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The sea anemone Bunodosoma caissarum toxin BcIII modulates the sodium
current kinetics of rat dorsal root ganglia neurons and is displaced in
a voltage-dependent manner
Emilio Salceda
a,1
, Omar Lo
´pez
a,1
, Andre
´J. Zaharenko
b,c,
*, Anoland Garateix
d
, Enrique Soto
a
a
Instituto de Fisiologı
´a, Universidad Auto
´noma de Puebla, 14 sur 6301, CU, San Manuel, Puebla, Pue., CP 72750, Mexico
b
Departamento de Fisiologia, Instituto de Biocie
ˆncias, Universidade de Sa
˜o Paulo, Rua do Mata
˜o, travessa 14, n: 321, CEP 05508-900, Sa
˜o Paulo, SP, Brazil
c
Centro de Biotecnologia, Instituto de Pesquisas Energe
´ticas e Nucleares (IPEN), Avenida Lineu Prestes, 2242, CEP 05508-000, Sa
˜o Paulo, SP, Brazil
d
Centro de Bioproductos Marinos (CEBIMAR), Agencia de Medio Ambiente, Ministerio de Ciencia, Tecnologı
´a y Medio Ambiente (CITMA), Calle Lomaentre 35 y 37, Alturas del Vedado,
10600, Ciudad de la Habana, Cuba
1. Introduction
Voltage-gated sodium channels (Na
+
channels) are integral
membrane proteins which consist of an
a
-subunit (able to form
functional ion channels when expressed in Xenopus oocytes) and
two auxiliary subunits,
b
1 (or
b
3) and
b
2. The
a
-subunit has four
homologous domains (I–IV) forming a ion pore; each domain has
six transmembrane segments (S1–S6); the S4 transmembrane
segments act as voltage sensors [6]. The intracellular loop between
domains III and IV has been related with the fast inactivation gate,
blocking the conduction pathway following channel activation [6].
A diversity of toxins and chemicals are known to either block or
modulate Na
+
channels by binding to specific receptor sites. At
least six neurotoxin receptor sites have been identified on the
mammalian sodium channel [5,45]. Among them, receptor site 3 is
a macrosite that involves the extracellular loops IS5–S6, IVS3–S4,
and IVS5–S6 of the ionic channel with an important participation
of the glutamic acid in position 1613 (rat brain Na
+
channel) [35] or
the aspartic acid in position 1612 (rat cardiac Na
+
channel) [1].
The family of site-3 neurotoxins comprise a structurally diverse
group of peptide toxins isolated from scorpions [4,25,33], sea
anemones [28,30], spiders [18,24], and wasps [23,36]. These
compounds increase the action potential duration by slowing
down the time course of the sodium channel fast inactivation
[32,37,40,41]. The binding affinity of site-3 toxins is decreased by
depolarization [4,16], and several electrophysiological studies
have shown that the dissociation rate of
a
-scorpion toxins is
voltage-dependent [3,4,7,14,26,27,34,35,46,49]. A similar voltage-
dependent dissociation for sea anemone toxins has been reported
[49,46], however this has been a less explored characteristic of sea
anemone toxins.
In this work we studied the electrophysiological effects of BcIII
(from the sea anemone Bunodosoma caissarum), a sodium channel
site-3 toxin, on macroscopic currents of rat dorsal root ganglia
Peptides 31 (2010) 412–418
ARTICLE INFO
Article history:
Received 22 August 2009
Received in revised form 3 December 2009
Accepted 3 December 2009
Available online 16 December 2009
Keywords:
Site-3 toxins
Voltage-gated sodium channels
Fast inactivation
CgNa
ATX-II
Neurotoxins
ABSTRACT
Sea anemone toxins bind to site 3 of the sodium channels, which is partially formed by the extracellular
linker connecting S3 and S4 segments of domain IV, slowing down the inactivation process. In this work
we have characterized the actions of BcIII, a sea anemone polypeptide toxin isolated from Bunodosoma
caissarum, on neuronal sodium currents using the patch clamp technique. Neurons of the dorsal root
ganglia of Wistar rats (P5–9) in primary culture were used for this stud y (n= 65). The main effects of BcIII
were a concentration-dependent increase in the sodium curre nt inactivation time course (IC
50
= 2.8
m
M)
as well as an increase in the current peak amplitude. BcIII did not modify the voltage at which 50% of the
channels are activated or inactivated, nor the reversal potential of sodium current. BcIII shows a voltage-
dependent action. A progressive acceleration of sodium current fast inactivation with longer
conditioning pulses was observed, which was steeper as more depolarizing were the prepulses. The
same was observed for other two anemone toxins (CgNa, from Condylactis gigantea and ATX-II, from
Anemonia viridis). These results suggest that the binding affinity of sea anemone toxins may be reduced
in a voltage-dependent manner, as has been described for
a
-scorpion toxins.
ß2009 Elsevier Inc. All rights reserved.
* Corresponding author at: Departamento de Fisiologia, Instituto de Biocie
ˆncias,
Universidade de Sa
˜o Paulo, Rua do Mata
˜o, travessa 14, n: 321, CEP 05508-900, Sa
˜o
Paulo, SP, Brazil. Tel.: +55 11 30917522; fax: +55 11 30917568.
E-mail addresses: esalceda@siu.buap.mx (E. Salceda), omar.lpz.r@gmail.com
(O. Lo
´pez), zaharenko@usp.br,a.j.zaharenko@ig.com.br (A.J. Zaharenko),
cebimar@infomed.sld.cu (A. Garateix), esoto@siu.buap.mx (E. Soto).
1
These authors contributed equally to this work..
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
0196-9781/$ – see front matter ß2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2009.12.005
Author's personal copy
(DRG) neurons. We found that under saturating concentration of
the toxin, BcIII unbind from its receptor site in a voltage-dependent
manner. Finally, we compare this latter result with those obtained
in the presence of two other site-3 neurotoxins, CgNa (from
Condylactis gigantea) and ATX-II (from Anemonia sulcata, now
called A. viridis).
2. Materials and methods
2.1. Biological materials
Wistar rats at postnatal day 5–9 of either gender were used for
the experiments. Animal care and procedures were in accordance
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals and the Reglamento de la Ley General de Salud
en Materia de Investigacio
´n para la Salud of the Secretarı
´a de Salud of
Mexico. All efforts were made to minimize animal suffering and to
reduce the number of animals used, as outlined in the ‘‘Guide to the
Care and Use of Laboratory Animals’’ issued by the National
Academy of Sciences.
2.2. Toxins
BcIII and CgNa were isolated and purified from the sea
anemones B. caissarum and C. gigantea as previously described
[31,44]. ATX-II was a gift from Professor L. Beress (Kiel, Germany).
Aliquots of stock solution (200
m
M) in deionized water were
prepared and stored in a freezer (20 8C). Before each experiment,
an aliquot was dissolved in the perfusion solution.
2.3. Cell preparation
Young Wistar rats (P5–9) of either gender were anesthetized
with ether and decapitated. DRG neurons were isolated and
cultured according to the procedure described previously [37].In
order to guarantee the viability of the cells under study and to
assure adequate spatial clamp, the neurons for recording were
selected to have a rounded or oval shape, not to be adhered to other
cells or to show any neurite outgrow, and to be refringent under
the phase-contrast microscope.
2.4. Electrophysiological recording
A coverslip with attached neurons was transferred to a 500
m
l
perfusion chamber mounted on the stage of an inverted phase-
contrast microscope (Nikon Diaphot, Tokyo, Japan). Cells were
bathed with an external solution containing 20 mM NaCl, 1 mM
MgCl
2
, 1.8 mM CaCl
2
, 45 mM TEA-Cl, 70 mM choline chloride,
10 mM 4-aminopyridine, and 5 mM HEPES (pH 7.4 adjusted with
HCl). Osmolarity was monitored by a vapor pressure osmometer
(Wescor, Logan, UT) and adjusted to 290 mOsm using dextrose.
A gravity-driven perfusion system maintained the external
solution flowing into the chamber at a rate of about 100
m
l/min. A
pair of glass capillaries placed approximately 40
m
m above the cell
under study continuously microperfused (10
m
l/min) external
solution or external solution plus toxin.
Patch pipettes were pulled from borosilicate glass capillaries
(TW120-3; WPI, Sarasota, FL), using a Flaming-Brown electrode
puller (P80/PC; Sutter Instruments, San Rafael, CA). They typically
had a resistance between 1 and 2.5 M
V
when filled with the
internal solution which was composed as follows: 10 mM NaCl,
100 mM CsF, 30 mM CsCl, 10 mM TEA-Cl, 8 mM EGTA, and 5 mM
HEPES (pH 7.2 adjusted with CsOH; osmolarity = 300 mOsm).
The whole-cell patch clamp technique was used to record ionic
currents with an Axopatch-1D amplifier (Molecular Devices,
Sunnyvale, CA). Command pulse generation and data sampling
were controlled by the PClamp 8.0 software (Molecular Devices)
using a 16-bit data acquisition system (Digidata 1320A, Molecular
Devices). Signals were low-pass filtered at 5 kHz and digitized at
20 kHz. Leakage and capacitive currents were digitally subtracted
using the P-P/n method; capacitance and series resistance (80%)
were electronically compensated. In the time course of an
experiment, seal and series resistance were continuously moni-
tored to guarantee stable recording conditions. Experiments were
made at room temperature (22–25 8C).
Experiments were rejected when, at the maximum peak
current, the voltage error exceeded 5 mV after compensation of
series resistance. No corrections were made for smaller values.
DRG neurons express a mixture of two sodium current (I
Na
)
subtypes: tetrodotoxin-sensitive (TTX-S; K
i
= 0.3 nM) and tetrodo-
toxin-resistant (TTX-R; K
i
= 100
m
M). The type of I
Na
in the cell
under study was determined before each experiment and only
those cells with <10% TTX-R I
Na
, as derived from a steady-state
inactivation profile, were used to determine the effects of BcIII on
TTX-S I
Na
.
It has been reported that both activation and steady-state
inactivation curves are shifted over time in whole-cell patch clamp
experiments [17,29]. In order to minimize the effects of time-
dependent shifts on our results, recordings were not initiated until
10–15 min after the whole-cell configuration was obtained.
2.5. Data analysis
Recordings were analyzed off-line with PClamp 8.0 and Origin
8.0 (OriginLab Corporation, Northampton, MA) software. Statistical
differences were determined using a Student’s ttest with a
significance level of P<0.05. Numerical data are presented as the
mean
SEM for at least four measurements, unless otherwise stated.
Concentration–response curve was obtained by measuring the
time constant of the inactivation (
t
h
) in sodium currents elicited by
a single-step voltage protocol, where 40 ms depolarizing test
pulses to 20 mV were applied from a holding potential (V
h
)of
100 mV every 8 s. Data were then plotted as a function of toxin
concentration and fit by the following function: y=A
1
+(A
2
A
1
)/
(1 + 10
((log IC50 x)p)
), where A
1
is the yvalue at the bottom
plateau, A
2
is the yvalue at the top plateau, log IC
50
is the xvalue
when the response is halfway between A
1
and A
2
, and pis the Hill
slope.
Current–voltage relationships and availability curves were
constructed using a standard double-pulse protocol in which, from
a holding potential of 100 mV, a 40 ms test pulse to 20 mV was
preceded by 40 ms prepulses between 120 and 70 mV at a
stimulus rate of 0.125 Hz. The peak amplitudes of the currents
were measured at the prepulse and converted to sodium chord
conductance using the following equation: G
Na
=I
Na
/(V
test
V
rev
),
where G
Na
is the sodium conductance, I
Na
is the sodium current
peak amplitude, V
test
is the test potential and V
rev
is the reversal
potential for the sodium current.
The steady-state inactivation curve (h
1
) was calculated by
dividing the current at a given prepulse by the maximum current
achieved in the test pulse (I/I
max
) and plotted as a function of the
prepulse potential. Both steady-state activation and inactivation
data were then fitted by a Boltzmann function.
To explore the state of the sodium channel on which BcIII exert
their actions, two depolarizing trains of 200 pulses to 20 mV from
aV
h
of 100 mV (pulse durationof 40 ms, pulse interval of 200 ms)
were applied. Between the first (control) and the second (test)
trains there was a rest period of 1 min, during which the cell was
held at a V
h
of 100 mV and perfused with 10
m
M BcIII.
Dissociation kinetics was measured with double-pulse proto-
cols. From a V
h
of 120 mV, a conditioning dissociation pulse to 0,
+60 or +100 mV with increasing durations was applied, returning
E. Salceda et al. / Peptides 31 (2010) 412–418
413
Author's personal copy
to 120 mV for 20 ms. I
Na
current was then elicited with a test
pulse to 20 mV. In order to normalize the change in the
maximum current amplitude and to quantify the degree of
inactivation removal by the toxin, the ratio between the current
at 5 ms after the start of the depolarization and the peak current
(I
5ms
/I
peak
) was calculated. I
5ms
/I
peak
gives an estimate of the
probability for the channels not to be inactivated after 5 ms [14]:a
zero value means full inactivation, a value of one means no
inactivation. The average values of I
5ms
/I
peak
from several
experiments were plotted as a function of the conditioning pulse
duration and fitted with a single-exponential function to obtain the
dissociation time constants (
t
off
).
3. Results
A total of 65 neurons were successfully patch-clamped for a
sufficient time to allow the study of the action of BcIII. The
capacitances of these cells formed a unimodal histogram with a
mean of 53.6
12.2 pF, which corresponds to a cell diameter of
about 41 mm.
The change in peak amplitude, and the activation and
inactivation time constants were calculated for I
Na
before and
about 2 min after toxin perfusion, time at which the maximum
effect was reached. Concentration-response curve was built using
0.3, 1, 3 and 10
m
M BcIII. For the rest of the experiments we used a
10
m
M toxin concentration because it was the concentration at
which the maximum effect was obtained.
The inactivation time course of the sodium current was well
fitted by a single-exponential function (correlation coefficient
95%). Applying the single-step voltage protocol described in
Section 2.5, the main effect of BcIII (10
m
M) was a significant
increase (n=5;P<0.05, Student’s ttest) on
t
h
of 49.2
7.3%; this
effect was concentration-dependent with an IC
50
= 2.8 0.02 mM
and a Hill slope coefficient of 0.76 0.04 (n= 20) (Fig. 1). In addition,
BcIII (10 mM) produced a significant increase (n=5; P<0.05,
Student’s ttest) in the current amplitude of 41 12%. The maximum
effect of BcIII on t
h
occurred within the first 2 min after perfusion with
toxin. Washout (5 min) removed 89 3% of its effect (10 mM). The
toxin did not affect the time course of I
Na
activation.
Current density versus voltage curves were obtained from
current–voltage relationships by normalizing ionic current ampli-
tude as a function of membrane capacitance (Fig. 2). Under control
conditions (n= 5), the maximum current density (93.4
15.9 pA/
pF) was achieved at 20 mV. Perfusion with 10 mM BcIII significantly
increased the maximum current density (132.0 15.8 pA/pF; n=5;
P<0.05, Student’s ttest). The increase in the current density caused
by BcIII was statistically significant in the voltage range between 20
and +10 mV. BcIII (10 mM) did not produce any significant change in
the reversal potential of I
Na
.
The sodium current chord conductance could be fitted by a
Boltzmann function yielding half-maximal activation potential
(V
1/2 act
)of30.8
0.9 mV under control conditions (n=5), and
32.6 1.6 mV in the presence of 10 mMBcIII(n= 5). The
calculated slopes were 6.5 0.4 and 5.3 0.6 mV, respectively
(Fig. 3A). These differences were not statistically significant
(P>0.05, Student’s ttest).
Steady-state inactivation profiles before and after BcIII (10
m
M)
were obtained with the double-pulse protocol detailed in Section
2.5.I/I
max
versus voltage curves were fitted by a Boltzmann
function. It was found that BcIII caused a nonsignificant
hyperpolarizing shift in the half-maximal inactivation potential
(V
1/2 inact
) from 60.1
0.5 mV under control conditions (n=5) to
64.8 2.4 mV in the presence of the toxin (n= 5). The calculated
slopes were 10.5 0.5 and 12.7 2.1 mV, respectively (Fig. 3B).
To explore the state of the sodium channel on which BcIII exerts
its action, two voltage pulse trains separated by a rest period were
applied (see Section 2.5). The inactivation time constant of the
sodium current evoked by each pulse was plotted against the pulse
number (Fig. 4). In control conditions
t
h
remained relatively
constant during the first 200 pulses. In contrast,
t
h
of the ionic
current produced by the first test pulse increased by 52.1
11.6%
following BcIII application, effect that was statistically significant
(n=3; P<0.05, Student’s ttest). The effectiveness of BcIII did not
increase by the remaining test pulses.
To investigate the possibility that BcIII (10
m
M) may unbind
from its receptor site in a voltage-dependent manner, a protocol of
depolarizing prepulses with increasing durations was applied. The
extent of fast inactivation removal was quantified by calculating
the ratio I
5ms
/I
peak
. It was found that the rate of displacement was a
function of the amplitude and duration of the prepulses, showing a
progressive acceleration of fast inactivation with longer condi-
tioning pulses (2.5–1000 ms) (Fig. 5). When prepulses to 0, +60 or
Fig. 1. BcIII increased the inactivation time course and the peak amplitude of
sodium current. (A) Representative experiment showing the effects of 10
m
M BcIII
about 1 min after toxin perfusion. Currents were elicited by a single-step voltage
protocol in which test pulses to 20 mV were applied from a V
h
of 100 mV every
8 s In this and the following figures, the dotted line indicates the zero current level.
Left inset is a zoom showing the increase in the current amplitude. Right inset
shows the mean 10
m
M BcIII effect on the inactivation time constant and its
recuperation after toxin washout. (B) Concentration–response curve of the effect of
BcIII (n= 20) on
t
h
. Data were fitted (solid line) by a dose–response function
yielding an IC
50
of 2.8
0.02 mM. In this and following graphs, the points represent
the mean standard error of the mean and the asterisks denote significant effects with
respect to control: **P<0.01, *P<0.05.
E. Salceda et al. / Peptides 31 (2010) 412–418
414
Author's personal copy
+100 mV were applied, the values of
t
off
were 285
42, 164 13,
and 113 8 ms, respectively (n= 3).
The kinetics of toxin dissociation has been studied for
a
-
scorpion toxins, but it has been less explored for sea anemone
toxins. Therefore, an interesting issue is to determine whether the
effect of other site-3 anemone toxins may be decreased by
depolarizing prepulses or, ultimately, whether they may be
removed from its receptor site as consequence of strong
depolarization. Thus, we decided to test whether the effects of
two other known site-3 neurotoxins (CgNa, from C. gigantea and
ATX-II, from A. sulcata (A. viridis)), at saturating concentrations, are
decreased during depolarization. It was found that the values of
t
off
were, for CgNa (10
m
M, n= 3): 457
55 and 265 33 ms for
conditioning prepulses of 0 and 60 mV, respectively; and for ATX-II
(10 mM, n= 3): 347 44 and 185 20 ms (data not shown). Results
show that these toxins also have a voltage-dependent dissociation
kinetics.
2
4. Discussion
In the present study the effects of the toxin BcIII, purified from
the venom of the sea anemone B. caissarum, on the sodium currents
in rat dorsal root ganglion cells were investigated using the whole-
cell patch clamp technique. The main action of BcIII on these
neurons was a slowing of the inactivation process of sodium
current, with no significant effects on activation kinetics. Action of
BcIII on I
Na
was dependent on the concentration with an IC
50
of
2.8
0.02 mM. BcIII showed a voltage-dependent action on the
slowing of the inactivation kinetics.
The increase in the inactivation time constant
t
h
is the most
notorious effect exerted by site-3 toxins [32,40,41]. The slowing of
inactivation in the presence of BcIII is consistent with the idea that
site-3 toxins destabilize the inactivated state of the sodium
channels. The effect of BcIII in our experiments was almost totally
removed by washout (5 min), in agreement with the data reported
by other authors about the reversibility of the effects of sea
anemone toxins upon vertebrate or insect Na
+
channels [2,9,40],in
contrast with the irreversible toxicity on crustacean Na
+
channels
[40].
Many gating models predict that the peak Na
+
current would be
greater when inactivation is slowed or removed. Our results are in
accordance with this prediction since BcIII increased the maximum
Na
+
current amplitude at all test potentials. It has been
demonstrated that scorpion
a
-toxins like Lqh-II, Lqh-III, and
Lqh
a
IT (purified from Leiurus quinquestriatus hebraeus) increased
the peak Na
+
currents of the rat skeletal muscle sodium channels
[14]. This effect could be a consequence of a prolonged open time of
individual Na
+
channels. However, other site-3 toxins do not affect
the peak sodium current or even decrease it. For example, many
scorpion
a
-toxins only slightly increase or decrease the peak
currents of Na
+
channels [19]. The same has been observed for ATX-
II and LqTx (from the scorpion L. quinquestriatus) when applied on
Na
v
1.2 channels [35]. Similarly, the sea anemone toxins BgII, BgIII
(both from Bunodosoma granulifera), ApC (from Anthopleura
elegantissima) and CgNa (from C. gigantea) did not affect the
current amplitude when applied to DRG neurons [37–39].
Therefore it is possible that some site-3 toxins may also produce
some degree of channel blockade.
Perfusion with BcIII did not produce a significant change in the
reversal potential, which indicates that the ion selectivity of Na
+
channels is not altered by this toxin. This is a marked difference
with respect to the actions of site-2 toxins such as batrachotoxin,
which also remove inactivation but significantly decrease the
selectivity of the sodium channel [22,47].
In previous works [37–39] we have shown that other anemone
toxins, when applied to DRG neurons, shifted the steady-state
inactivation curve to hyperpolarizing values and caused a decrease
in the voltage dependence of sodium channel inactivation by
increasing the slope factor of the h
1
curve. This latter effect has
been also observed with site-3 toxins from scorpions [14], and
wasps [36]. In the present work we have found that, in the presence
of BcIII, there was a consistent tendency to shift to the left (i.e., to
more hyperpolarized potentials) the voltage at which half of the
channels are inactivated; however, this change was not statisti-
cally significant. The absence of effect on V
1/2 inact
is not an
exclusive property of BcIII: ATX-II does not induce a significant
shift of the h
1
curve when applied on hH1 (human heart subtype 1)
or rSkM1 (rat skeletal muscle subtype 1) Na
+
channels [11]. Thus,
some of the effects of site-3 toxins could be highly dependent on
the sodium channel isoform and not on the characteristics of the
toxins themselves. In fact, two previous studies about the actions
of three anemone toxins (including BcIII) on different Na
+
channels
subtypes (from Na
v
1.1 to Na
v
1.7) have shown differences in
the manner in which each toxin affect the diverse isoforms of the
voltage-gated sodium channel, including their actions on the
steady-state voltage-dependent inactivation [31,48].
To explore the state of the sodium channel on which BcIII exert
its effects, a post-resting voltage protocol was applied. In the
presence of the toxin, the
t
h
of the current elicited by the first pulse
Fig. 2. Effects of BcIII on the current density versus voltage curve. (A) Sodium
currents produced at different potentials (for clarity, only the first 4 ms of each
record is presented) in the absence (top) and in the presence (bottom) of 10
m
M
BcIII. Currents were produced by 40 ms voltage pulses to the potentials indicated on
top from a holding potential of 100 mV. BcIII affected the currents at all the shown
voltages. (B) Current density versus voltage relationships (n= 5). Perfusion with
10
m
M BcIII (closed circles) did not produce significant changes either on the
voltage at which the maximum current density was reached or on the reversal
potential.
2
In our experimental conditions, the viability of the neurons was greatly reduced
when long prepulses at +100 mV were applied. For this reason, and because our
supply of toxins was limited, CgNa and ATX-II were not tested at this voltages.
E. Salceda et al. / Peptides 31 (2010) 412–418
415
Author's personal copy
after a rest period increased as compared to the
t
h
measured in the
control train. This result indicate that BcIII has a high affinity for
the closed state of the channel, as has been suggested for other site-
3 toxins [13,14,39] and, accordingly, the toxin shows no preference
for the sodium channel in the open state.
It has been reported that the binding affinity of site-3 toxins is
decreased by depolarization [4,16], and a number of electrophysi-
ological studies have shown that the rate at which
a
-scorpion
toxins dissociate from its receptor site is voltage-dependent
[3,4,7,14,26,27,34,35,46,49]. Only a few of these works have shown
an analogous voltage-dependent dissociation for sea anemone
toxins (generally for ATX-II, from A. viridis [46,49]), and since it has
been proposed that nonidentical amino acids of the IVS3–S4 linker
in the Na
+
channel participate in
a
-scorpion and sea anemone
toxins binding to overlapping sites [35], a question to be answered
is whether sea anemone toxins may also be removed from its
receptor site as a consequence of depolarization. It was found that,
with some quantitative differences probably attributable to the
charge of the toxin molecules, the effect of BcIII, CgNa and ATX-II
on
t
h
was progressively decreased with longer conditioning pulses,
and that the acceleration of fast inactivation was steeper as more
depolarizing were the prepulses. As has been pointed elsewhere
[13], several factors may affect the toxin affinity to voltage-gated
Na
+
channels including the channel activation, fast and slow
inactivation and membrane voltage. In our experiments BcIII did
not affect the channel activation and hence there is only a small
probability of a direct coupling between the toxin binding and the
molecular substrate of Na
+
channel activation. Toxins could be
removed by membrane voltage only by being in a position where
they could sense the electrical field across the membrane, i.e.,
immersed into the membrane structure itself [3]; however, given
the structural characteristics of site-3 toxins (a compact folding
stabilized by three disulphide bridges, and patches of exposed
negatively charged residues), this seems an unlikely explanation.
Studies using liposomes containing phosphatidylserine and
phosphatidylcholine showed that ApB toxin (from Anthopleura
xanthogrammica) does not immerse into the membrane, contrary
to the
b
-scorpion peptides, suggesting that the ApB interaction
with sodium channel takes place in the extracellular region of the
channel [43]. Most probably, structural torsions of the sodium
channels upon strong depolarization may contribute to displace
the peptides from their binding sites. Our experiments do not
discard, however, the possibility of a toxin interaction with the
slow inactivated state, but a direct experimental proof about this is
difficult to achieve [13].
From the structural point of view, not only the positively
charged amino acid residues (K/R35, K36) are important for
binding of the sea anemone Type 1 toxins to sodium channels
[21,31,35]. Comparing the primary sequence of CgNa, ATX-II and
BcIII (Fig. 6), a negatively charged patch (D37 and E38) in CgNa do
not affect the potency of this toxin, neither its mode of action on
voltage-gated sodium channel. As a consequence, we may assume
that the docking of each peptide to the channels should have the
Fig. 3. Effects of BcIII on the voltage-dependence of I
Na
conductance and steady-state inactivation. (A) G/G
max
(normalized conductance) versus voltage curves before and after
10
m
M BcIII. Continuous curves were obtained by fitting the data with a Boltzmann function yielding half-maximal activation of 30.8
0.9 mV under control conditions
(n= 5), and 32.6 1.6 mV in the presence of 10 mM BcIII (n= 5). The calculated slopes were 6.5 0.4 and 5.3 0.6 mV, respectively. (B) Steady-state inactivation before and after
10 mM BcIII. The steady-state inactivation parameter (h
1
) was determined using the two-pulse protocol described in Section 2.5. BcIII caused a nonsignificant hyperpolarizing shift
in the half-maximal inactivation potential from 60.1 0.5 mV under control conditions (n=5)to64.8 2.4 mV in the presence of the toxin (n= 5). The calculated slopes were
10.5 0.5 and 12.7 2.1 mV, respectively.
Fig. 4. BcIII acts on voltage-gated sodium channels in the closed state. (A) The toxin
was applied at the beginning of the rest period (1 min) represented by the break. (B)
Temporal course of the inactivation time constant
t
h
in a typical experiment. The
maximum effect of BcIII was already evident from the first pulse after the rest
period. The bar indicates perfusion with 10
m
M BcIII.
E. Salceda et al. / Peptides 31 (2010) 412–418
416
Author's personal copy
contribution of distinct amino acids in the contact surface. This
aspect should be considered in more detail in future studies.
Further experiments are necessary to determine whether our
observations have the same biophysical meaning that has been
suggested for
a
-scorpion toxins by Campos et al. [3]. However,
given that BcIII, CgNa and ATX-II modify the fast inactivation by
binding to the closed state of Na
+
channels, and that site-3 toxin
binding mainly occurs in the S3–S4 extracellular linker in domain
IV of the Na
+
channel [35,8] and evidence associates the S4
segment in domain IV with the fast inactivation process
[10,12,15,20,42], it is plausible that when depolarizing prepulses
are applied, the electrical field on DIV-S4 reduces the binding
strength of the receptor site.
Acknowledgments
Part of this work was supported by CONACyT (Consejo Nacional
de Ciencia y Tecnologı
´a, Me
´xico) grant I0110/127/08 and by the
CNPq (Conselho Nacional de Desenvolvimento Cientı
´fico e
Tecnolo
´gico) grant 563874/2005-8.
References
[1] Benzinger GR, Kyle JW, Blumenthal M, Hanck DA. A specific interaction
between the cardiac sodium channel and site-3 toxin Anthopleurin B. J Biol
Chem 1998;273:80–4.
[2] Bosmans F, Aneiros A, Tytgat J. The sea anemone Bunodosoma granulifera
contains surprisingly efficacious and potent insect-selective toxins. FEBS Lett
2002;532:131–4.
[3] Campos FV, Coronas FIV, Beira
˜o PSL. Voltage-dependent displacement of the
scorpion toxin Ts3 from sodium channels and its implication on the control of
inactivation. Br J Pharm 2004;142:1115–22.
[4] Catterall WA. Membrane potential-dependent binding of scorpion toxin to the
action potential Na
+
ionophore. Studies with a toxin derivative prepared by
lactoperoxidase-catalyzed iodination. J Bio Chem 1977;252:8660–8.
[5] Catterall WA. Structure and function of voltage-gated ion channels. Annu Rev
Biochem 1995;64:493–531.
[6] Catterall WA. From ionic currents to molecular mechanisms: the structure and
function of voltage-gated sodium channels. Neuron 2000;26:13–25.
[7] Catterall WA, Ray R, Morrow CS. Membrane potential dependent binding of
scorpion toxin to action potential Na
+
ionophore. Proc Natl Acad Sci USA
1976;73:2682–6.
[8] Ceste
`le S, Caterall WA. Molecular mechanisms of neurotoxin action on voltage-
gated sodium channels. Biochimie 2000;82:883–92.
[9] Ceste
`le S, Kopeyan C, Oughideni R, Mansuelle P, Granier C, Rochat H. Biochem-
ical and pharmacological characterization of a depressant insect toxin from the
venom of the scorpion Buthacus arenicola. Eur J Biochem 1997;243:93–9.
[10] Chahine M, George Jr AL, Zhou M, Ji S, Sun W, Barchi RL, et al. Sodium channel
mutations in paramyotonia congenita uncouple inactivation from activation.
Neuron 1994;12:281–94.
[11] Chahine M, Plante E, Kallen RG. Sea anemone toxin (ATX II) modulation of
heart and skeletal muscle sodium channel
a
-subunits expressed in tsA201
cells. J Membrane Biol 1996;152:39–48.
[12] Chanda B, Bezanilla F. Tracking voltage-dependent conformational changes in
skeletal muscle sodium channel during activation. J Gen Physiol 2002;120:
629–45.
[13] Chen H, Heinemann SH. Interaction of scorpion
a
-toxins with cardiac sodium
channels: binding properties and enhancement of slow inactivation. J Gen
Physiol 2001;117:505–18.
[14] Chen H, Gordon D, Heinemann SH. Modulation of cloned skeletal muscle
sodium channels by the scorpion toxins Lqh II, Lqh III and Lqh
a
IT. Pflu
¨gers
Arch 2000;439:423–32.
[15] Chen LQ, Santarelli V, Horn R, Kallen RG. A unique role for the S4 segment of
domain 4 in the inactivation of sodium channels. J Gen Physiol 1996;108:549–
56.
[16] Couraud F, Rochat H, Lissitzky S. Binding of scorpion and sea anemone
neurotoxins to a common site related to the action potential Na
+
ionophore
in neuroblastoma cells. Biochem Biophys Res Commun 1978;83:1525–30.
[17] Fernandez JM, Fox AP, Krasne S. Membrane patches and whole-cell mem-
branes: a comparison of electrical properties in rat clonal pituitary (GH3) cells.
J Physiol 1984;356:565–85.
[18] Fletcher JI, Chapman BE, Mackay JP, Howden ME, King GF. The structure of
versutoxin (
d
-atracotoxin-Hv1) provides insights into the binding of site 3
neurotoxins to the voltage-gated sodium channel. Structure 1997;5:1525–35.
[19] Gordon D, Martin-Euclaire MF, Ceste
`le S, Kopeyan C, Carlier E, Khalifa RB, et al.
Scorpion toxins affecting sodium current inactivation bind to distinct homol-
ogous receptor sites on rat brain and insect sodium channels. J Biol Chem
1996;271:8034–45.
[20] Horn R, Ding S, Gruber HJ. Immobilizing the moving parts of voltage-gated ion
channels. J Gen Physiol 2000;116:461–76.
[21] Khera PK, Blumenthal KM. Importance of highly conserved anionic residues
and electrostatic interactions in the activity and structure of the cardiotonic
polypeptide anthopleurin B. Biochemistry 1996;35:3503–7.
[22] Khodorov BI. Chemicals as tools to study nerve fiber sodium channels: effect of
batrachotoxin and some local anesthetics. Prog Biophys Mol Biol 1978;45:
153–74.
Fig. 5. The effect of BcIII is gradually removed by depolarization. (A) Superimposed current traces for a toxin-dissociation experiment in the presence of BcIII (10
m
M). The
inset shows the pulse protocol used. The superimposed traces show the progressive acceleration of inactivation caused by prepulses of different durations. For clarity, only
five traces (2.5, 20, 50, 300 and 700 ms) are shown. Arrows show the currents obtained after conditioning pulses of 2.5 and 700 ms. (B) Dissociation curves obtained at the
indicated prepulse voltages. Curves were fitted using a single-exponential function (continuous line) yielding the time constant,
t
off
, for each conditioning voltage. The
horizontal lines indicate the mean
1 SD in the absence of toxin.
Fig. 6. Primary sequence alignment of the Type 1 sodium channel toxin BcIII (on top) with analogous peptide toxins ATX-II and CgNa. Black, gray and white backgrounds
indicate identical, homologous and different amino acids, respectively. Cysteines are bonded as C1–C5, C2–C4 and C3–C6.
E. Salceda et al. / Peptides 31 (2010) 412–418
417
Author's personal copy
[23] Kinoshita E, Maejima H, Yamaoka K, Konno K, Kawai N, Shimizu E, et al. Novel
wasp toxin discriminates between neuronal and cardiac sodium channels. Mol
Pharmacol 2001;59:1457–63.
[24] Little MJ, Zappia C, Gilles N, Connor M, Tyler MI, Martin-Eauclaire MF, et al.
d
-
Atracotoxins from Australian funnel-web spiders compete with scorpion
a
-
toxin binding but differentially modulate alkaloid toxin activation of voltage-
gated sodium channels. J Biol Chem 1998;273:27076–83.
[25] Meves H, Simard JM, Watt DD. Interactions of scorpion toxins with the sodium
channel. Ann NY Acad Sci 1986;479:113–32.
[26] Mozhayeva GN, Naumov AP, Grishin EV, Soldatov NM. Effect of the toxins of
the scorpion Buthus eupeus on the sodium channels of the membrane of the
node of Ranvier. Biophysics 1979;4:242–9.
[27] Mozhayeva GN, Naumov AP, Nosyreva ED, Grishin EV. Potential-dependent
interaction of toxin from venom of the scorpion Buthus eupeus with sodium
channels in myelinated fibre: voltage clamp experiments. Biochim Biophys
Acta 1980;597:587–602.
[28] Narahashi T, Moore JW, Shapiro BI. Condylactis toxin: interaction with nerve
membrane ionic conductances. Science 1969;163:680–1.
[29] Nicholson GM, Willow M, Howden MEH, Narahashi T. Modification of sodium
channel gating and kinetics by versutoxin from the Australian funnel-web
spider Hadronyche versuta.Pflu
¨gers Arch 1994;428:400–9.
[30] Norton RS. Structure and structure–function relationships of sea
anemone proteins that interact with the sodium channel. Toxicon 1991;
29:1051–84.
[31] Oliveira JS, Redaelli E, Zaharenko AJ, Cassulini RR, Konno K, Pimenta DC, et al.
Binding specificity of sea anemone toxins to Na
v
1.1–1.6 sodium channels:
unexpected contributions from differences in the IV/S3–S4 outer loop. J Biol
Chem 2004;279:33323–35.
[32] Pelhate M, Hue B, Sattelle DB. Pharmacological properties of axonal sodium
channels in the cockroach Periplaneta americana L. II. Slowing of sodium
current turnoff by Condylactis toxin. J Exp Biol 1979;83:49–58.
[33] Possani LD, Becerril B, Delepierre M, Tytgat J. Scorpion toxins specific for Na
+
channels. Eur J Biochem 1999;264:287–300.
[34] Ray R, Catterall WA. Membrane potential dependent binding of scorpion
toxin to the action potential sodium ionophore. Studies with a 3-(4-
hydroxy 3-[
125
I] iodophenyl) propionyl derivative. J Neurochem 1978;
31:397–407.
[35] Rogers JC, Qu Y, Tanada TN, Scheuer T, Catterall WA. Molecular determinants
of high affinity binding of
a
-scorpion toxin and sea anemone toxin in the S3–
S4 extracellular loop in domain IV of the Na
+
channel
a
subunit. J Biol Chem
1996;271:15950–62.
[36] Sahara Y, Gotoh M, Konno K, Miwa A, Tsubokawa H, Robinson HPC, et al. A new
class of neurotoxin from wasp venom slows inactivation of sodium current.
Eur J Neurosci 2000;12:1961–70.
[37] Salceda E, Garateix A, Soto E. The sea anemone toxins BgII and BgIII prolong the
inactivation time course of the tetrodotoxin-sensitive sodium current in rat
dorsal root ganglion neurons. J Pharmacol Exp Ther 2002;303:1067–74.
[38] Salceda E, Garateix A, Aneiros A, Salazar H, Lo
´pez O, Soto E. Effects of ApC, a sea
anemone toxin, on sodium currents of mammalian neurons. Brain Res
2006;1110:136–43.
[39] Salceda E, Pe
´rez-Castells J, Lo
´pez-Me
´ndez B, Garateix A, Salazar H, Lo
´pez O,
et al. CgNa, a type I toxin from the giant Caribbean sea anemone Condylactis
gigantea shows structural similarities to both type I and II toxins, as well as
distinctive structural and functional properties. Biochem J 2007;406:67–76.
[40] Salgado VL, Kem WR. Actions of three structurally distinct sea anemone toxins
on crustacean and insect sodium channels. Toxicon 1992;30:1365–81.
[41] Shapiro BI, Lilleheil G. The action of anemone toxin on crustacean neurons.
Comp Biochem Physiol 1969;28:1225–41.
[42] Sheets MF, Kyle JW, Kallen RG, Hanck DA. The Na
+
channel voltage sensor
associated with inactivation is localized to the external charged residues of
domain IV, S4. Biophys J 1999;77:747–57.
[43] Smith JJ, Alphy S, Seibert AL, Blumenthal KM. Differential phospholipid binding
by site 3 and site 4 toxins. Implications for structural variability between
voltage-sensitive sodium channel domains. J Biol Chem 2005;280:11127–33.
[44] Sta
¨ndker L, Beress L, Garateix A, Christ T, Ravens U, Salceda E, et al. A new toxin
from the sea anemone Condylactis gigantea with effect on sodium channel
inactivation. Toxicon 2006;48:211–20.
[45] Strichartz G, Rando T, Wang GK. An integrated view of the molecular toxinol-
ogy of sodium channel gating in excitable cells. Annu Rev Neurosci 1987;10:
237–67.
[46] Strichartz GR, Wang GK. Rapid voltage-dependent dissociation of scorpion
a
-
toxins coupled to Na channel inactivation in amphibian myelinated nerves. J
Gen Physiol 1986;88:413–35.
[47] Wang SY, Mitchell J, Tikhonov DB, Zhorov BS, Wang GK. How batrachotoxin
modifies the sodium channel permeation pathway: computer modeling and
site-directed mutagenesis. Mol Pharmacol 2006;69:788–95.
[48] Wanke E, Zaharenko AJ, Redaelli E, Schiavon E. Actions of sea anemone type 1
neurotoxins on voltage-gated sodium channel isoforms. Toxicon 2009;54:
1102–11.
[49] Warashina A, Fujita S, Satake M. Potential-dependent effects of sea anemone
toxins and scorpion venom on crayfish giant axon. Pflu
¨gers Arch 1981;391:
273–6.
E. Salceda et al. / Peptides 31 (2010) 412–418
418
