Response to “Sodium current inhibition by nanosecond pulsed electric field (nsPEF)—fact or artifact?” by Verkerk et al.

It was nice to learn that our studies of nanosecond pulsed electric field (nsPEF) effects on membrane currents [Nesin et al., 2012; Nesin and Pakhomov, 2012] gained the attention of scientists outside the immediate field of bioelectromagnetics. The insight and constructive comments from scientists representing diverse areas are most welcome and help to identify the next research steps. Also, answering to critical comments gives the authors extra opportunity to convey more details about already published experimental data. The comments by Verkerk et al. [2012] start with a basic introduction to the patch clamp method. They reiterate a well-known fact that the command voltage (Vc) is distributed between the series resistance of the pipette (Rs) and the cell membrane resistance (Rm), so that the clamped membrane potential (Vm) is actually less than Vc. This difference can be negligible for Rs Rm, but may cause measurement errors when Rs is too high and/or Rm is too low. These considerations are thoroughly known by patch clamp practitioners and are emphasized in every relevant textbook (e.g., Molleman [2002]); hence, the reiteration appears somewhat redundant for a journal article. Next, Verkerk et al. point to the effect of increasing the leak current (Ileak) by nsPEF. The increased Ileak reflects lower Rm and increased deviation of Vm from Vc. For a Vc of 80 mV (which we used as a holding potential), the development of Ileak 1⁄4 2,500 pA translates into Vm depolarization from 80 to 70 mV, and holding the cell at a more depolarized Vm increases the inactivation of INa. Thus, Verkerk et al. hypothesize that the inhibition of INa by nsPEF was caused by an error in setting the holding membrane potential because of the huge Ileak. This concern could be legitimate if Verkerk et al. used the right numbers. Regretfully, they did not. Instead, they arbitrarily chose a very large Ileak value of 2,500 pA, which has little relevance to the reported experiments. Why? There is no explanation in their paper. Apparently, using this heavily exaggerated value was the only way to support the ‘‘artifact hypothesis.’’ If we use the actual and typical experimental values of Ileak and estimate the artifacts using Figure 1 in Verker et al. paper, it becomes evident that the potential artifacts were too small even to be detected; or, in other cases, they were much smaller than the observed nsPEF effects. Let us take a look at the actual Ileak values measured for Vc of 80 mV. In Figure 2B [Nesin et al., 2012], Ileak is only 50 pA (1.8 kV/cm nsPEF) or 200 pA (3 kV/cm). In Figure 2C, Ileak is also about 50 pA. Using Figure 1 in the article by Verkerk et al., the respective error in the holding voltage was just 1 mV (noise!) and the inhibition of INa was 1– 2% (also just noise). In actuality, INa was inhibited by as much as 30–60%. Therefore, the ‘‘artifact hypothesis’’ by Verkerk et al. is irrelevant and fails to explain these experimental data. Most of the other figures (Figs. 4–6 from Nesin et al. [2012], and Figs. 1, 3, and 5 from Nesin and Pakhomov [2012]) show data from cells that were ‘‘patched’’ prior to nsPEF exposure. Therefore, Ileak