here the authors also admit that there is a role for KV currents in shaping the inward current, since they hypothesize that a shift in the reversal potential is due to the increased influence of these currents. Even though the reversal potential is measured at a more depolarized voltage (~+40 mV) than +10 mV, this highlights the problems with interpretation of voltage clamp data from currents that are not isolated.

Reviewer #2: This research article addresses the effects of twin nanosecond electric pulses (NEP) on sodium ion (Na+) conductance (INa) in cultured adrenal chromaffin cells. Whole, single bovine cells isolated from a fresh adrenal medulla are voltage clamped and then exposed to NEP. The inhibition and recovery dynamics of INa in cells were compared for exposure to no pulse (control), single, and twin pairs of 5 ns pulses. Variation of electric field amplitude (5 MV/m, 8 MV/m, or 10 MV/m) and time interval between NEP pairs (10 ms, 100 ms, 1 s, 5 s, or 30 s) produced differential inhibition and recovery effects. All NEP exposures inhibited INa before recovery to some extent. Twin NEP exposures increased percent inhibition of maximal conductance over comparable single NEP exposures (except 5 MV/m and 10 ms interval), while twin NEP exposures with lower amplitudes and shorter intervals enhanced recovery. The mechanism underlying twin NEP inhibition and recovery of INa does not depend on a shift in voltage-dependence of steady-state activation and inactivation, suggesting that NEP exposure does not interfere with voltage sensors. Nor does NEP exposure change the reversal potential of inward current, suggesting ion selectivity and perhaps the structure of ion conduction pathways are maintained. Since maximal Na+ conductance was inhibited, the hypothesis that twin NEP exposure reduces the number of active channels is adopted to explain the mechanism of inhibition. As an initial experiment toward addressing this mechanistic hypothesis, lipid rafts are disrupted by universally depleting cholesterol within cells before exposure to NEP. Although there was minimal effect of cholesterol depletion on the response of cells to single NEP, it enhanced inhibition and nearly eliminated recovery of INa following twin NEP exposure. The important conclusion is that tuning of 5 ns electric pulse exposures can be used to modulate sodium ion channel activity in cells. The impact of this conclusion and the overall study make this well-written article appropriate for publication in PLOS ONE.

Minor revisions are recommended in order to address the following comments.

- All data does not appear fully available. Only mean values are presented within the manuscript and figures. Please make full data available either as supporting information or in a public repository.

- No primary experimental evidence linking caveolae to the results is presented, and methyl-b-cyclodextrin universally depletes cholesterol within cells. Cholesterol impacts numerous membrane properties and functions in addition to fusion, trafficking and recycling, such as bilayer fluidity and the second messenger molecule, as mentioned in the Discussion. Therefore, it is recommended to reword line 368 (perhaps “investigate” instead of “determine”), and to remove “capable of disrupting caveolae integrity” in line 506.

- Regarding the influence of cholesterol depletion on cellular responses to 10 ns and 600 ns electric pulses, consider citing this 2016 publication by Cantu et al. (https://doi.org/10.1016/j.bbamem.2016.07.006).

- Capitalization of the title is inconsistent with the others in Reference 47 (lines 710-713).

Reviewer #3: The manuscript by Yang et al., describes the effects of dual high intensity pulses on voltage-gated sodium channel currents in adrenal chromaffin cells. There are interesting findings on the recovery of the sodium channel current in response to dual rather than single NEPs and the effects of depletion of membrane cholesterol. The manuscript is generally well written but requires clarification on the methodology and statistical tests used which could determine whether some conclusions are in agreement with the data presented.

1) Throughout the manuscript, the authors conclude that there are differences in the magnitude of inhibition and / or recovery of voltage-gated Na+ channel currents in chromaffin cells exposed to dual NEPs in comparison to those exposed to a single NEP. However, with the exception of the data presented in figure 6, there is no mention of which statistical tests were used to conclude that the responses were significantly different. Could the authors provide the results from the statistical tests in the manuscript text and figures?

2) The authors concluded that there was an additive effect on inhibition only when the interval between the NEPs exceeded 100ms. With regards to this, could the authors clarify a couple of points in the methodology? In the methods, the authors state that a single NEP or pair of NEPs was applied between the 20th and 21st voltage steps with an interval of 0.5s between the time the NEP was delivered and recording of currents.

a) Did the voltage step depolarizations to +10mV every 3s continue during the interval between the two NEPs? In figures 1B, 3B and 4B (all 30s NEP intervals) there look to be data points that indicate the step depolarizations were continued in between exposure to the two NEPs.

b) Does the 0.5s interval between the time the NEP was delivered refer to the first NEP? If there is an interval of 0.5s between the first NEP of the pair and the subsequent step depolarization to record INa, is the second NEP exposure after the 21st voltage step for any cells in which the interval between two NEPs is 1s or greater? In addition, is the time between exposure to the 2nd NEP and the next step depolarization to record INa equal or different for each dual NEP exposure interval? For example, in the 10ms data is the 2nd NEP-voltage step interval ~ 490ms and in the 1s data the same interval ~ 2.5s?

It would aid the reader if the authors could depict this protocol, indicating after which voltage step the second NEP exposure occurred and the time interval between NEP exposure and the next step depolarization.

c) If the 2nd NEP-voltage step time interval is unequal, does varying the interval between a single NEP, of different electric field strengths, and a voltage step to record INa affect the voltage-gated sodium channel inhibition observed?

Minor comments:

1) In figures 1-4 is the same control or 1 pulse data set used in all panels? Could the authors state in the figure legends if this is the case.

2) Legend for Figure 1 – I think page 10 line 209 should read voltage step to +10mV rather than -10mV.

3) It would be useful to the reader to see some representative sodium current traces.

4) Could the authors provide more detail in the methods on the double pulse protocol used to determine steady state inactivation including conditioning pulse duration, test pulse duration and voltage step, interval between pulses. It would also be useful to the reader to provide a schematic of this protocol and the steady state activation protocol alongside the relevant data sets.

5) PLOS does not permit references to data shown. In the instance where this refers to the reversal potential, it may be helpful for the readers to see the IV curves that were used to determine steady state activation and conductance rather than remove the reference to this data.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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2 Apr 2020

All comments were addressed in the point by point response to all three reviewers.

Submitted filename: Yang et al 2020 R1 Response to Reviewers_Final.docx

Click here for additional data file.

7 May 2020

PONE-D-19-29052R1

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses

PLOS ONE

Dear Dr. Leblanc,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewers 2 and 3 were unable to provide additional comments in the timeframe provided but in looking through your responses I found them to satisfy those previous critiques.  Reviewer 1 notes that you have done an excellent job revising the manuscript and has a few remaining comments that would be helpful to address.  These are mostly minor issues involving clarification and perhaps rewording of the discussion. The reviewer does suggest an experiment to determine if acute methyl beta cyclodextrin alters the sodium channel current, suggesting this is important when implicating lipid rafts in the altered response to the nanosecond pulses.  This would be useful data to provide.  However, if you are unable to do so in a reasonable time frame due to current work restrictions then please consider alternative approaches to addressing this concern.  For example, is there evidence in the literature, or can the conclusion be tempered / reworded to reflect the possibility that acute effects of the drug on the channel could alter interpretation.

We would appreciate receiving your revised manuscript by Jun 21 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PONE-D-19_29052_R1

The authors have done an excellent job responding to the previous criticisms. The new experiments now make it clear that NaV channels are affected by the high intensity electric pulses (NEP). The other major issues have also been resolved.

The double NEPs present several different effects over a single NEP. First, the size of the NaV current reduction is generally larger, as one would expect. Second, there is a recovery of NaV current amplitude, but only if the two pulses are ≤ 100 ms apart. Interestingly, this recovery is actually an over-recovery, since the current amplitude is even larger than the current would be under control conditions, at least for twin NEPs 10 ms apart and < 10 MV/m. Indeed, the lack of full recovery with 10 MV/m NEPs at 10 ms interval suggests a different mechanism involved to prevent recovery. The data are very interesting. The problem is that the Discussion does clearly present these ideas. The speculated mechanisms involved in the initial reduction of current are presented, but the ‘paradoxical’ recovery is not. It would be helpful to the reader if the authors would re-organize the Discussion to talk about the NaV current inhibition by NEPs, including the possible mechanisms, and then the paradoxical recovery, again including possible mechanisms. Further, the enhancement of the current beyond that in control (at 9 min) needs a mechanistic discussion. The recovery is discussed, but that discussion comes before the presentation of the mechanisms for the NaV current inhibition, and therefore there is an absence of mechanisms for the recovery.

One experiment that needs to be added is the effect of acutely applied MβCD on the NaV current. The authors show the effect of a 30 min MβCD pretreatment and conclude that the associated effects are consistent with lipid raft involvement, but this conclusion is only valid if the acute application shows no or significantly smaller effects. It is generally agreed that the 30 min incubation period is required for the disruption of lipid rafts by MβCD.

Other comments

1. Figure 1. Why is there apparently no CaV current when NMDG was used to replace Na+? Is there a small inward current in TTX that could be attributed to CaV channels? Clarification would be helpful.

2. Figure 6 legend. For the panels showing recovery, the measurement was taken at 9 min after the NEPs, but that time is not mentioned in the figure legend. It is mentioned in the text but should be added to the legend. In this figure, there is no mention of significant differences. It is not clear if the any of the presented valued in each of the graphs are different from one another.

3. Line 367. It is stated that K-gluconate was replaced with aspartate, which is not true. The replacement compound was actually Cs-aspartate, at least that is what I could glean from the Methods section. This needs to be corrected.

4. The description of the experiments measuring the leak current need to be clarified. Were these experiments done in the same cells used for the NaV current measurements? It is also not clear why figure 8 presents discrete points for the leak measurement instead of a continuous line, as would be recorded from a ramp. If these points were measured from the ramp, it should be clear how the measurement was done. For example, were a range of values averaged for each point? If so, how wide was the voltage range covered?

5. The conclusion seems to focus solely on the single NEP, since it describes only the NaV inhibition. There needs to be discussion here of the recovery as well. In addition, there needs to be discussion of the potential selectivity of the NEPs, since the focus here is on pain management. It seems very likely that other NaV channels will also be affected, which would impact other sensory axons.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

15 May 2020

Those are specifically addressed in two separate documents (Cover Letter and Response to Reviewer #1).

Submitted filename: Yang et al 2020 R2 Response to Reviewer #1 Final.docx

Click here for additional data file.

20 May 2020

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses

PONE-D-19-29052R2

Dear Dr. Leblanc,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

29 May 2020

PONE-D-19-29052R2

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses

Dear Dr. Leblanc:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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PLOS ONE