Response to "Sodium current inhibition by nanosecond pulsed electric field (nsPEF)--fact or artifact?" by Verkerk et al

Article (PDF Available)inBioelectromagnetics 34(2) · February 2013with5 Reads
DOI: 10.1002/bem.21756 · Source: PubMed
Bioele c tromagnetics 34:165 ^16 6 (2013)
R esponse
Response to ‘ ‘ Sodium Current Inhibition by
Nanosecond Pulsed Electric Field (nsPEF)Fact
or Artifact?’byVerkerket al.
Andrei G . Pakho mo v*
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk,Virginia
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. [2012] start
with a basic introduction to the patch clamp method.
They reiterate a well-known fact that the command
voltage (V
c
) is distributed between the series resis-
tance of the pipette (R
s
) and the cell membrane resis-
tance (R
m
), so that the clamped membrane potential
(V
m
) is actually less than V
c
. This difference can be
negligible for R
s
R
m
, but may cause measurement
errors when R
s
is too high and/or R
m
is too low.
These considerations are thoroughly known by patch
clamp practitioners and are emphasized in every rele-
vant 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 (I
leak
) by nsPEF. The
increased I
leak
reflects lower R
m
and increased devia-
tion of V
m
from V
c
.ForaV
c
of 80 mV (which we
used as a holding potential), the development of
I
leak
¼ 2,500 pA translates into V
m
depolarization
from 80 to 70 mV, and holding the cell at a more
depolarized V
m
increases the inactivation of I
Na
.
Thus, Verkerk et al. hypothesize that the inhibition
of I
Na
by nsPEF was caused by an error in setting
the holding membrane potential because of the huge
I
leak
.
This concern could be legitimate if Verkerk
et al. used the right numbers. Regretfully, they did
not. Instead, they arbitrarily chose a very large I
leak
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 I
leak
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 I
leak
values mea-
sured for V
c
of 80 mV. In Figure 2B [Nesin et al.,
2012], I
leak
is only 50 pA (1.8 kV/cm nsPEF) or
200 pA (3 kV/cm). In Figure 2C, I
leak
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 I
Na
was 1–
2% (also just noise). In actuality, I
Na
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, I
leak
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: andrei@pakhomov.net,
apakhomo@odu.edu
Received for review 31 July 2012; Accepted 20 August 2012
DOI 10.1002/bem.21756
Published online 18 September 2012 in Wiley Online Library
(wileyonlinelibrary.com).
ß 2012 Wiley Periodicals,Inc.
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 I
Na
(two- to sixfold) was observed concurrently
with I
leak
values between 400 and 1,400 pA (note
that I
leak
values in Fig. 6 are shown for 90 mV and
are 15–25% greater than at 80 mV). A typical I
leak
value in cells that show a two- to sixfold inhibition
of I
Na
can be conservatively estimated to be about
1,000 pA for a 80 mV V
c
.
For 1,000 pA I
leak
, Figure 1 in the Verkerk
et al. article predicts the reduction of V
m
from
80 mV to 76 mV and a 10% decrease in I
Na
.
While these values are slightly above the ‘noise’
level, they are far below the actual effect of I
Na
inhi-
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 I
Na
minutes after
I
leak
had recovered. Somehow, Verkerk et al. again
ignore the fact that the I
leak
in these experiments was
only 50 pA to start with. They further speculate that
‘the voltage dependency of I
Na
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 I
Na
inactivation’ 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 I
Na
may decrease with I
leak
as low as
50 pA, or may increase despite I
leak
as high as
1,500 pA. Poor correlation between I
leak
and the
inhibition of I
Na
does 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 V
m
by V
c
.
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 I
Na
, I
Ca
, and of certain but
not other types of I
K
(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
collaboration.
REFER ENCES
Molleman A. 2002. Patch clamping: An introductory guide to
patch clamp electrophysiology. Padstow, Cornwall, Great
Britain: John Wiley & Sons.
Nesin V, Pakhomov AG. 2012. Inhibition of voltage-gated Na
þ
current by nanosecond pulsed electric field (nsPEF) is not
mediated by Na
þ
influx or Ca
2þ
signaling. Bioelectromag-
netics 33:443–451.
Nesin V, Bowman AM, Xiao S, Pakhomov AG. 2012. Cell
permeabilization and inhibition of voltage-gated Ca
2þ
and
Na
þ
channel currents by nanosecond pulsed electric field.
Bioelectromagnetics 33:394–404.
Pakhomov AG, Pakhomova ON. 2010. Nanopores: A distinct
transmembrane passageway in electroporated cells. In:
Pakhomov AG, Miklavcic D, Markov MS, editors. Ad-
vanced electroporation techniques in biology in medicine.
Boca Raton: CRC Press. pp 178–194.
Verkerk AO, van Ginneken ACG, Ronald Wilders R. 2012.
Sodium current inhibition by nanosecond pulsed electric
field (nsPEF)—Fact or artifact? Bioelectromagnetics (this
issue).
166 Pakhomov
Bioelectromagnetics
  • [Show abstract] [Hide abstract] ABSTRACT: Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca(2+) and Na(+) channels (I(Ca) and I(Na)) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I(Ca) and I(Na). Concurrently, nsPEF increased a non-inactivating "leak" current (I(leak)), presumably due to the formation of nanoelectropores or larger pores in the plasma membrane. The nsPEF effects were similar in cells that were exposed intact and subsequently brought into the whole-cell recording configuration, and in cells that were first brought into the whole-cell configuration and then exposed. Although both I(leak) and the inhibition of VG currents were enhanced at higher E-field levels, these two nsPEF effects showed relatively weak correlation with each other. In some cells, I(leak) increased 10-fold or more while VG currents remained unchanged. At longer time intervals after exposure (5-15 min), I(Ca) and I(Na) could remain inhibited although I(leak) had largely recovered. The causal relation of nsPEF inhibitory effects on VG currents and permeabilization of the plasma membrane is discussed.
    Article · Jul 2012
  • [Show abstract] [Hide abstract] ABSTRACT: In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na(+) current (I(Na)) resulted from (i) reduction of the transmembrane Na(+) gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca(2+)-dependent mechanism. We found that a single 300 ns electric pulse at 1.6-5.3 kV/cm triggered sustained Na(+) influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a Sodium Green Dye. In the whole-cell patch clamp configuration, this influx was efficiently buffered by the pipette solution so that the increase in the intracellular concentration of Na(+) ([Na](i)) did not exceed 2-3 mM. [Na](i) increased uniformly over the cell volume and showed no additional peaks immediately below the plasma membrane. Concurrently, nsPEF reduced VG I(Na) by 30-60% (at 4 and 5.3 kV/cm). In control experiments, even a greater increase of the pipette [Na(+)] (by 5 mM) did not attenuate VG I(Na), thereby indicating that the nsPEF-induced Na(+) influx was not the cause of VG I(Na) inhibition. Similarly, adding 20 mM of a fast Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the pipette solution did not prevent or attenuate the inhibition of the VG I(Na) by nsPEF. These findings point to possible Ca(2+)-independent downregulation of the VG Na(+) channels (e.g., caused by alteration of the lipid bilayer) or the direct effect of nsPEF on the channel.
    Article · Sep 2012
  • [Show abstract] [Hide abstract] ABSTRACT: In two recent publications in Bioelectromagnetics it has been demonstrated that the voltage-gated sodium current (I(Na) ) is inhibited in response to a nanosecond pulsed electric field (nsPEF). At the same time, there was an increase in a non-inactivating "leak" current (I(leak) ), which was attributed to the formation of nanoelectropores or larger pores in the plasma membrane. We demonstrate that the increase in I(leak) , in combination with a residual series resistance, leads to an error in the holding potential in the patch clamp experiments and an unanticipated inactivation of the sodium channels. We conclude that the observed inhibition of I(Na) may be largely, if not fully, artifactual. Bioelectromagnetics. © 2012 Wiley Periodicals, Inc.
    Full-text · Article · Feb 2013
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