It was nice to learn that our studies of nanosec-
ond pulsed electric field (nsPEF) effects on mem-
brane 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 scien-
tists representing diverse areas are most welcome
and help to identify the next research steps. Also,
answering to critical comments gives the authors ex-
tra opportunity to convey more details about already
published experimental data.
The comments by Verkerk et al.  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 resis-
tance of the pipette (Rs) and the cell membrane resis-
tance (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 rele-
vant textbook (e.g., Molleman ); hence, the
reiteration appears somewhat redundant for a journal
Next, Verkerk et al. point to the effect of
increasing the leak current (Ileak) by nsPEF. The
increased Ileakreflects lower Rmand increased devia-
tion of Vmfrom Vc. For a Vcof ?80 mV (which we
used as a holding potential), the development of
Ileak¼ 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 INaby nsPEF was caused by an error in setting
the holding membrane potential because of the huge
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 exagger-
ated value was the only way to support the ‘‘artifact
hypothesis.’’ If we use the actual and typical experi-
mental values of Ileakand 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 Ileakvalues mea-
sured for Vcof ?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, Ileakis 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 INawas 1–
2% (also just noise). In actuality, INawas 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. , and Figs. 1, 3, and 5 from Nesin and
Pakhomov ) show data from cells that were
‘‘patched’’ prior to nsPEF exposure. Therefore, Ileak
Grant sponsors: National Cancer Institute (R01CA125482);
National Institute of General Medical Sciences (R01GM088303);
Air Force Office of Scientific Research (LRIR 09RH09COR).
*Correspondence to: Andrei G. Pakhomov, 4211 Monarch Way,
Suite 300 Norfolk, VA 23508. E-mail: firstname.lastname@example.org,
Received for review 31 July 2012; Accepted 20 August 2012
Published online 18 September 2012 in Wiley Online Library
was measured much sooner after nsPEF (in 10–20 s),
and its values typically were higher. In most of these
experiments, and in most cells, a profound reduction
in INa (two- to sixfold) was observed concurrently
with Ileak values between 400 and 1,400 pA (note
that Ileakvalues in Fig. 6 are shown for ?90 mV and
are 15–25% greater than at ?80 mV). A typical Ileak
value in cells that show a two- to sixfold inhibition
of INacan be conservatively estimated to be about
1,000 pA for a ?80 mV Vc.
For 1,000 pA Ileak, Figure 1 in the Verkerk
et al. article predicts the reduction of Vm from
?80 mV to ?76 mV and a 10% decrease in INa.
While these values are slightly above the ‘‘noise’’
level, they are far below the actual effect of INainhi-
bition by nsPEF. Therefore, the ‘‘artifact hypothesis’’
by Verkerk et al. again fails to explain the experi-
mental data, although may account for a minor
portion of the nsPEF effect.
Closer to the end of their comments, Verkerk
et al. specifically discuss Figure 2C [Nesin et al.,
2012], which shows a still inhibited INaminutes after
Ileakhad recovered. Somehow, Verkerk et al. again
ignore the fact that the Ileakin these experiments was
only 50 pA to start with. They further speculate that
‘‘the voltage dependency of INa inactivation shifts
toward more negative potentials in time after cells
are patch clamped.’’ However, there is no ‘‘in time’’
factor here. Apparently, Verkerk et al. ignored the
notion (second paragraph on the same page) that
‘‘the whole-cell configuration was formed 30–60 s
prior to the scheduled data collection at 5, 10, or
15 min after exposure.’’ In other words, the cells
were left ‘‘untouched’’ until immediately before the
measurements, so any discussions about the shift of
‘‘voltage dependency of INainactivation’’ due to the
prolonged holding of cells under patch clamp condi-
tions are not applicable.
Notably, Verkerk et al. omitted any discussion
of important Figure 7 [Nesin et al., 2012], which
shows that INa may decrease with Ileak as low as
50 pA, or may increase despite Ileak as high as
1,500 pA. Poor correlation between Ileak and the
inhibition of INadoes not fit with the hypothesis of
Verkerk et al.
It might also be useful for Verkerk et al. to take
a look at Figure 9.5 in one of the referenced papers
[Pakhomov and Pakhomova, 2010]. Using a potentio-
metric fluorescent dye concurrently with whole-cell
patch clamp, we demonstrated that, within the stud-
ied limits, nsPEF exposure did not alter the accuracy
of controlling Vmby Vc.
The data and arguments provided above are
more than adequate to rule out the artifact hypothesis
proposed by Verkerk et al. This hypothesis fails to
explain the experimental data on all accounts, and
that is why we did not discuss this type of artifacts in
the original experimental papers. The nsPEF-induced
inhibition of voltage-gated INa, ICa, and of certain but
not other types of IK(unpublished) is an intriguing
and complex phenomenon that awaits detailed analy-
sis. If Verkerk and coworkers are genuinely interest-
ed in this topic, they are most welcome to join the
effort. We are open for ideas and proposals for
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