Fig 1.
NEP exposure of a chromaffin cell.
(A) Representative waveform of a 5 ns, 8 MV/m pulse. The inset shows the simulated E-field distribution between the electrodes and at the level of the patch clamped cell on a color-coded scale as viewed from the side (top) and from above (bottom) the electrodes. (B) Photomicrograph of a patched chromaffin cell located between the electrode tips that are positioned 40 μm above the bottom of the dish.
Fig 2.
I-V relationships for early peak inward and late outward currents.
(A) The voltage step protocol consisted of 50 ms steps ranging from –70 to +80 mV, applied in 10 mV increments every 2 s. (B) Corresponding traces of the early inward and late outward current elicited by the voltage step protocol. (C) I-V relationship for the peak inward current (arrow in (B)) obtained for each of 6 successive voltage step protocols applied to the same cell. The interval between each protocol was 3 min for a total elapsed time of 21 min. (D) Corresponding I-V relationship for the outward current of the same cell shown in (C). Current represents the mean value of the late outward current for the interval between the arrows in (B). The results are representative of 5 cells.
Fig 3.
Effect of eliminating Na+ and Ca2+ on the I-V relationships for inward and outward currents.
The voltage step protocol was the same as described in Fig 2 for obtaining inward (A) and outward (B) currents. The I-V relationship was first obtained in the presence of normal BSS, then after the external bath solution was changed to BSS in which extracellular Na+ was absent (NMDG+) and finally to BSS in which both extracellular Na+ and Ca2+ were absent (NMDG+-Ca2+free). The data represent the mean value of 4 cells ± SE.
Fig 4.
Inward and outward currents evoked by a constant-voltage step protocol.
(A) The constant-voltage step protocol consisted of applying a 100 ms voltage step to +10 mV from a holding potential. (B) Control traces of inward and outward current obtained by applying a total of 200 voltage step protocols, with a 3 s interval between each protocol. The inset shows an expanded view of the peak inward current. (C) Representative traces of inward and outward current following exposure of a cell to a single 5 ns pulse applied at an E-field of 5 MV/m, and in (D), at an E-field of 10 MV/m. In both (C) and (D), the pulse was applied between the 20th and 21st voltage step protocols, with an interval of 0.5 s between the time the NEP was delivered and recording of currents. The arrow indicates Ileak, which was recorded with a delay of 8 ms after the pulse [22].
Fig 5.
Effect of a single 5 ns pulse at different E-field amplitudes on peak inward current.
(A) Time course of the changes in peak inward current for an unexposed cell (control) compared to a cell exposed to a 5 ns, 5 MV/m pulse, obtained by using the constant-voltage step protocol described in Fig 4. Normalized current represents the magnitude of peak inward current normalized to the mean of the peak inward current recorded for the 8 voltage step protocols that immediately preceded the 21st voltage step protocol. Data are expressed as the mean ± SE (control, n = 11; 5 MV/m, n = 9). (B) Time course of the changes in peak inward current for a cell exposed to a 5 ns pulse at E-fields of 8 MV/m and 10 MV/m. Data are expressed as the mean ± SE (8 MV/m, n = 10; 10 MV/m, n = 11).
Fig 6.
Effect of a single 5 ns pulse on the voltage-dependence of steady-state inactivation and activation of INa.
(A) Steady-state inactivation of INa was determined by holding cells at –70 mV and applying a series of 1 s conditioning potentials ranging from –100 mV to –20 mV in 10 mV increments, with each voltage step followed by a constant 100 ms test pulse to +10 mV to record INa (inset). A 5 MV/m pulse was delivered and after 1 s the voltage step protocol repeated. INa in each case was normalized to their respective maximal values and plotted as a function of the conditioning potential. The data were fitted to a Boltzmann function (see Methods) and expressed as the mean ± SE (n = 4). (B) Steady-state activation of Na+ at an E-field of 5 MV/m. Steady-state activation of INa was determined by holding cells at –70 mV and applying 50 ms steps ranging from –70 mV to +40 mV in 10 mV increments every 2 s (inset). A 5 MV/m pulse was delivered and after 1 s the voltage step protocol repeated. Peak Na+ current in each case was converted to conductance (see Methods), normalized to their respective maximal values and fitted to a Boltzmann function. Data are expressed as the mean ± SE (n = 6). (C) Same as in (A) for a pulse applied at an E-field of 8 MV/m (n = 6). (D) Same as in (B) for a pulse applied at an E-field of 8 MV/m (n = 5).
Table 1.
Decrease in INa caused by Ileak versus the NEP.
Fig 7.
Effect of a single 5 ns pulse at different E-field amplitudes on maximal Na+ conductance.
The protocol (inset) was the same as that for generating steady-state activation of INa. Peak INa was converted to conductance as already described and relative conductance obtained by normalizing the conductance both before and after the pulse to the maximum conductance before the pulse. The data were fitted to a Boltzmann function (see Methods) and expressed as the mean ± SE. (A) Pulse applied at an E-field of 5 MV/m (n = 6) and (B) at 8 MV/m (n = 5).
Fig 8.
Effect of single 5 ns pulse at different E-field amplitudes on late outward current.
(A) Time course of the changes in mean outward current for an unexposed cell (control) compared to a cell exposed to a 5 ns, 5 MV/m pulse, obtained by using the constant-voltage step protocol described in Fig 4A in which the voltage was stepped from –70 mV to +10 mV for 100 ms. Normalized current represents the mean magnitude of the late outward current (see Fig 4B) normalized to the mean of the late outward current recorded for the 8 voltage step protocols that immediately preceded the 21st voltage step protocol. Data are expressed as the mean ± SE (Control, n = 11; 5 MV/m, n = 9). (B) Time course of the changes in mean outward current for a cell exposed to a 5 ns pulse at E-fields of 8 MV/m and 10 MV/m (8 MV/m, n = 3/10; 10 MV/m, n = 6/11). In both (A) and (B), the results were obtained from the same cells as those shown in Fig 5 for peak inward current.
Fig 9.
Effect of a single 5 ns pulse at different E-field amplitudes on late outward current.
Time course of the changes in mean outward current for an unexposed cell (control) compared to a cell exposed to a 5 ns pulse applied at E-fields of 5, 8 and 10 MV/m. The constant-voltage step protocol consisted of stepping the voltage from –70 mV to +80 mV for 100 ms (inset). Normalized current represents the same as that described in Fig 8. Results are expressed as the mean ± SE (Control, n = 4; 5 MV/m, n = 5; 8 MV/m, n = 5; 10 MV/m, n = 5).