Báo cáo khoa học: Voltage-gated sodium channel isoform-specific effects of pompilidotoxins doc

Voltage-gated sodium channel isoform-specific effects of
pompilidotoxins
Emanuele Schiavon
1,
*, Marijke Stevens
2,
*, Andre
´
J. Zaharenko
3
, Katsuhiro Konno
4
, Jan Tytgat
2
and Enzo Wanke
1
1 Dipartimento di Biotecnologie e Bioscienze, Universita
`
di Milano-Bicocca, Milan, Italy
2 Laboratory of Toxicology, University of Leuven, Belgium
3 Center of Biotechnology, Instituto de Pesquisas Energe
´
ticas e Nucleares & Depto. de Fisiologia, Instituto de Biocie
ˆ
ncias, Universidade de
Sa˜o Paulo, Brazil
4 Institute of Natural Medicine, University of Toyama, Japan
Introduction
Voltage-gated sodium channels (VGSCs) play a major
role in the generation and propagation of action poten-
tials in all excitable tissues. They are composed of a

pore-forming a-subunit associated with up to four
known different b-subunits. To date, nine mammalian
Na
v
channel isoforms (Na
v
1.1–Na
v
1.9) have been func-
tionally characterized. They are differentially distributed
in the central and peripheral nervous system, in skeletal
Keywords
channel isoforms; ion channels; sodium
channel inactivation; toxin binding; wasp
toxins
Correspondence
E. Wanke, Dipartimento di Biotecnologie e
Bioscienze Universita
`
di Milano-Bicocca,
Piazza della Scienza 2, 20126 Milano, Italy
Fax: +39 02 64483565
Tel: +39 02 64483303
E-mail:
*These authors contributed equally to this
work
(Received 13 October 2009, revised 13
November 2009, accepted 4 December
2009)
doi:10.1111/j.1742-4658.2009.07533.x

Pompilidotoxins (PMTXs, a and b) are small peptides consisting of 13
amino acids purified from the venom of the solitary wasps Anoplius samari-
ensis (a-PMTX) and Batozonellus maculifrons (b-PMTX). They are known
to facilitate synaptic transmission in the lobster neuromuscular junction,
and to slow sodium channel inactivation. By using b-PMTX, a-PMTX and
four synthetic analogs with amino acid changes, we conducted a thorough
study of the effects of PMTXs on sodium current inactivation in seven
mammalian voltage-gated sodium channel (VGSC) isoforms and one insect
VGSC (DmNa
v
1). By evaluating three components of which the inactivat-
ing current is composed (fast, slow and steady-state components), we could
distinguish three distinct groups of PMTX effects. The first group concerned
the insect and Na
v
1.6 channels, which showed a large increase in the steady-
state current component without any increase in the slow component.
Moreover, the dose-dependent increase in this steady-state component was
correlated with the dose-dependent decrease in the fast component. A sec-
ond group of effects concerned the Na
v
1.1, Na
v
1.2, Na
v
1.3 and Na
v
1.7 iso-
forms, which responded with a large increase in the slow component, and
showed only a small steady-state component. As with the first group of

effects, the slow component was dose-dependent and correlated with the
decrease in the fast component. Finally, a third group of effects concerned
Na
v
1.4 and Na
v
1.5, which did not show any change in the slow or steady-
state component. These data shed light on the complex and intriguing
behavior of VGSCs in response to PMTXs, helping us to better understand
the molecular determinants explaining isoform-specific effects.
Abbreviations
AFT-II, Anthopleura fuscoviridis toxin; ATX-II, Anemonia sulcata toxin; Bc-III, Bunodosoma caissarum toxin; Cn2, Centruroides noxius toxin;
PMTX, pompilidotoxin; a-PMTX, a-pompilidotoxin; b -PMTX, b-pompilidotoxin; TFA, trifluoroacetic acid; TTX, tetrodotoxin; VGSC, voltage-
gated sodium channel.
918 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS
muscle, and in cardiac muscle [1]. In insects, only three
orthologous sodium channel isoforms have been suc-
cessfully expressed in Xenopus oocytes so far, namely
DmNa
v
1, BgNa
v
1, and MdNa
v
1. DmNa
v
1 (isolated
from Drosophila melanogaster) was isolated first. This
channel is encoded by the para gene, and is coexpressed
with TipE for fully functional expression [2,3].

VGSCs are associated with a broad range of chan-
nelopathies and channel-related diseases. Channelopa-
thies result from mutations in channel genes and cause
a channel dysfunction. They are, for instance, mani-
fested as cardiac [4], neuronal [5] and pain disorders
[6], as well as various forms of epilepsy and febrile sei-
zures [7]. Not only can mutations in genes cause a
channel dysfunction, but aberrant expression of VGSC
is also a contributory factor in a growing series of
channel-related diseases. For example, upregulation of
Na
v
1.6 was recently shown to play an important role
in multiple sclerosis [8]. Furthermore, elevated levels
of VGSCs are observed in Alzheimer patients [9] and
various types of cancer [10].
To date, little is understood about the components
of the venom of solitary wasps. However, solitary
wasps such as the spider wasps (Pompilidae) are
known to paralyze their prey instead of killing it, as
they make a nest for their larvae so that they can feed
on living prey. Hence, their venom promises to contain
interesting neurotoxins acting on VGSCs. In the search
for such new neurotoxins, Konno et al. [11,12] identi-
fied two new compounds, named a-pompilidotoxin
(a-PMTX) and b-pompilidotoxin (b-PMTX), from the
spider wasps Anoplius samariensis and Batozonel-
lus maculifrons, respectively. Although they originate
from two different species of the Pompilidae family,
they differ in only one amino acid at position 12.

During further characterization, this single amino acid
difference (Arg12 for b-PMTX versus Lys12 for
a-PMTX) appeared to be responsible for a difference
in potency, as b-PMTX appeared to be five times as
potent as a-PMTX in lobster neuromuscular junctions
[12,13]. In structure–activity studies, the location of
three other basic residues at positions 1, 3 and 12 was
found to be crucial for toxin action [13], but none
of the synthesized analogs was more potent than
b-PMTX. Simultaneously, Sahara et al. [14] reported
that a-PMTX slows down the inactivation in tetrodo-
toxin (TTX)-sensitive VGSCs of rat trigeminal neu-
rons. In chimeric channel studies with Na
v
1.2 and
Na
v
1.5 from rats, b-PMTX selectively targeted the
Na
v
1.2 channel, and its binding site was localized in
the S3–S4 extracellular loop of domain 4 at Glu1616
[15]. b-PMTX effects were compared with those of
Anemonia sulcata toxin (ATX-II), which is also known
to slow inactivation. Differential actions were
observed, although their tertiary structures showed
similarities [16,17]. More recently, Grieco and Raman
[18] described the ability of b-PMTX to increase or
induce resurgent currents in Purkinje cells expressing
or lacking Na

v
1.6, respectively.
By using a series of six pompilidotoxin (PMTX) ana-
logs (Table 1) [16], we studied the effects of PMTXs on
sodium current inactivation in seven mammalian VGSC
isoforms and one insect VGSC (DmNa
v
1) (Table 2). By
evaluating three components of which the recorded cur-
rent is composed (fast, slow and steady-state compo-
nents), we could distinguish three distinct groups of
PMTX effects. Additionally, this work is unique in
including extensive characterization of b-PMTX and
analogs over the tested VGSC isoforms.
Results
Screening of peptides for robustness of the
slowing of the inactivation process both in
mammalian isoforms and in one insect isoform
As only a limited amount of each compound was
available, we initially decided to use a simple and fast
method to detect the robustness of the effects. To
investigate whether a peptide is able to bind to the ion
Table 1. Primary sequence alignment of b-PMTX and its analogs,
tested in this study. Normal type and bold type indicate identical
and homologous amino acids, respectively.
b-PMTX RIKIGLFDQLSRL-NH
2
3Rb-PMTX RIRIGLFDQLSRL-NH
2
1K3Rb-PMTX KIRIGLFDQLSRL-NH

2
1Kb-PMTX KIKIGLFDQLSRL-NH
2
a-PMTX RIKIGLFDQLSKL-NH
2
1Ka-PMTX KIKIGLFDQLSKL-NH
2
Table 2. Position and amino acid sequences of hNav1.x and para ⁄ -
TipE (DmNav1) channels. Segments 3 and 4 of domain IV and its
corresponding extracellular linker are shown. Identical amino acids
are in normal type, homologous amino acids are in bold type, and
different amino acids are in italic.
Position IV S3
Linker
IV S4
Navl.1 1616 IVGMFLAELIEKYFVSPTLFRVI
Navl.2 1606 IVGMFLAELIEKYFVSPTLFRVI
Navl.3 1601 IVGMFLAEMIEKYFVSPTLFRVI
Navl.4 1628 IVGLALSDLIQKYFVSPTLFRVI
Navl.5 1603 IVGTVLSDIIQKYFFSPTLFRVI
Navl.6 1597 IVGMFLADIIEKYFVSPTLFRVI
Navl.7 1579 IVGMFLADLIETYFVSPTLFRVI
para 1694 ILGLVLSDIIEKYFVSPTLLRVV
E. Schiavon et al. Pompilidotoxins and voltage-gated Na
v
isoforms
FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS 919
channel protein and cause slowing of the physiological
fast inactivation process, we routinely measured the
sodium current traces at 20 ms (30 s for the insect

isoform) after eliciting the current, i.e. about 20 times
after the end of the fast inactivation (test voltage of
0 mV). Determining the ratio between this current and
the peak current (I
20
⁄ I
peak
) is the standard procedure
for quantification of these effects independently of
the mode of action, either for a slow or a steady-state
component increase [19].
In Fig. 1, a 3D plot illustrates the average I
20
⁄ I
peak
ratio for both the eight isoforms and the PMTX ana-
logs. DmNa
v
1 ⁄ TipE was only tested with b-PMTX.
Almost no effects were seen for Na
v
1.4 and Na
v
1.5, and
an increase in potency from Na
v
1.7 up to Na
v
1.1
through Na

v
1.3 and Na
v
1.2 was observed. Of all synthe-
sized PMTX analogs, b-PMTX generally showed the
largest effects on the tested isoforms. Consequently, we
decided to use b-PMTX for subsequent experiments, as
it was also previously demonstrated to be the most
potent PMTX toxin [13], and a sufficient amount of this
compound was available. Although the I
20
⁄ I
peak
proce-
dure has been frequently used in many laboratories, it
cannot easily show whether the effects produced by the
toxin peptide are more related to a change in the inacti-
vation time constant of the toxin-bound channels, or to
an incomplete process of the inactivation machinery
caused by the toxin itself [20,21].
An in-depth investigation of the three components
of the sodium current was performed both on seven
mammalian and one insect VGSC with b-PMTX. As
illustrated in Fig. 2, under control conditions, the fast
component, A
f
, was generally large. In contrast, the
slow (A
s
) and steady-state (A

ss
) components were very
low or negligible (see Experimental procedures). Dur-
ing toxin action, the fast and slow time constants
(s
f
and s
s
) did not change significantly, but, the ampli-
tudes of the three components did. Moreover, these
components interchanged their relative amplitudes, so
the ratio between each component and the total ampli-
tude (T) is illustrated in Figs 3–6 in a dose-dependent
manner. Collectively, with these data, we studied in
detail how the PMTXs affected the sodium currents in
each of the tested VGSC isoforms.
Na
v
1.1, Na
v
1.2, Na
v
1.3 and Na
v
1.7 dose–response
relationships for fast, slow and steady-state
components and their voltage-dependent activation
The results are divided into Figs 3 and 4 for Na
v
1.1,

Na
v
1.2 and Na
v
1.7, and for Na
v
1.3, respectively,
Fig. 1. Effects of different analogs of PMTX on the eight VGSC iso-
forms. The ratio I
20 ms
⁄ I
peak
is plotted, on the z-axis, for the six dif-
ferent analogs of PMTX and the eight VGSC isoforms. A maximal
concentration of 46 l
M was used (n = 4 for each data point). See
[17] and Experimental procedures for details of the sequences of
the other peptides.
Fig. 2. Dissection analysis of the sodium currents to separate the
fast, slow and steady-state components. A representative response
obtained with b-PMTX and its quantitative analysis are shown. The
macroscopic current traces in controls and in the presence of
b-PMTX are shown as a line plus open squares and open circles,
respectively. The best-fitted decomposed components of the mac-
roscopic current (fast, slow, and steady-state) for the b-PMTX trace
are shown as lines. Inset: the same, but zoomed-in to reveal only
the early currents and the beginning of the fitting. The fast, slow
and steady-state components are indicated as lines.
Pompilidotoxins and voltage-gated Na
v

isoforms E. Schiavon et al.
920 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS
according to their similarity with regard to the
observed effects. Figures 3 and 4 show, for each repre-
sentative isoform: (a) the typical time course of the
currents; (b) the fast and slow fractional component
change versus toxin concentration; (c) the A
ss
⁄ T dose–
response relationship; and (d) the voltage-dependent
conductances of each component.
In Fig. 3, these data are presented for Na
v
1.2
together with the exemplary recordings from cells
expressing Na
v
1.1 and Na
v
1.7, as they exhibit similar
effects. For these three isoforms, s
f
, the time constant
of the fast component, lay in the range from
0.4 ± 0.02 to 0.7 ± 0.03 ms (n = 4 for each type). In
contrast, s
s
, the time constant of the slow component,
which was about 4.3 ± 0.3 ms in controls (but its
amplitude was well below 0.5%), increased up to

8.5 ± 0.02 ms (n = 4 for each type) and 15 ±
0.02 ms (n = 4 for each type) when tested at low or
high concentrations, i.e. from about 10 up to 140 lm.
These values were not consistently increasing in the
membrane voltage range between )20 and +30 mV
(not shown).
In Fig. 3B, the dose–response relationship for Na
v
1.2
is presented, describing the exchange between the
amount of channels that are successively bound by the
increased number of peptide molecules. The A
f
and A
s
data crossover at about 50% suggests a concentration
value of about 30 lm. In Fig. 3C, the concentration-
dependent increase of the A
ss
component is described,
and it was found that that this effect was relatively
small, amounting to a maximum of < 20% of the total
current. To investigate whether the emergence of the
slow component was voltage-dependent, conductances
were determined for a range of membrane voltages, at
a fixed concentration of 45 lm. Theoretically, we would
expect the A
f
⁄ T plot obtained in the presence of toxin
to follow the behavior seen in controls, as this ampli-

tude originates from the unbound channels. The nor-
malized data do reveal such a type of curve for the
A
f
⁄ T plot. The A
s
⁄ T component, in contrast, remains
constant after having reached its maximum at )20 mV.
The results for Na
v
1.3 are presented in Fig. 4. The A
s
time constants were not significantly different from
those mentioned above for the other isoforms. It can be
Fig. 3. Effects on the Na
v
1.1, Na
v
1.2 and Na
v
1.7 isoforms. (A) Data from three exemplary cells expressing Na
v
1.1, Na
v
1.2 and Na
v
1.7 chan-
nels. Superimposed traces elicited at )5 mV from a holding potential of )110 mV in controls and during perfusion of b-PMTX at 46 and
140 l
M. (B) Fractional change of the fast component (triangles) and slow component (circles) versus [b-PMTX] for Na

v
1.2 currents (n = 4).
(C) Dose–response curve of the fractional steady-state component (n = 4) for Na
v
1.2 currents. The line is the best fit with a Hill curve with
EC
50
, power and maximum value equal to 21 ± 2.4 lM, 1.1 ± 0.13, and 0.16, respectively. (D) Conductance–voltage plots obtained in con-
trols (solid squares) and in the presence of toxin (solid circles and triangles) with 46 l
M b-PMTX, for Na
v
1.2 currents. The fractional conduc-
tances of the slow components (A
s
⁄ T, closed circles) and the fast components (A
f
⁄ T, solid inverted triangles) are plotted against the applied
membrane voltage. The same data are also shown resized to 1 (open circles, open triangles).
E. Schiavon et al. Pompilidotoxins and voltage-gated Na
v
isoforms
FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS 921
noticed that, in Fig. 4B, the crossover between the
amplitudes of A
s
and A
f
occurs at about 100 instead of
20 lm, as shown in Fig. 3 for Na
v

1.2. Moreover, the
predicted EC
50
of the A
ss
component is also around
100 lm, instead of the value of 21 lm seen for Na
v
1.2 in
Fig. 3. The voltage-dependent conductance of the slow
component started to decrease in the region from )10
to +30 mV, after displaying a peak at )20 mV. This
effect was somewhat unexpected, as it could suggest
that the binding of the toxin could be voltage-dependent
by itself. All together, these data suggest that the toxin
action shows complex behavior and depends on the par-
ticular amino acid sequence of each isoform.
Effects on the sodium currents of Na
v
1.6 are
characterized by a robust voltage-dependent
steady-state component and the absence of the
slow component
The effects of the toxin on Na
v
1.6 currents are remark-
ably different from those described above for other
isoforms. As illustrated in Fig. 5A, the slow compo-
nent was not detected at all and the fast component
completely disappeared at 45 and 140 lm. In contrast,

a steady-state component appeared early after activa-
tion, with a maximal amplitude of about one-third of
the fast peak observed in controls. This is illustrated in
Fig. 5B, which confirms that the population of toxin-
bound channels is in equilibrium with the population
of unbound channels – here represented by the steady-
state component instead of the slow component, as
compared with Figs 3 and 4. To evaluate the A
ss
com-
ponent, its dose–response relationship was defined, as
outlined in Fig. 5C. At a fixed concentration of 45 lm,
the A
ss
component also appeared to demonstrate volt-
age-dependent behavior, as well as the A
f
(Fig. 5D).
This surprising result underlines the peculiarities of
this isoform, which are already known: (a) the possibil-
ity of generating a resurgent current in control condi-
tions in cells expressing this isoform (cerebellar
Purkinje cells, [22]); and (b) the ability of Centruro-
ides noxius toxin (Cn2), a scorpion b-toxin, to induce a
resurgent current, both in cells expressing Na
v
1.6 chan-
nels and in cerebellar neurons [23]. Moreover, Cn2 has
been reported to cause a strong hyperpolarizing shift
in the voltage dependency of the activation.

This coincidence prompted us to design experiments
to examine whether b-PMTX and Cn2 could eventu-
ally compete for the same site on the channel. Specifi-
cally, we investigated whether the Na
v
1.6 channels
were still targeted by Cn2 if b-PMTX is already bound
by the channel, i.e. whether the two toxins act via dif-
ferent binding sites. The results of these experiments,
presented in Fig. 6, recapitulate the Cn2 effects and
the b-PMTX effects as if they were produced by two
independent pathways. In these experiments, peptide
concentrations in the range of their EC
50
values were
used. In Fig. 6A,B, the superimposed traces of the cur-
rents are shown at )30 and +20 mV, in the absence
(Fig. 6A) and presence (Fig. 6B) of b-PMTX. Only at
+20 mV was there clear evidence that b-PMTX
induced the A
ss
component, as would be expected from
the data shown in Fig. 5. When Cn2 was added after
addition of b-PMTX (Fig. 6C), a large peak current
did arise at )30 mV, owing to a hyperpolarizing shift
of the activation, which was produced by the scorpion
b-toxin (a blocking effect at +20 mV also occurred;
Fig. 4. Effects on Na
v
1.3. (A) Superimposed traces elicited at

)5 mV from a holding potential of )110 mV in controls and during
perfusion of b-PMTX at 46 and 140 l
M. (B) Fractional change of the
fast and steady-state components versus [b-PMTX] (n = 4). (C)
Dose–response curve of the fractional steady-state component
(n = 4). The line is the best fit with a Hill curve with EC
50
, Hill coef-
ficient and maximum value equal to 99 ± 1 l
M, 1.11 ± 0.06, and
0.21, respectively. (D) Conductance–voltage plots obtained in con-
trols (closed squares) and in the presence of toxin with 46 l
M
b-PMTX (solid circles and triangles). The fractional conductances of
the fast components (A
f
⁄ T, solid inverted triangles) and the slow
components (A
s
⁄ T, solid circles) are plotted against the applied
membrane voltage. The same data are also shown resized to 1
(open symbols).
Pompilidotoxins and voltage-gated Na
v
isoforms E. Schiavon et al.
922 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS
Fig. 5. Effects on Na
v
1.6. (A) Superimposed
traces elicited at )5 mV from a holding

potential of )110 mV in controls and during
perfusion of b-PMTX at 46 and 140 l
M. (B)
Fractional change of the fast and steady-state
components versus [b-PMTX] (n = 4). (C)
Dose–response curve of the fractional slow
component (n = 4) obtained at three different
membrane potentials of )20, )5 and
+25 mV. The three lines are the best fit with
Hill curve parameters (EC
50
, Hill coefficient),
as follows: 43.2 ± 1.7 l
M, 1.2 ± 0.05
()20 mV); 30 ± 1 l
M, 1.5 ± 0.06 ()5 mV);
23.6 ± 1.7 l
M, 1.6 ± 0.13 (+25 mV). (D)
Conductance–voltage plots obtained in
controls (solid squares) and in the presence
of toxin with 46 l
M b -PMTX (triangles). The
fractional conductances of the fast compo-
nents (A
f
⁄ T, solid inverted triangles) and the
steady-state components (A
ss
⁄ T, solid
triangles) are plotted against the applied

membrane voltage. The same data are also
shown resized to 1 (open triangles).
Fig. 6. The competition between b-PMTX and Cn2. Upper panel. Superimposed traces of currents elicited at )90, )30 and +20 mV from a
holding potential of )100 mV. The four panels show the three traces, for the same cell, from left to right, in controls and in the presence of
b-PMTX [46 l
M (note the steady-state component)], after subsequent addition of 36 nM Cn2 and 1 min after starting the washout of
b-PMTX. The washout of Cn2 has a time course of more than 20 min. Lower left panel. The normalized conductance–voltage plots obtained
in controls (open squares) and in the presence of Cn2 (open circles). Note the left-shift induced increase in conductance in the region below
)30 mV (n = 3). Lower right panel. The ratio of the steady-state conductance with respect to peak control conductance (measured at
+10 mV) is plotted against the applied membrane potential in the four conditions as shown in the upper panels. Note the large increase in
the steady-state component produced also at membrane potentials below the threshold of activation of )30 mV (n = 3).
E. Schiavon et al. Pompilidotoxins and voltage-gated Na
v
isoforms
FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS 923
see [23]). In both traces of Fig. 6C, a steady-state com-
ponent was also present. Consecutively, a rapid wash-
out of b-PMTX was performed, with Cn2 still present.
As a result, the steady-state component immediately
disappeared (Fig. 6D). Nevertheless, we did not see
additional effects when both toxins were present. To
validate the recordings, an analysis of the voltage
dependence of normalized peak conductances in con-
trols and in the presence of Cn2 is shown in Fig. 6E,
and the conductance ratios (G
ss
⁄ G
control
) derived from
the previous four experiments (Fig. 6F) clearly con-

firmed that the two toxins should work by an indepen-
dent mechanism.
Effects on the sodium currents of the insect
isoform show a higher affinity with respect to
those observed in Na
v
1.6
In order to test the effects of the peptide not only on
mammalian isoforms, but also on insect sodium chan-
nels, we were forced to use another expression system,
namely the oocyte expression system. The effects of
the toxin on the DmNa
v
1 ⁄ TipE isoform are shown in
Fig. 7. These data qualitatively recapitulated the pep-
tide action shown in Fig. 5 for Na
v
1.6.
In Fig. 7A, the superimposed traces suggest com-
plete removal of the fast component at the expense of
the steady-state one. The data shown in Fig. 7B,C con-
firm quantitatively that the action follows a dose–
response relationship, with an EC
50
about 10-fold
lower than that presented in Fig. 5 for Na
v
1.6. The
interesting difference was the fact that, contrary to any
expectation, the final amplitude of the currents pro-

duced by the toxin could be greater than the original
values present in controls.
Na
v
1.4 and Na
v
1.5 effects and voltage-dependent
activation
⁄
inactivation at maximal concentration
Although the data presented in Fig. 2 for Na
v
1.4 and
Na
v
1.5 excluded any slow and steady-state components
in the action of b-PMTX, we nevertheless wanted to
Fig. 7. Effects on the insect isoform DmNa
v
1 ⁄ TipE (expression in X. laevis oocytes). (A) Superimposed whole cell sodium current traces in
the presence of 0.5, 3 and 50 l
M b-PMTX and in the control situation in oocytes expressing the DmNa
v
1 ⁄ TipE insect channel. An arrow indi-
cates the zero-current level. Currents were elicited at 0 mV from a holding potential of )90 mV. (B) Fractional change of the fast and steady-
state components of the current versus the applied b-PMTX concentrations (n = 2–7). (C) Dose–response curve of the fractional steady-state
components obtained at membrane potentials of )20, 0, and +20 mV (n = 2–7). Curves were fitted with Hill curve parameters (EC
50
, Hill
coefficient) as follows: 18.5 ± 15.1 l

M, 1.9 ± 1.6 (+20 mV); 4.6 ± 0.5 lM, 2.7 ± 0.5 (0 mV); 5.8 ± 0.6 lM, 2.03 ± 0.25 ()20 mV). (D) Con-
ductance–voltage plots obtained in controls (solid squares) and in the presence of toxin with 10 l
M b-PMTX (triangles). The fractional con-
ductances of the fast (A
f
⁄ T, solid inverted triangles) and steady-state (A
ss
⁄ T, solid triangles) components of the current are plotted against
the applied voltage. The same data are also shown resized to 1 (open triangles).
Pompilidotoxins and voltage-gated Na
v
isoforms E. Schiavon et al.
924 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS
investigate whether other effects could be observed by
examining in detail the voltage-dependent activation
and inactivation. These results are given Fig. S1. Exem-
plary traces for both types of channels did not reveal
any effect on the slow or steady-state component,
although some very small inhibitory effects were seen
for the cardiac (Na
v
1.5) isoform. In Fig. S1B, a small
hyperpolarizing shift of the steady-state fast inactiva-
tion of Na
v
1.5 was observed, and could probably be
responsible for the small inhibition seen in Fig. S1A.
Summary of the effects on the slow and
steady-state components in Na
v

1.1, Na
v
1.2,
Na
v
1.3, Na
v
1.6, Na
v
1.7 and DmNa
v
1 ⁄ TipE
Collectively, the previous data suggest that b-PMTX is
able to recognize the insect as well as the mammalian
Na
v
1.1, Na
v
1.2, Na
v
1.3, Na
v
1.6 and Na
v
1.7 isoforms,
but that the exerted actions are consistently different.
To summarize, Fig. 8 provides an overview of the dif-
ferent effects on the tested VGSC isoforms, for the
increase in the slow component as well as in the
steady-state component. Excluding Na

v
1.4 and Na
v
1.5,
for which we did not obtain significant results, and
based on the obtained results, two divergent groups of
effects can be distinguished. The first group concerns
the insect and Na
v
1.6 isoforms. Both showed a large
increase in the steady-state component without any
increase in the slow component. The second group
concerns Na
v
1.1, Na
v
1.2, Na
v
1.3, and Na
v
1.7, which
showed a large increase in the slow component, with
only a small steady-state component increase. These
data could suggest that the two isoform groups inter-
act in different ways with the toxin by offering differ-
ent binding sites.
Discussion
It is very common for a number of peptides purified
from various animal venoms to potentially alter the
inactivation process that occurs in VGSCs during the

physiological upstroke of the action potential. Undeni-
ably, the inactivation of VGSCs is their most vulnera-
ble kinetic feature, as it is influenced, being mostly
slowed or abolished, by many kinds of chemical sub-
stances such as drugs and toxins, and by mutations.
These mutations often encompass only a single amino
acid in the channel’s sequence. The sequences of the
VGSCs, as well as their transmembrane topology, have
been understood for a while. However, relating struc-
tural channel components to a profound function still
remains a difficult task, and has not been accom-
plished to date. Nevertheless, site-directed mutagenesis
is increasingly being applied to resolve this issue. The
molecular exploration of hereditary diseases has
also contributed significantly to the identification of
relevant regions.
According to the well-known definition derived
through the extensive work conducted by Ce
`
stele and
Catterall [24], ‘site 3¢ is considered to be responsible
for a large part of the toxin-induced effects on the
inactivation of VGSCs known to date. This site is
located in domain 4 in the extracellular linker between
segments S3 and S4. It is the target site for sea anem-
one, scorpion, spider and wasp toxins. These toxins
are peptides with different structures and lengths.
In the current study, eight different VGSC isoforms
were tested for their selective response to PMTXs,
which are known to cause slowing of the inactivation

of VGSCs. This study resembles previous work con-
ducted by Oliveira et al. [25], in which it was demon-
strated that the 47–48 amino acid peptides from sea
anemones (type 1 toxins) very selectively targeted some
isoforms more than others. Additionally, one natural
single amino acid substitution [K36A, between ATX-II
and Anthopleura fuscoviridis toxin (AFT-II)] is suffi-
cient to cause large diversities in potency and selectiv-
ity [25]. The results of the current study demonstrate
that the PMTXs were also able to discriminate
between the different VGSCs, classifying the tested
VGSCs into three groups according to their PMTX-
induced kinetics.
Fig. 8. Three-dimensional plot of the normalized increase of the
slow or steady-state components seen in the different isoforms.
Data were obtained at a concentration of 46 l
M (n = 5).
E. Schiavon et al. Pompilidotoxins and voltage-gated Na
v
isoforms
FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS 925
For Na
v
1.1, Na
v
1.2, Na
v
1.3, and Na
v
1.7, the addi-

tion of b-PMTX mainly resulted in a large increase in
the slow component of current inactivation and, to a
minor extent, a small increase in a steady-state compo-
nent. These data quantitatively demonstrate an almost
perfect dose–response relationship between two popu-
lations of channels, those bound by the peptide and
those unbound. This produces a net decrease in the
fast component and a corresponding increase in the
slow component of the inactivation. The maximal
effects seen at the level of the steady-state component,
represented by the ratio A
ss
⁄ T, did not reach a value
of 0.2, and the voltage-dependent inactivation curves
also did not show any significant effect (data not
shown).
A completely different biophysical effect was
revealed by experiments performed on Na
v
1.6 and
the DmNa
v
1 ⁄ TipE. No slow component increase was
noticed in these experiments. In contrast, the steady-
state component largely increased, reaching a value of
1(A
ss
⁄ T). Moreover, its growth was comparable to the
decrease in the fast component. This suggests that, in
this isoform, the action of the peptide was accompa-

nied by a mechanism somewhat different from that
observed in the other isoforms. In addition, previous
studies demonstrated that Na
v
1.6 and Na
v
1.3 are also
prone to produce a large steady-state component under
the action of AFT-II and Bunodosoma caissarum toxin
(Bc-III) [25], although in this case these toxins also
produced a slow inactivation component.
The data for DmNa
v
1 shown in Fig. 7A are very
striking, because they bear a strong resemblance to
data obtained in earlier experiments. In those experi-
ments, enzymes capable of irreversibly removing the
short peptide responsible for the inactivation mecha-
nism were injected intracellularly [26,27]. In more
recent, analogous, experiments, a similar effect was
shown with the use of a scorpion a-toxin [28] and the
sea anemone toxins BgII and BgIII (isolated from
Bunodosoma granulifera) [29]. Similarly to what is seen
in Fig. 7A, the currents of the DmNa
v
1 ⁄ TipE channels
also did not show any A
s
component upon BgII ⁄ BgIII
application.

Na
v
1.6 has distinctive properties, because it is excep-
tionally sensitive to the actions of other neurotoxins,
such as scorpion b -toxins [23]. This led us to investi-
gate competition between b-PMTX and Cn2. As
shown in Fig. 6, it became clear that the two peptides
do not significantly interact, as the increase in the
steady-state component was also present in those
channels bound by Cn2, which opened at voltages
lower than the threshold values typical of the normal
conditions.
In another study, a chimera channel was constructed
by inserting domain 2 of DmNa
v
1 channels in the rat
brain rBIIA (rNa
v
1.2) sodium channel [30]. It is
important to mention that this chimera exhibited insect
channel properties in the activation of the rBIIA chan-
nel, and also determined the selectivity of the excit-
atory AahIT scorpion toxin from Androctonus australis
hector over insect channels. The same study demon-
strated the unexpected involvement of domain 2 in the
inactivation process, even though scorpion a-toxins
had not changed their binding ability because of the
presence of the insect domain 2 in the rat channel.
Considering our present results on the coapplication of
Cn2 and b-PMTX, this is one more indication that

b-PMTX would not bind to domain 2 (the target of
Cn2).
As expected from previous results reported by
Kinoshita et al. [15], Na
v
1.4 and Na
v
1.5 did not show
any effect on the slow component or the steady-state
component of inactivation. In general, the dose–
response EC
50
values of the slow or steady-state com-
ponent illustrated in Figs 3, 4, 5 and 7 correlated well
with the data showing the crossover of the populations
of toxin-bound and toxin-unbound channels. Indeed,
Na
v
1.2 and Na
v
1.6 were the isoforms with the best
affinity in the range from 20 to 30 lm;Na
v
1.7 had an
intermediate value of about 55–60 lm (data not
shown), and Na
v
1.3 had the lowest affinity, at about
100 lm.
The described specificity of the b-PMTX-induced

effects on the tested isoforms can be explained by the
model previously obtained from a study by Kinoshita
et al. [15]. They suggested that specific site-directed
mutagenesis in the rBII (rNa
v
1.2) isoform was able to
eliminate an increase in the so-called modification ratio
(in our case: I
20
⁄ I
peak
). This amino acid modification
involved a Glu fi Gln mutation at position 1616,
which is exactly reproduced in natural sequences of the
Na
v
1.4 and Na
v
1.5 isoforms, and is a fingerprint of
these channels (presented in Table 2; human isoforms
and invertebrate channels are aligned). Consequently,
our results confirmed that Na
v
1.4 and Na
v
1.5 did not
show any effect in the slow or steady-state component,
as shown in Fig. 2 and Fig. S1. In contrast, the differ-
ence in amino acid sequence of the Na
v

1.6 and
DmNa
v
1 isoforms with respect to the Na
v
1.1, Na
v
1.2,
Na
v
1.3 and Na
v
1.7 group can be restricted to the DI
sequence versus EL, EM or DL for the Na
v
1.1, Na
v
1.2,
Na
v
1.3 and Na
v
1.7 group. Moreover, DmNa
v
1 insect
channels present the sequence DIIE, similar to the
sequence starting at Asp1604 in Na
v
1.6. Considering
the preferential affinity of b-PMTX, the individual or

combined role of these residues still remains unclear.
Pompilidotoxins and voltage-gated Na
v
isoforms E. Schiavon et al.
926 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS
Within the last 15 years, much research has been
conducted on ‘site 3 mutagenesis’, as well as chimera
channels, in order to analyze the alleged binding sites
of scorpion a -toxin and b-toxin and type 1 sea anem-
one toxins in VGSCs [30–32]. Researchers discovered
that the sea anemone toxin ATX-II and scorpion
a-toxins bind to a common extracellular site. In addi-
tion, they found that the negatively charged Glu1613
in Na
v
1.2 was crucial for toxin binding [31]. More
recently, Heinemann and co-workers [32] contributed
greatly to the field by scrutinizing the role of the
E ⁄ DE, E ⁄ Q and KYFV equivalent in positions 1623,
1626 and 1627, 1628, 1629 and 1630 of Na
v
1.1
(Table 2), respectively. The assayed toxins consisted of
a quantity of scorpion a-toxins (Lqh-2, Lqh-3, and
LqhaIT) from Leiurus quinquestriatus hebraeus as well
as d-SVIE conotoxin from Conus striatus. Each of
these peptides binds to site 3 of VGSCs and impairs
the inactivation process in a similar way as b-PMTX.
Taking into consideration the current results, com-
bined with previously discussed site-directed mutagene-

sis findings [30–32] and studies on chimera channels
[15,30], it has become clear that certain ‘microhetero-
geneous residues’ in S3–S4 linker segments have a very
specific affect on toxin interactions. However, even
with the current knowledge of the S3 and S4 segments
of domain 4, it still remains unclear whether other
topological components of the VGSCs may be
involved in the binding of toxins that impair the inacti-
vation process and increase steady-state components.
Further study on chimeras and site-directed mutagene-
sis will undoubtedly prove beneficial in addressing this
issue.
Experimental procedures
Peptide synthesis
Peptides were synthesized by a stepwise solid-phase method
using Fmoc chemistry with TGS-RAM resin (Rapp Poly-
mere GmbH, Tu
¨
bingen, Germany) on a Shimadzu PSSM-8
peptide synthesizer (Shimadzu Corp., Kyoto, Japan). All
Fmoc-l-amino acids were purchased from Nova Biochem.
The side chain protective groups were t-butyloxycarbonyl
for Lys, 2,2,5,7,8-pentamethylchroman-6-sulfonyl for Arg,
and t-butyl for Thr. Cleavage of the peptide from the resin
was achieved by treatment with a mixture of trifluoroacetic
acid (TFA) ⁄ phenol ⁄ thioanisole ⁄ 1,2-ethanedithiol ⁄ ethyl-
methyldisulfide ⁄ H
2
O (80 : 5 : 5 : 3 : 2 : 5, by volume),
using 10 mLÆg

)1
resin at room temperature for 8 h. After
removal of the resin by filtration and washing twice with
TFA, the combined filtrate was added dropwise to diethyl
ether at 0 °C and then centrifuged at 3000 r.p.m. for
10 min. The crude synthetic peptide obtained was purified
by semipreparative RP-HPLC using YMC-PAK ODS
(20 · 150 mm; Yamamura Kagaku, Kyoto, Japan) with
isocratic elution of 28–30% CH
3
CN ⁄ H
2
O ⁄ 0.1% TFA at a
flow rate of 7 mLÆmin
)1
. The homogeneity and the
sequence were confirmed by HPLC and MALDI-TOF MS
[17]. Besides b-PMTX, the synthetic peptides used were:
3Rb (same as b-PMTX except for a Lys fi Arg change at
position 3), 1Kb (same as b-PMTX except for an Arg fi
Lys change at position 1), a-PMTX, 1K3Rb (same as
b-PMTX except for Arg fi Lys and Lys fi Arg changes
at positions 1 and 3, respectively), and 1Ka (same as
a-PMTX except for an Arg fi Lys change at position 1).
Human Na
v
1.x isoforms
Cell culture
HEK293 cell lines stably expressing human Na
v

1.1, Na
v
1.2,
Na
v
1.3, Na
v
1.5 and Na
v
1.6 (generously donated by Glaxo-
SmithKline, Medicines Research Centre, Stevenage, UK)
were cultured in modified DMEM supplemented with 10%
fetal bovine serum as previously described [25]. Na
v
1.4-
expressing cells were obtained by stable transfection of a
plasmid containing the hNa
v
1.4 construct (a kind gift from
D. Conti-Camerino, University of Bari, Italy). Na
v
1.7-
expressing cells were obtained by transient transfection of a
plasmid containing the hNa
v
1.7 construct (a kind gift from
F. Hofmann through A. Wada, University of Miyazaki,
Japan). Approximately 2 · 10
4
cells were transfected with

2 lg of the hNa
v
1.7 vector along with 0.2 lg of green
fluorescent protein in a pEGFP-C1 vector (Clontech, Euro-
clone, Milan, Italy), using the Lipofectamine reagent kit
(Invitrogen, Milan, Italy), following the instructions of the
manufacturer. Currents were recorded 24–72 h following
transfection.
Solutions and drugs
The standard extracellular solution contained: 70 mm NaCl,
67 mm N-methyl-d-glucamine, 1 mm CaCl
2
, 1.5 mm MgCl
2
,
5mm Hepes, and 10 mmd-glucose (pH 7.40). The standard
pipette solution contained: 105 mm CsF, 27 mm CsCl,
5mm NaCl, 2 mm MgCl
2
,10mm EGTA, and 10 mm
Hepes (pH 7.30). About 6–8% of the cells expressing the
Na
v
1.6 channel clone had a persistent sodium current, as
reported by Burbidge et al. [33]. We systematically tested
these cells, and discarded those showing incomplete inacti-
vation (a residual current after 250 ms of < 0.1% of the
peak sodium current). Known quantities of the toxins were
dissolved in the extracellular solution just before the start
of the experiments. TTX (Sigma, Milan, Italy) was used at

300 nm on the Na
v
1.1, Na
v
1.2, Na
v
1.3, Na
v
1.4, Na
v
1.6 and
Na
v
1.7 currents, and the resulting traces were subtracted
from the control traces to obtain the TTX-sensitive
currents; the Na
v
1.5 clone (which has a much higher TTX
E. Schiavon et al. Pompilidotoxins and voltage-gated Na
v
isoforms
FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS 927
ID
50
than 100 nm) never showed any significant contami-
nating potassium currents at the test potentials. The extra-
cellular solutions were delivered through a remote-
controlled nine-hole (0.6 mm) linear positioner placed near
the cell being studied. The average response time was 2–3 s.
Patch-clamp recordings

The currents were recorded at room temperature using a
MultiClamp 700A (Axon Instruments, Crisel, Rome, Italy),
as previously described [25]; pipette resistance was about
1.3–2.1 MX; cell capacitance and series resistance errors
were carefully (85–90%) compensated for before each run
of the voltage clamp protocol, in order to reduce voltage
errors to < 5% of the protocol pulse. The P ⁄ N leak proce-
dure was routinely used. pclamp 8.2 (Axon Instruments)
and origin 7 (OriginLab, Northampton, MA, USA) were
routinely used during data acquisition and analysis. All
data regarding activation were obtained using a holding
potential of )90 mV, a 100 ms preconditioning pulse of
)120 mV (to completely remove fast inactivation), and a
7 ms test pulse from )80 to +40 mV. For steady-state
inactivation, the 200 ms preconditioning pulse was varied
from )120 to +10 mV, and the test pulse was )20 or
)10 mV. To obtain the conductance–voltage data, the peak
currents were divided by the driving force (V
M
+ 67) and
normalized using the value in the range +10 to +30.
Insect sodium channel isoforms
Xenopus laevis oocyte expression system
For expression in X. laevis oocytes, cDNA encoding
DmNa
v
1 ⁄ TipE was subcloned into vector pGH19-13-5, and
the TipE cDNA was subcloned into vector pGH19. Vectors
were linearized with NotI, and in vitro transcription was
performed using the T7 mMESSAGE-mMACHINE tran-

scription kit (Ambion, Monza, Italy). Stage V–VI oocytes
were harvested from anesthetized female X. laevis frogs as
described previously [34]. Oocytes were injected with 50 nL
of cRNA (1 : 1 ratio for DmNa
v
1 and TipE subunits) at a
concentration of 1 ngÆnL
)1
, using a microinjector (Drum-
mond Scientific, Rochester, USA). The oocytes were incu-
bated in a solution containing 96 mm NaCl, 2 mm KCl,
1.8 mm CaCl
2
,2mm MgCl
2
, and 5 mm Hepes (pH 7.4),
supplemented with 50 mgÆL
)1
gentamicin sulfate and
180 mgÆL
)1
theophylline.
Electrophysiological recordings
Two-electrode voltage-clamp recordings were performed at
room temperature (18–22 °C), using a Geneclamp 500
amplifier (Axon Instruments) controlled by a pclamp data
acquisition system (Axon Instruments). Whole cell currents
from oocytes were recorded 4–5 days after injection. The
bath solution composition was 96 mm NaCl, 2 mm KCl,
1.8 mm CaCl

2
,2mm MgCl
2
, and 5 mm Hepes (pH 7.4).
Voltage and current electrodes were filled with 3 m KCl.
Resistances of both electrodes were maintained between 0.5
and 0.7 MX. The elicited currents were filtered at 2 kHz
and sampled at 10 kHz, using a four-pole low-pass Bessel
filter. To eliminate the effect of the voltage drop across the
bath grounding electrode, the bath potential was actively
controlled by a two-electrode bath clamp. Leak subtraction
was performed using a )P ⁄ 4 protocol.
Representative whole cell currents were elicited every 5 s
by a 100 ms voltage pulse to 0 mV, starting from a holding
potential of )90 mV. The fractional components of the cur-
rents were plotted against the logarithm of the applied con-
centrations. To assess the concentration dependency of the
b-PMTX-induced effects, dose–response curves of the
steady-state component were constructed and fitted with
the Hill equation to obtain EC
50
values (i.e. the toxin con-
centration that produces 50% of the maximum effect) at
three voltages. Conductances of the fractional components
were shown as a function of voltage. Each experiment was
performed at least twice (n ‡ 2), and a maximum of seven
times. All data were analyzed with origin (OriginLab
Corp., USA), and presented as mean ± standard error of
the mean.
Data analysis

We followed the procedure previously described [25].
Briefly, we analyzed the data on the assumption that each
sodium current trace is the sum of two exponential decay-
ing components, which are the slow (s) and the fast (f)
component, and a steady-state (ss) component. As a param-
eter for these components, their amplitude, as calculated by
clampfit (Axon Instruments), was used. Under control
conditions, the amplitude of the fast component (A
f
) was
generally large, and the amplitudes of the slow (A
s
) and
steady-state (A
ss
) components were very low or negligible.
During toxin action, either a large increase in A
s
or a large
increase in A
ss
occurred, depending on the isoform. The
large increase in A
s
was occasionally associated with a
smaller increase in A
ss
. The average value of the fast time
constant (s
f

) found in control traces was used as a fixed
parameter during the fitting procedure of the currents
obtained under the action of the peptide. We systematically
observed that the quality of fitting of the toxin-induced cur-
rents was the same whether we used a ‘free’ or fixed (con-
trol) value of s
f
. An example of how we analyzed the
observed effects and obtained these theoretical components
and their corresponding macroscopic current traces is fully
shown in Fig. 2.
This strongly suggests that the currents recorded in the
presence of toxin were always the sum of two types of cur-
rent: those deriving from toxin-bound channels (modified),
Pompilidotoxins and voltage-gated Na
v
isoforms E. Schiavon et al.
928 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS
and those deriving from toxin-free channels (not modified
and thus equivalent to control channels). Therefore, we
examined about 80 ms of each trace in controls, and com-
puted A
f
and its time constant (s
f
) with the fitting proce-
dure present in clampfit (Axon Instruments). On the
contrary, in the presence of the toxin, we retained s
f
of con-

trols, and computed, on the one hand, the amplitude of the
fast-inactivating component originating from the unbound
channels, namely A
f
, and on the other hand, the A
ss
com-
ponent and its s
s
, and the A
s
component, both originating
from the toxin-bound channels [25].
Acknowledgements
We thank B. Billen for critical discussions and N. Van
Nuffelen for reading the manuscript and making inci-
sive comments. This study was partially supported by
grants from the Italian Ministero dell’Universita
`
e
della Ricerca Scientifica e Tecnologica (MIUR-
PRIN2005-2001055320, MIUR-FIRB2001-
RBNE01XMP4-002, and MIUR-FISR2001-0300179)
and the Universita
`
di Milano-Bicocca to E. Wanke,
and by grants from the Italian Ministero dell’Univer-
sita
`
e della Ricerca Scientifica e Tecnologica to A.

Zaharenko (MIU-PRIN2003-2005), and partially spon-
sored by grants G.0330.06 and G.0257.08 (F.W.O.
Vlaanderen) and UAP p6 ⁄ 31 (Interuniversity Attrac-
tion Poles Program–Belgian State–Belgian Science Pol-
icy) to J. Tytgat. M. Stevens was sponsored by the
Wetenschappelijk Onderzoek Multiple Sclerose
(W.O.M.S. vzw). E. Schiavon was a PhD student of
physiology at the Department of Biotechnologies and
Biosciences of the University of Milano-Bicocca.
References
1 Catterall WA, Goldin AL & Waxman SG (2005) Inter-
national Union of Pharmacology. XLVII. Nomencla-
ture and structure–function relationships of voltage-
gated sodium channels. Pharmacol Rev 57, 397–409.
2 Dong K (2007) Insect sodium channels and insecticide
resistance. Invert Neurosci 7, 17–30.
3 King GF, Escoubas P & Nicholson GM (2008) Peptide
toxins that selectively target insect Na(V) and Ca(V)
channels. Channels (Austin) 2, 100–117.
4 Ackerman MJ (2004) Cardiac channelopathies: it’s in
the genes. Nat Med 10, 463–464.
5 Kullmann DM (2002) The neuronal channelopathies.
Brain 125, 1177–1195.
6 Dib-Hajj SD, Binshtok AM, Cummins TR, Jarvis MF,
Samad T & Zimmermann K (2009) Voltage-gated
sodium channels in pain states: role in pathophysiology
and targets for treatment. Brain Res Rev 60, 65–83.
7 Mantegazza M, Gambardella A, Rusconi R, Schiavon
E, Annesi F, Cassulini RR, Labate A, Carrideo S,
Chifari R, Canevini MP et al. (2005) Identification of

an Nav1.1 sodium channel (SCN1A) loss-of-function
mutation associated with familial simple febrile seizures.
Proc Natl Acad Sci USA 102, 18177–18182.
8 Waxman SG (2008) Mechanisms of disease: sodium
channels and neuroprotection in multiple sclerosis –
current status. Nat Clin Pract Neurol 4, 159–169.
9 Kim DY, Carey BW, Wang H, Ingano LA, Binshtok
AM, Wertz MH, Pettingell WH, He P, Lee VM, Woolf
CJ et al. (2007) BACE1 regulates voltage-gated sodium
channels and neuronal activity. Nat Cell Biol 9,
755–764.
10 Fiske JL, Fomin VP, Brown ML, Duncan RL & Sikes
RA (2006) Voltage-sensitive ion channels and cancer.
Cancer Metastasis Rev 25, 493–500.
11 Konno K, Miwa A, Takayama H, Hisada M, Itagaki
Y, Naoki H, Yasuhara T & Kawai N (1997) Alpha-
pompilidotoxin (alpha-PMTX), a novel neurotoxin
from the venom of a solitary wasp, facilitates transmis-
sion in the crustacean neuromuscular synapse. Neurosci
Lett 238, 99–102.
12 Konno K, Hisada M, Itagaki Y, Naoki H, Kawai N,
Miwa A, Yasuhara T & Takayama H (1998) Isolation
and structure of pompilidotoxins, novel peptide neuro-
toxins in solitary wasp venoms. Biochem Biophys Res
Commun 250, 612–616.
13 Konno K, Hisada M, Naoki H, Itagaki Y, Yasuhara T,
Nakata Y, Miwa A & Kawai N (2000) Molecular deter-
minants of binding of a wasp toxin (PMTXs) and its
analogs in the Na+ channels proteins. Neurosci Lett
285, 29–32.

14 Sahara Y, Gotoh M, Konno K, Miwa A, Tsubokawa
H, Robinson HP & Kawai N (2000) A new class of
neurotoxin from wasp venom slows inactivation of
sodium current. Eur J Neurosci 12, 1961–1970.
15 Kinoshita E, Maejima H, Yamaoka K, Konno K,
Kawai N, Shimizu E, Yokote S, Nakayama H &
Seyama I (2001) Novel wasp toxin discriminates
between neuronal and cardiac sodium channels. Mol
Pharmacol 59, 1457–1463.
16 Kawai N & Konno K (2004) Molecular determinants of
two neurotoxins that regulate sodium current inactivation
in rat hippocampal neurons. Neurosci Lett 361, 44–46.
17 Konno K & Kawai N (2004) Pompilidotoxins. Novel
peptide neurotoxins blocking sodium channel inactiva-
tion from solitary wasp venom. Curr Med Chem Cent
Nerv Syst Agents 4, 139–146.
18 Grieco TM & Raman IM (2004) Production of resur-
gent current in NaV1.6-null Purkinje neurons by slow-
ing sodium channel inactivation with beta-
pompilidotoxin. J Neurosci 24, 35–42.
19 Leipold E, Lu S, Gordon D, Hansel A & Heinemann
SH (2004) Combinatorial interaction of scorpion toxins
E. Schiavon et al. Pompilidotoxins and voltage-gated Na
v
isoforms
FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS 929
Lqh-2, Lqh-3, and LqhalphaIT with sodium channel
receptor sites-3. Mol Pharmacol 65, 685–691.
20 Ulbricht W (2005) Sodium channel inactivation: molec-
ular determinants and modulation. Physiol Rev 85,

1271–1301.
21 Wanke E, Zaharenko AJ, Redaelli E & Schiavon E
(2009). Actions of sea anemone type 1 neurotoxins on
voltage-gated sodium channel isoforms. Toxicon 54,
1102–1111.
22 Raman IM & Bean BP (1997) Resurgent sodium
current and action potential formation in dissociated
cerebellar Purkinje neurons. J Neurosci 17, 4517–
4526.
23 Schiavon E, Sacco T, Cassulini RR, Gurrola G, Tempia
F, Possani LD & Wanke E (2006) Resurgent current
and voltage sensor trapping enhanced activation by a
beta-scorpion toxin solely in Nav1.6 channel. Signifi-
cance in mice Purkinje neurons. J Biol Chem 281,
20326–20337.
24 Ce
`
stele S & Catterall WA (2000) Molecular mechanisms
of neurotoxin action on voltage-gated sodium channels.
Biochimie 82, 883–892.
25 Oliveira JS, Redaelli E, Zaharenko AJ, Cassulini RR,
Konno K, Pimenta DC, Freitas JC, Clare JJ & Wanke
E (2004) Binding specificity of sea anemone toxins to
Nav 1.1–1.6 sodium channels: unexpected contributions
from differences in the IV ⁄ S3–S4 outer loop. J Biol
Chem 279, 33323–33335.
26 Armstrong CM, Bezanilla F & Rojas E (1973) Destruc-
tion of sodium conductance inactivation in squid axons
perfused with pronase. J Gen Physiol 62, 375–391.
27 Rojas E & Rudy B (1976) Destruction of the sodium

conductance inactivation by a specific protease in per-
fused nerve fibres from Loligo. J Physiol 262, 501–531.
28 Arnon T, Potikha T, Sher D, Elazar M, Mao W, Tal T,
Bosmans F, Tytgat J, Ben-Arie N & Zlotkin E (2005)
BjalphaIT: a novel scorpion alpha-toxin selective for
insects – unique pharmacological tool. Insect Biochem
Mol Biol 35, 187–195.
29 Bosmans F, Aneiros A & Tytgat J (2002) The sea
anemone Bunodosoma granulifera contains surprisingly
efficacious and potent insect-selective toxins. FEBS Lett
532, 131–134.
30 Shichor I, Zlotkin E, Ilan N, Chikashvili D, Stuhmer
W, Gordon D & Lotan I (2002) Domain 2 of Drosoph-
ila para voltage-gated sodium channel confers insect
properties to a rat brain channel. J Neurosci 22,
4364–4371.
31 Rogers JC, Qu Y, Tanada TN, Scheuer T & Catterall
WA (1996) Molecular determinants of high affinity
binding of alpha-scorpion toxin and sea anemone toxin
in the S3–S4 extracellular loop in domain IV of the
Na+ channel alpha subunit. J Biol Chem 271, 15950–
15962.
32 Leipold E, Hansel A, Olivera BM, Terlau H &
Heinemann SH (2005) Molecular interaction of
delta-conotoxins with voltage-gated sodium channels.
FEBS Lett 579, 3881–3884.
33 Burbidge SA, Dale TJ, Powell AJ, Whitaker WR, Xie
XM, Romanos MA & Clare JJ (2002) Molecular
cloning, distribution and functional analysis of the
NA(V)1.6 voltage-gated sodium channel from human

brain. Brain Res Mol Brain Res 103, 80–90.
34 Liman ER, Tytgat J & Hess P (1992) Subunit stoichi-
ometry of a mammalian K+ channel determined by
construction of multimeric cDNAs. Neuron 9, 861–871.
Supporting information
The following supplementary material is available:
Fig. S1. Effects on the activation and inactivation in
Na
v
1.4 and Na
v
1.5 isoforms.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
Pompilidotoxins and voltage-gated Na
v
isoforms E. Schiavon et al.
930 FEBS Journal 277 (2010) 918–930 ª 2010 The Authors Journal compilation ª 2010 FEBS