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The Sea Anemone Toxins BgII and BgIII Prolong the
Inactivation Time Course of the Tetrodotoxin-Sensitive Sodium
Current in Rat Dorsal Root Ganglion Neurons
EMILIO SALCEDA, ANOLAND GARATEIX, and ENRIQUE SOTO
Instituto de Fisiologı´a, Universidad Auto´ noma de Puebla, Me´ xico (Em.S., En.S.); and Instituto de Oceanologı´a, Ministerio de Ciencia,
Tecnologia y Medio Ambieute, La Habana, Cuba (A.G.)
Received May 23, 2002; accepted September 4, 2002
ABSTRACT
We have characterized the effects of BgII and BgIII, two sea
anemone peptides with almost identical sequences (they only
differ by a single amino acid), on neuronal sodium currents
using the whole-cell patch-clamp technique. Neurons of dorsal
root ganglia of Wistar rats (P5-9) in primary culture (Leibovitz⬘s
L15 medium; 37°C, 95% air/5% CO
2
) were used for this study
(n⫽154). These cells express two sodium current subtypes:
tetrodotoxin-sensitive (TTX-S; K
i
⫽0.3 nM) and tetrodotoxin-
resistant (TTX-R; K
i
⫽100
M). Neither BgII nor BgIII had
significant effects on TTX-R sodium current. Both BgII and BgIII
produced a concentration-dependent slowing of the TTX-S
sodium current inactivation (IC
50
⫽4.1 ⫾1.2 and 11.9 ⫾1.4
M, respectively), with no significant effects on activation time
course or current peak amplitude. For comparison, the concen-
tration-dependent action of Anemonia sulcata toxin II (ATX-II), a
well characterized anemone toxin, on the TTX-S current was
also studied. ATX-II also produced a slowing of the TTX-S
sodium current inactivation, with an IC
50
value of 9.6 ⫾1.2
M
indicating that BgII was 2.3 times more potent than ATX-II and
2.9 times more potent than BgIII in decreasing the inactivation
time constant (
h
) of the sodium current in dorsal root ganglion
neurons. The action of BgIII was voltage-dependent, with sig-
nificant effects at voltages below ⫺10 mV. Our results suggest
that BgII and BgIII affect voltage-gated sodium channels in a
similar fashion to other sea anemone toxins and
␣
-scorpion
toxins.
Voltage-dependent sodium channels have receptor sites
that may be recognized by different groups of neurotoxins
(Adams and Olivera, 1994; Trainer et al., 1994). For this
reason, this kind of compound has been shown to be a useful
pharmacological tool for studying the functional and struc-
tural mapping of sodium channel proteins (Catterall, 2000).
Toxins that act on sodium channels can be classified in two
main groups according to their pharmacological effects on the
channel: blockers (e.g., tetrodotoxin and
-conotoxin) and
modulators (e.g., batrachotoxin,
␣
- and

-scorpion toxins, and
sea anemone toxins). The latter is further divided into sev-
eral classes based upon the effects on channel activation and
inactivation kinetics (Narahashi, 1998).
A large number of neurotoxins have modulatory actions on
Na
⫹
channel function by modifying the processes linked to
channel activation and inactivation, ionic selectivity, and
other properties involved in action potential generation. At
least six neurotoxin receptor sites have been identified on the
mammalian sodium channel (Strichartz et al., 1987; Catter-
all, 1995). Anemone peptide neurotoxins and
␣
-scorpion tox-
ins share receptor site 3 on sodium channels (Couroud et al.,
1978; Catterall, 1995, 2000; Gordon et al., 1998), which in-
volves the extracellular loops IS5-S6, IVS3-S4, and IVS5-S6
of the ionic channel (Rogers et al., 1996). Anemone toxins are
generally smaller than the structurally unrelated
␣
-scorpion
toxins and have three rather than four disulfide bridges
(Norton, 1997); nevertheless, both groups bind to site 3, ex-
hibit similar pharmacological properties, displace one an-
other from their binding site, and their main effect is to delay
channel inactivation, resulting in a prolongation of the action
potential.
Bunodosoma granulifera is an anemone species very com-
mon at the Cuban seashores. Several active compounds with
pharmacological actions on ionic channels have been isolated
from its secretions (Aneiros et al., 1993; Loret et al., 1994;
Dauplais et al., 1997; Salinas et al., 1997; Alessandri-Haber
et al., 1999; Garateix et al., 2000). Among them, BgII and
BgIII are two peptide toxins (molecular masses: 5072 and
5073, respectively) with almost identical sequences (they
only differ by a single amino acid), causing toxicity in mice
This work was supported by CONACyT Grant E120.1869/2000
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.038570.
ABBREVIATIONS: DRG, dorsal root ganglion; TTX-S, tetrodotoxin-sensitive; TTX-R, tetrodotoxin-resistant; L15, Leibovitz⬘s L15 medium;
h
,
inactivation time constant; ATX-II, Anemonia sulcata toxin II; G
Na
, peak sodium conductance.
0022-3565/02/3033-1067–1074$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 38570/1026540
JPET 303:1067–1074, 2002 Printed in U.S.A.
1067
when injected intracerebroventricularly and markedly differ-
ent binding to rat brain synaptosomes; both effects are
higher for BgII (Loret et al., 1994).
In this work, we have characterized the effects of BgII and
BgIII toxins on neuronal sodium currents. Rat dorsal root
ganglion (DRG) neurons were chosen for this study since
these cells express two sodium current subtypes: a tetrodo-
toxin-sensitive (TTX-S) sodium current, which is readily
blocked by TTX (K
I
⫽0.3 nM), and a tetrodotoxin-resistant
(TTX-R) sodium current, which is highly resistant to TTX (K
i
⫽100
M) (Roy and Narahashi, 1992; Novakovik et al.,
2001). To our knowledge, this is the first electrophysiological
characterization of the action of these toxins on neuronal
cells.
Materials and Methods
Animal care and procedures were carried out in accordance with
the Declaration of Helsinki. The number of animals used for this
work was kept to the minimum necessary for a meaningful interpre-
tation of the data.
Toxins. BgII and BgIII were isolated and purified from sea anem-
one B.granulifera, as previously described (Aneiros et al., 1993;
Loret et al., 1994). Some experiments were performed with ATX-II
obtained from the sea anemone Anemonia sulcata (a gift from Pro-
fessor L. Beress, Kiel, Germany). TTX was obtained from Sigma-
Aldrich (St. Louis, MO). Aliquots of stock solution in deionized water
were prepared and stored in a freezer (⫺20°C). Before each experi-
ment, they were dissolved in the perfusion solution.
Cell Preparation. Young Wistar rats (P5-9) of either sex were
anesthetized with ether and subsequently decapitated. DRGs were
isolated from the vertebral column and incubated (30 min at 37°C) in
Leibovitz⬘s L15 medium (L15) (Invitrogen, Carlsbad, CA) containing
1.125 mg/ml trypsin and 1.125 mg/ml collagenase (both from Sigma-
Aldrich). Following enzyme treatment, the ganglia were washed 3
times with sterile L15 and mechanically dissociated. The cells were
then plated onto 35-mm culture dishes (Corning, Corning, NY) con-
taining 12 ⫻10-mm glass coverslips (Corning) previously coated
with poly-D-lysine (Sigma-Aldrich). Neurons were incubated 4 to 6 h
in a humidified atmosphere (95% air/5% CO
2
at 37°C, using a CO
2
water-jacketed incubator; Nuaire, Plymouth, MN) to allow the iso-
lated cells to settle and adhere to the coverslips. The incubation
medium (pH 7.4) contained L15, 15.7 mM NaHCO
3
(Merck, Naucal-
pan, Mexico), 10% fetal bovine serum, 2.5
g/ml Fungizone (both
from Invitrogen), 100 U/ml penicillin (Lakeside, Toluca, Mexico), and
15.8 mM HEPES (Sigma-Aldrich).
Electrophysiological Recording. A coverslip with attached
neurons was transferred to a 500-
l perfusion chamber mounted on
the stage of an inverted phase-contrast microscope (Nikon Diaphot,
Tokyo, Japan). Cells were bathed with an external solution contain-
ing 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.
The pH of this solution was adjusted to 7.4 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 around 100
l/min. In addition to this perfusion system, a
double-barrel array built up with borosilicate glass capillaries
(TW120–3; WPI, Sarasota, FL) was placed approximately 40
m
above the cell under study; each barrel was coupled to an indepen-
dent syringe driven by a Baby Bee pump (BAS, West Lafayette, IN).
Through this system, the neuron was continuously microperfused
(10
l/min) with external solution or with external solution plus
toxin. Some experiments were designed to study the effects of BgII or
BgIII on TTX-R sodium currents. To achieve this, TTX (300 nM) was
added to both bath and microperfusion solutions.
The whole-cell patch-clamp technique was used to record ionic
currents. Patch pipettes were pulled from borosilicate glass capillar-
ies (TW120–3; WPI), using a Flaming-Brown electrode puller (P80/
PC; Sutter Instruments, San Rafael, CA), which had resistances of
0.9 to 1.8 M⍀when filled with internal solution. Pipette solution
contained 10 mM NaCl, 100 mM CsF, 30 mM CsCl, 10 mM TEA-Cl,
8 mM EGTA, and 5 mM HEPES. The pH of this solution was
adjusted to 7.3 with CsOH. Osmolarity was adjusted to 300 mOsm.
The internal solution was filtered on the day of use with a 0.22-
m
pore size syringe filter (Millipore, Bedford, MA).
To measure ionic currents, an Axopatch-1D amplifier (Axon In-
struments, Foster City, CA) was used. Command pulse generation
and data sampling were controlled by the PClamp 8.0 software (Axon
Instruments) using a 16-bit data acquisition system (Digidata
1320A; Axon Instruments). Signals were low-pass filtered at 5 kHz
and digitized at 20 kHz. Leakage and capacitive currents were dig-
itally subtracted with the P-P/2 method. Capacitance and series
resistance (80%) were electronically compensated. Experiments were
rejected when, at the maximum peak current, the voltage error
exceeded 5 mV after compensation of series resistance. No correc-
tions were made for smaller values.
The type of sodium current present in the cell under study was
determined before each experiment. In accordance with the criterion
used by Strachan et al. (1999), only those cells with ⬍10% TTX-R
sodium current, as derived from a steady-state inactivation profile,
were accepted to determine the effects of BgII and BgIII on TTX-S
sodium currents. Experiments were performed at room temperature
(23–25°C).
Data Analysis. Recordings were analyzed off-line using PClamp
8.0 and Origin software (Microcal Software, Northampton, MA).
Statistical differences were determined using a Student’sttest with
p⬍0.05. Curve-fitting routines were performed using a nonlinear
least-squares method. Numerical data are presented as the mean ⫾
S.E. for at least four measurements.
Concentration-response curves were obtained by measuring the
parameters under study in sodium currents elicited by a single-step
voltage protocol, where 40 ms depolarizing test pulses to ⫺10 mV
were applied from a holding potential of ⫺90 mV every 8 s. Data
were then plotted as a function of toxin concentration and fit by the
following function.
y⫽A1⫹A2⫺A1
1⫹10共log IC50 ⫺x兲䡠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.
To test whether BgII or BgIII showed a use-dependent action,
from a holding potential of ⫺90 mV repetitive pulses to ⫺10 mV were
applied at frequencies of 0.1, 1 and 5 Hz. Current-voltage relation-
ships and availability curves were constructed using a standard
double-pulse protocol; from a holding potential of ⫺100 mV, a 40-ms
test pulse to ⫺10 mV was preceded by 40-ms prepulses between
⫺100 and 70 mV (time interval between sweeps ⫽8 s).
The peak amplitudes of the currents were measured at the pre-
pulse and converted to sodium conductance by means of the following
equation:
GNa ⫽INa
Vtest ⫺Vrev
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 poten-
tial for the sodium current. Normalized conductance curves were
then plotted and fit by a Boltzmann distribution with the following
function.
GNa
Gmax ⫽1
1⫹exp
冉
Vtest ⫺V1/ 2
k
冊
1068 Salceda et al.
where G
max
is the maximum sodium conductance, V
1/2
is the test
potential that activates 50% of the sodium channels, kis the slope of
the curve, and the other terms are as above.
The steady-state inactivation parameter (h
⬁
) was calculated by
dividing the current achieved following a given prepulse by the
maximum current achieved in the test pulse. The results of such
operation were plotted as a function of the prepulse potential and
were fitted by the following Boltzmann function.
h⬁⫽1
1⫹exp
冉
Vpre ⫺V1/ 2 inact
k
冊
where V
pre
is the prepulse potential, V
1/2 inact
is the prepulse poten-
tial at which h
⬁
is 0.5, and kis the slope of the curve at this
mid-point.
The effects of BgII and BgIII on the rate at which sodium channels
recover from inactivation were investigated using a two-pulse proto-
col with a variable interpulse interval (⌬t) as follows: from a holding
potential of ⫺100 mV, a 40-ms conditioning prepulse to ⫺10 mV was
used to inactivate sodium channels, after which a 2.5-ms depolariz-
ing test pulse to ⫺10 mV was applied. The interpulse interval be-
tween the conditioning and the test pulses was varied between 1.25
and 80 ms. The peak current recorded during the test pulse was
normalized against the current amplitude during the conditioning
prepulse and plotted as a function of ⌬t. Data obtained from this
protocol were fitted by a single exponential function.
Results
A total of 154 neurons (mean capacity ⫽52.5 ⫾18 pF, S.D.)
were successfully voltage-clamped for a sufficient time to
allow the study of BgII, BgIII, and ATX-II actions. The ca-
pacitances of the DRG neurons used for this study formed a
unimodal histogram with the mean, which corresponds to a
cell diameter of about 41
m. This distribution represents
only the cells selected for recording, basically neurons with a
medium size cell body (probably A

neurons).
The percentage of change in peak amplitude and activation
and inactivation time constants were calculated for ionic
currents from both TTX-S and -R sodium currents before and
about 1 min after perfusion with toxin. For the TTX-S cur-
rent subtype, concentration-response curves were obtained
using concentrations of 0.1, 0.3, 1, 3, 10, and 30
M. For the
rest of the experiments in this study, we used a 10
M toxin
concentration.
To study the effects of BgII and BgIII on TTX-R sodium
currents, TTX (300 nM) was added to both bath and mi-
croperfusion solutions (n⫽15). This procedure made evident
the existence of two subtypes of TTX-R current: 1) a slowly
activating (
⫽0.46 ⫾0.2 ms) and slowly inactivating (
⫽
4.55 ⫾1.96 ms) current that activates at about ⫺40 mV (n⫽
7); and 2) a current that fails to inactivate, giving rise to a
large late component (n⫽8). These two types of TTX-R
currents coincide with those previously described in DRG
neurons (Bossu and Feltz, 1984; Baker and Wood, 2001).
Neither 10
M BgII (n⫽9) nor 10
M BgIII (n⫽6) had
significant effects (p⬎0.05, Student’sttest) on either TTX-R
sodium current subtypes (Fig. 1).
Both BgII (n⫽87) and BgIII (n⫽22) produced a concen-
tration-dependent effect on the TTX-S sodium current inac-
tivation time course, with no significant effects (p⬎0.05,
Student’sttest) on activation time course or current peak
amplitude (Fig. 2). The inactivation time course of TTX-S
sodium currents was adjusted with an exponential function
over the following 10 ms after peak current. In the presence
of 3
M BgII, the inactivation time constant (
h
) increased
97.6 ⫾35.4%, whereas a 10
M toxin concentration produced
154.2 ⫾17.4% increase in
h
. The IC
50
value for BgII was
4.1 ⫾1.2
M, with a fixed slope value of 1.0. In contrast, the
action of BgIII was less effective; at 3
M,
h
was increased
15.2 ⫾8.5%, whereas at 10
M,
h
increased 60.9 ⫾13.0%.
The IC
50
value for BgIII experiments was 11.9 ⫾1.4
M,
with a fixed slope of 1.0.
To compare the effect of these toxins with ATX-II, a well
Fig. 1. Effects of 10
M BgII (A and C) and BgIII (B and D) on TTX-R sodium currents (n⫽15). Recordings were made in presence of 300 nM TTX.
Currents were elicited by a single-step voltage protocol (40-ms depolarizing test pulses to ⫺10 mV from a holding potential of ⫺90 mV every 8 s). Two
subtypes of TTX-R current were found, partially inactivating currents with a time constant
⫽4.55 ⫾1.96 ms (A and B; n⫽7) and very slow
activating currents (
⫽1.38 ⫾0.2 ms) failing to inactivate (C and D; n⫽8). For each case, two superimposed traces are presented, under control
conditions and approximately 2 min after toxin perfusion. Neither BgII (n⫽9) nor BgIII (n⫽6) produced significant effects on either TTX-R sodium
current subtypes.
BgII and BgIII Anemone Toxins 1069
characterized sea anemone toxin, an experimental series was
done to study the effect of ATX-II on the DRG neurons (Fig.
2, C and D). ATX-II increased the inactivation time constant
of the Na
⫹
TTX-S current with an IC
50
value of 9.6 ⫾1.3
M,
indicating that BgII is 2.3 more potent than ATX-II and 2.9
times more potent than BgIII in decreasing the
h
of the Na
⫹
current. The maximum effect of both BgII and BgIII on the
TTX-S sodium current inactivation time course was always
achieved within the first minute after perfusion with either
toxin, and once reached, it was stable throughout the expo-
sure period to these agents (about 100 s). Washout of toxin
effects was complete for both BgII and BgIII at 10
M and
took less than a minute (see inserts in Fig. 2). The slowing in
the inactivation time course elicited by 10
M BgII (n⫽4)
was voltage-dependent, whereas it was not for 10
M BgIII
(n⫽4). Analysis of
h
versus voltage curves showed that the
effect of 10
M BgII on the inactivation time constant of the
TTX-S Na
⫹
current was significant (p⬍0.05, Student‘st
test) only for voltages under ⫺10 mV. The increase in
h
induced by BgII at ⫺10 mV was 64% smaller than that
produced at ⫺30 mV. At voltages above ⫺10 mV, although
there is a tendency of
h
to increase, the change was not
significant. The action of both toxins was not use-dependent
(data not shown).
From current-voltage relationships, current density versus
voltage curves were obtained by normalizing ionic current
amplitudes as a function of membrane capacity (Fig. 3). Un-
der control conditions, the maximum current density
(⫺138 ⫾24 pA/pF) was achieved at ⫺10 mV. Perfusion with
10
M BgII (Fig. 3A) produced a nonsignificant decrease (p⬎
0.05, Student’sttest) in the current density (⫺101 ⫾12
pA/pF). Perfusion with BgIII (Fig. 3B) produced a nonsignif-
icant increase in the current density. The reversal potential
remained unchanged in the presence of toxins, and it was
Fig. 2. Concentration-response curves of the effect of BgII, BgIII, and ATX-II. A and B, typical experiments showing the effects of 10
M BgII and BgIII
on TTX-S sodium currents, respectively. Traces were obtained by a single-step voltage protocol (40-ms depolarizing test pulses to ⫺10 mV from a
holding potential of ⫺90 mV every 8 s). Toxins produced a marked slowing of the inactivation process. The insets show the time course of toxin effects
on
h
. Bars in the inserts indicate the time interval of toxin perfusion around the cell. C and D, concentration-response curves of the effect of ATX-II
(n⫽32; open circles), BgII (n⫽87; triangles), and BgIII (n⫽22; filled circles) on
h
. Data were fit by (solid lines) the dose-response function described
under Materials and Methods. The IC
50
values calculated from these experiments were 4.1 ⫾1.2
M (BgII), 9.6 ⫾1.3 (ATX-II), and 11.9 ⫾1.4
M
(BgIII). Asterisks denotes a significant effect of BgII and BgIII with respect to its controls. ⫹⫹ denotes significant effects with respect to ATX-II action
(Student’sttest; p⬍0.05). Points represent the mean ⫾standard error of the mean.
1070 Salceda et al.
consistent with the one calculated from the Nernst equation
(18 mV).
The peak sodium conductance (G
Na
) at several potential
values was calculated as a chord conductance from the cor-
responding peak current. Normalized conductance curves
were then fitted by a Boltzmann distribution and the corre-
sponding V
1/2
and slope were calculated for each curve. Fig-
ure 4 shows the voltage dependence of G/G
max
under control
conditions and after perfusion with 10
M of either BgII or
BgIII. The mean value of V
1/2
for control experiments was
⫺22 ⫾0.8 mV. No significant differences (p⬎0.05, Student’s
ttest) in the G
Na
were produced when BgII or BgIII were
applied.
Measurements of the voltage dependence of h
⬁
(steady-
state inactivation parameter) were made using a two-pulse
protocol. It was found that 10
M of either BgII or BgIII
caused a significant (p⬍0.05, Student’sttest) hyperpolar-
izing shift in the voltage at which half of the channels are
inactivated (V
1/2 inact
), from a control value of ⫺61 ⫾0.8 mV
to ⫺73 ⫾0.9 mV after BgII and ⫺69 ⫾1.4 mV after BgIII
application (Fig. 5). The calculated slopes were 7.7 ⫾0.6 mV
(control), 8.8 ⫾0.8 mV (BgII), and 10 ⫾1.6 mV (BgIII). The
slope of the curve for BgIII indicates that steady-state inac-
tivation became significantly less voltage-dependent (p⬍
0.05, Student‘sttest). The hyperpolarization shift of the Na
⫹
current implies that at ⫺70 mV the current availability in
the presence of BgIII is 82% of the control and that in the
presence of BgII it is 70% of the control value.
It has been shown that the recovery rate of the TTX-S
current could be described by the sum of two exponential
functions: a fast component (
f
) with a time constant of a few
milliseconds and a slow component (
s
) with a time constant
in the order of several hundred milliseconds. Elliot and Elliot
(1993) pointed out the inherent difficulty in the characteriza-
tion of the slow component, which would require the use of
interpulse intervals lasting several seconds. For this reason,
we defined
hrec
as the time required to reach 63% of reacti-
vated channels. Under control conditions,
hrec
was 15.5 ⫾2
Fig. 3. Effects of BgII and BgIII on the current density versus voltage
relationships. Curves were obtained by normalizing TTX-S current am-
plitudes to membrane capacity. Ionic currents were obtained by a voltage
protocol in which 40-ms pulses between ⫺100 and 70 mV were applied
from a holding potential of ⫺100 mV. Under control conditions (n⫽4 for
each case), the maximum current density was achieved at ⫺10 mV. BgII
(10
M) (A; n⫽4) produced a nonsignificant decrease in the current
density at this voltage (⫺101 ⫾12 pA/pF). When 10
M BgIII (B; n⫽4)
was perfused, the maximum current density was larger (173 ⫾44 pA/pF),
although not significantly when compared with control values (p⬎0.05;
Student’sttest).
Fig. 4. Effects of BgII and BgIII on the voltage dependence of sodium
conductance. A, typical experiments from which activation curves were
obtained. B, the voltage dependence of G
Na
under control conditions and
after perfusion with BgII (n⫽4) or BgIII (n⫽4), each at a 10
M
concentration. Data were fit by a Boltzmann function (solid lines). The
mean value of V
1/2
for control experiments was ⫺22 ⫾0.8 mV. After 10
M BgII application, the V
1/2
was ⫺24 ⫾0.9 mV. After perfusion with 10
M BgIII, the V
1/2
was ⫺25 ⫾1 mV. The slope for the three curves were
also very similar, around 6.5 mV.
BgII and BgIII Anemone Toxins 1071
ms. No significant effects (p⬎0.05, Student’sttest) were
observed when 10
M BgII (n⫽5) or 10
M BgIII (n⫽4)
were applied. The values of
hrec
were 17.5 ⫾1.2 and 16.4 ⫾
1.8 ms, respectively (data not shown).
Discussion
In the present work, we have made a comparative study of
the effects of BgII, BgIII, and ATX-II on neuronal sodium
currents. Our experiments show that the main effect exerted
by both BgII and BgIII is a concentration-dependent slowing
of the inactivation process of TTX-S sodium current, with no
significant effects either on activation kinetics or current
peak amplitude. No significant effects were observed on
TTX-R sodium currents.
In our experimental conditions, BgII was about 3 times
more potent than BgIII, a finding that is consistent with a
previous study in mice by Loret et al. (1994), showing that
BgII is more toxic than BgIII when injected intracerebroven-
tricularly. These authors showed that the higher toxicity of
BgII is correlated to a higher binding competition with the
␣
-scorpion toxin AaH-II (from Androctonus australis Hector)
in rat brain synaptosomes despite their lack of sequence
homology. An interesting fact worth mentioning is that BgII
and BgIII amino acid sequences are almost identical, differ-
ing only by a single amino acid; in BgIII, at position 16, an
aspartic acid replaces the asparagine of BgII. BgII and BgIII
exhibit a higher similarity with type 1 sea anemone toxins
like ATX-II, ApA, or ApB (both from Anthopleura xantho-
grammica) than with type 2 toxins like ShI (from Stichodac-
tyla helianthus). The asparagine in position 16 is a very
conservative residue among type 1 toxins. Our results rein-
force the idea that this amino acid residue plays a central role
in the action of these toxins (Loret et al., 1994; Goudet et al.,
2001).
The electrophysiological effects of ATX-II and
␣
-scorpion
toxins on macroscopic TTX-S sodium currents include the
inhibition of the Na
⫹
channel inactivation (Pelhate et al.,
1984; Neumcke et al., 1985), the presence of sodium currents
after prolonged depolarization (Warashina and Fujita, 1983),
and voltage-dependent action (Strichartz and Wang, 1986).
The effects of both BgII and BgIII are very similar. Never-
theless, only BgII showed a voltage-dependent action. As it
was described for ATX-II, it seems to show more affinity for
its binding site at hyperpolarized than at depolarized poten-
tials (El-Sherif et al., 1992). In contrast, BgIII action was not
voltage-dependent. According to Rogers et al. (1996), sea
anemone toxin binding is less voltage-dependent than
␣
-scor-
pion toxins, suggesting that they have fewer binding contacts
outside of the IVS3-S4 loop, so it is subjected to less steric or
torsional distortion when the channel is depolarized. The
absence of voltage-dependent action observed for BgIII could
be explained by a modification of the electrostatic interac-
tions with the sodium channel due to the presence of an
additional negative charge at the toxin molecule. In the lit-
erature, there are reports indicating a voltage-dependent
action of ATX-II (Lawrence and Catterall, 1981; Strichartz
and Wang, 1986) and
␣
-PMTX (pompilidotoxin, a site 3 neu-
rotoxin from wasp venom) (Sahara et al., 2000) and also a
voltage-independent action (Vincent et al., 1980; Isenberg
and Ravens, 1984). At the moment, there is not a clear
explanation for these discrepancies, but it is possible that
isoform or species-specific differences in gating among so-
dium channels could be playing a role in this respect. The fact
that BgII and BgIII actions were not use-dependent suggests
that these toxins show no preference for the open state of the
sodium channel.
BgIII caused a decrease in the voltage dependence of so-
dium channel inactivation (i.e., significantly increased the
slopes of steady-state inactivation). This result is in agree-
ment with other studies involving site 3 neurotoxins (Gal-
lagher and Bluementhal, 1994; Cahine et al., 1996; Chen et
al., 2000).
In contrast with the lack of effect of BgII and BgIII on the
TTX-R currents in DRG neurons, BgII produced a significant
slowing of the inactivation and an increase in the slope of the
inactivation curve in ventricular cardiomyocytes Na
⫹
cur-
rents that are TTX-R (Goudet et al., 2001). Moreover, accord-
ing to our experiments, in the presence of BgII or BgIII,
steady-state inactivation curves of DRG neurons showed a
shift to the left (i.e., to more hyperpolarized potentials) in the
voltage at which half of the channels are inactivated. This
Fig. 5. Effects of BgII and BgIII on steady-state sodium current inacti-
vation. A, typical records from which inactivation curves were obtained.
B, steady-state inactivation profiles under control conditions (n⫽8) and
during perfusion of 10
M BgII (n⫽4) or 10
M BgIII (n⫽4). The
steady-state inactivation (h
⬁
) was determined using the two-pulse proto-
col shown in the inset. Data were plotted as a function of the prepulse
potential and fit by a Boltzmann function. A 10
M concentration of
either BgII or BgIII caused a significant hyperpolarizing shift in the
V
1/2 inact
, from ⫺61 ⫾0.8 mV (control) to ⫺73 ⫾0.9 mV (BgII) and ⫺69 ⫾
1.4 mV (BgIII).
1072 Salceda et al.
effect is shared by several site 3 toxins (Gordon et al., 1996).
BgII and BgIII applied on cloned hH1 sodium channels pro-
duced a depolarizing shift in the steady-state inactivation
curves but no shift at all when tested on rat ventricular
cardiomyocytes (Goudet et al., 2001). Neither BgII nor BgIII
showed a significant effect on sodium current recovery from
inactivation. These data are in agreement with the results
reported by Goudet et al. (2001) in rat ventricular cardiomy-
ocytes.
Several studies have revealed that sea anemone toxins
bind with higher affinity to cardiac sodium channels than to
neuronal ones (El-Sherif et al., 1992; Roden et al., 2002). The
discrepancies between the actions of BgII and BgIII on car-
diac and neuronal cells could be the result of tissue- or
species-specific differences. TTX-R currents in DRG neurons
are mainly due to type 1.8 and 1.9 Na
⫹
channels, whereas in
the heart, the 1.5 subunit is mainly expressed (Novakovik et
al., 2001; Dib-Hajj et al., 2002).
Site 3 sodium channel toxins have been suggested to slow
the open state to inactivated state transition rate
(Warashina and Fujita, 1983; Strichartz and Wang, 1986;
Schreibmayer et al., 1987; Kirsch et al., 1989), but this phe-
nomenon is difficult to study with macroscopic currents be-
cause current decay at a wide range of voltages represents a
combination of delayed channel openings and channel inac-
tivation (El-Sherif et al., 1992). Nevertheless, both the slow-
ing of inactivation and the reduction in voltage dependence of
steady-state inactivation observed in our experiments sug-
gest that the general explanation proposed by Rogers et al.
(1996) regarding the putative mechanism of action of site 3
toxins is also applicable to BgII and BgIII: 1) the toxin re-
ceptor site undergoes a conformational change that is re-
quired for fast inactivation; 2) bound toxin slows this confor-
mational change and, as a consequence, slows the
inactivation process; and 3) since site 3 neurotoxins bound
across the IVS3-S4 extracellular loop of the sodium channel
and that translocation of IVS4 segment may be required for
the inactivation gate to close, anemone toxins could be slow-
ing or blocking such translocation and thus hindering inac-
tivation. Slowing of the inactivation, however, would in-
crease current density, an effect that has not been observed
in our experiments; thus we cannot exclude that BgII and
BgIII may also produce some degree of channel occlusion.
The existence of several peptides from different species
that bind site 3 of the Na
⫹
channel is relevant from an
evolutionary point of view, indicating that this site is con-
served among species, thus constituting a target for poison-
ous toxins to act. Additionally, the analysis of peptide se-
quences of toxins acting on this site may allow the
identification of the elements of the sequence that are essen-
tial for binding to site 3 of the Na
⫹
channel. This could be of
particular relevance in the case of BgII and BgIII, toxins that
only differ by a single amino acid.
Acknowledgments
We are profoundly in debt to Professor Abel Aneiros for the kind
gift of BgII and BgIII toxins and with Professor L. Beress for kindly
supplying ATX-II. We are also grateful to M. Sa´nchez-Alvarez for
proofreading the English version.
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Address correspondence to: Dr. Emilio Salceda, Instituto de Fisiologı´a,
Universidad Auto´noma de Puebla, Apartado Postal 406, Puebla, Pue., CP
72001, Me´xico. E-mail: esalceda@siu.buap.mx
1074 Salceda et al.
