From the
At least six topologically separated neurotoxin receptor sites
have been identified on sodium channels that reveal strong allosteric
interactions among them. We have studied the allosteric modulation
induced by veratridine, binding to receptor site 2, and brevetoxin
PbTx-1, occupying receptor site 5, on the binding of
Voltage-dependent sodium channels are integral plasma membrane
proteins responsible for the rapidly rising phase of action potentials
in most excitable tissues. As a critical element in nerve excitability,
sodium channels serve as specific targets for many neurotoxins. These
toxins occupy different receptor sites on the sodium channel and have
been used as tools for functional mapping and characterization of the
channel (reviewed in Catterall(1986, 1992)).
At least six neurotoxin
receptor sites have been identified by direct radio-toxin binding on
the rat brain sodium channel (). Receptor site 1 binds the
water-soluble heterocyclic guanidines tetrodotoxin and saxitoxin. These
toxins inhibit sodium conductance by occluding the extracellular
opening of the ion pore. Lipid-soluble alkaloid toxins that bind at
receptor site 2, such as veratridine and batrachotoxin (BTX),
Although the
identification and characterization of the distinct receptor sites have
been predominantly performed using vertebrate-excitable preparations
(Catterall, 1980, 1986; Strichartz et al., 1987), insect
neuronal membranes have been shown to possess similar receptor sites.
The presence of receptor sites 1-4 has been indicated by the
binding of
Toxins that differentiate between sodium channels of various phyla
have also been described. The most studied examples are the
insect-selective toxins derived from scorpion venoms (Zlotkin, 1987).
Two groups of scorpion toxins that modify sodium conductance
exclusively in insect neuronal preparations have been studied; the
excitatory toxins, which induce repetitive firing in insect nerves, and
the depressant toxins, which depolarize the nerve membrane and block
the sodium conductance in insect axons (Pelhate and Zlotkin, 1982;
Zlotkin et al., 1985, 1991). These toxins bind selectively to
insect sodium channels at two distinct receptor sites and therefore
indicate the existence of unique features in the structure of insect
channels, as compared with their mammalian counterparts (Gordon et
al., 1984, 1992, 1993; Moskowitz et al., 1994).
Sodium
channels from various excitable tissues and animal phyla contain a
major
Although the sodium channel receptor sites are
topologically separated, there are strong allosteric interactions among
them (Catterall, 1986; Strichartz et al., 1987; Sharkey et
al., 1987; Baden 1989; Trainer et al., 1993; Gordon and
Zlotkin, 1993; Fainzilber et al., 1994). The phenomenon that
one neurotoxin binding at its receptor site is able to alter, or
modulate, the binding of another toxin bound at a distinct (and perhaps
distant) receptor site emphasizes the dynamic interactivity of the
different structural regions within the sodium channel protein. Thus,
differences in the allosteric modulation that may be detected between
two neurotoxin receptor sites on different channels may indicate the
presence of functionally relevant structural differences between them.
Such differences may be responsible for the different coupling between
the neurotoxin receptor sites. Since the neurotoxins modify the gating
properties of the sodium channels, elucidation of their interactions
will contribute to the understanding of their mode of action and the
mechanism of gating on the structural and dynamic levels of sodium
channel function.
These considerations provided the overall
motivation for the present study. As a first step in the above
presumption, we have studied the allosteric modulation induced by
veratridine (binding to receptor site 2) and brevetoxin PbTx-1
(occupying receptor site 5) on the binding of
We have used AaH II, the most active
Our
comparative approach reveals that the two groups of lipid-soluble
activators of sodium channels, brevetoxin and veratridine, modulate the
binding of an
Rat brain synaptosomes (100 µg of protein/ml) or insect
synaptosomes (P
Normal
physiological saline had the following composition (in mM):
NaCl, 200; KCl, 3.1; CaCl
Preliminary experiments revealed that the specific binding of
Fig. 1A demonstrates that increasing concentrations
of brevetoxin PbTx-1 inhibit about 70% of the specific binding of AaH
II. The concentration of brevetoxin that inhibits 50% of the binding
(IC
Previous studies have demonstrated that veratridine enhanced
the binding of radiolabeled
On this background, we have examined the
effects of veratridine on the negative allosteric interaction observed
between PbTx-1 and AaH II binding sites (Fig. 1). Veratridine is
able to increase the specific binding of
As can be
seen in , veratridine at higher concentrations (100
µM and above, see Fig. 2A) induces a
significant shift in the IC
Thus, it appears that brevetoxin and veratridine are inducing
opposite allosteric modulations at receptor site 3; the former induces
a negative modulation and the latter a positive modulation. Moreover,
the combination of the two allosteric modulators follows in reversing
the effects of each, resulting in AaH II binding levels around control
with no additions (Fig. 2). It should be noted, however, that
each modulator is active on its own way even at the highest
concentration of the other (Fig. 2), indicating that their
contrasting effects on AaH II binding are not due to competition or
inhibition of their binding, in accordance with previous studies
(Catterall and Gainer, 1985; Sharkey et al., 1987; Trainer et al., 1993).
In contrast to the vast information on allosteric
interactions among different receptor sites in mammalian sodium
channels, very little is known on allosteric modulations among
neurotoxin receptor sites in insect sodium channels. To study the
allosteric interactions between PbTx-1 and veratridine at the
We used
To elucidate the mechanism by which
PbTx-1 alters the normal ionic conductance of the axon, voltage clamp
experiments were performed. Brevetoxin PbTx-1 did not alter the
activation and inactivation properties of the Na
The
activity of PbTx-1 has been detected under small depolarization pulses,
from -70 to -55 mV (Fig. 4C), when no sodium
currents are normally detected. Application of PbTx-1 induces the
appearance of two sodium currents: 1) a discrete permanent inward
current at the holding potential of -70 to -60 mV (not
always seen), present also after the end of the voltage pulses to
-10 mV (Fig. 4B, panel2); and
2) a more detectable, slowly developing sustained (not inactivating)
Na
Thus, the electrophysiological activity of brevetoxin
PbTx-1 on cockroach axon is similar to the previously reported activity
on other preparations (Westerfield et al., 1977; Parmentier et al., 1978; Huang et al., 1984; Baden, 1989).
The present study examines, for the first time, the
allosteric interactions that occur among three distinct, identified
receptor sites on sodium channels in different animal phyla in mammals
and in insects. Modulation at the
The disagreement revealed between our results
and the previous one using PbTx-1 and PbTx-2 may be attributed to 1)
the different neuronal preparations used, which are expected to express
different sodium channel subtypes in their membranes; 2) structural
differences in the two
Other brevetoxin analogs, which have
been reported to be inactive on
Alkaloid toxins, such as veratridine, are able to
increase the affinity of the
The
alterations in sodium channel structure by brevetoxin are likely to be
exerted to wider regions implied to be on the extracellular side, where
different scorpion toxins are suggested to bind (Thomsen and Catterall,
1989; Gordon et al., 1992). This notion is supported by the
positive allosteric modulation observed by brevetoxin on the
voltage-independent binding of Lqh
The differences in
allosteric interactions observed between locust and cockroach may
result either from inherent differences in the sodium channel
structures or because the relevant neurotoxins may be binding to
different sodium channel subtypes, supposed to be present in cockroach
CNS membranes. The latter implies that brevetoxin and veratridine are
able to selectively discriminate between different sodium channel
subtypes, and those subtypes that bind the lipid-soluble activators do
not bind the Lqh
Electrophysiological data using isolated cockroach giant axons are
in agreement with previous observations of the effect of brevetoxin on
squid axon and on a ventral nerve cord of a crayfish (Westerfield et al., 1977; Parmentier et al., 1978) and confirm
that 1) PbTx-1 is active on the sodium conductance at micromolar
concentrations, and no specific action has been detected on potassium
permeability; 2) brevetoxin depolarizes the insect axonal membrane and
induces repetitive firing of normal action potentials at high frequency
with no effect on the inactivation of the sodium currents (Fig. 4); and 3) voltage clamp experiments have shown that
brevetoxin induced sodium currents at negative potentials, when
normally no sodium current is detected. These sodium currents develop
slowly and do not inactivate (Fig. 4C). The inward
constant sodium current recorded at -70 to -60 mV in the
presence of PbTx-1 may correspond to a limited number of sodium
channels that remain open for a long time or that do not inactivate at
all when their receptor site 5 is occupied by brevetoxin.
Alternatively, the toxin may modify all the channels and give way to a
long living, low conductance-modified state, as previously shown for
veratridine and BTX effects (Barnes and Hille, 1988; Quandt and
Narahashi, 1982). At present, we cannot distinguish between these
possibilities, and patch clamp experiments will be necessary. The
relatively higher concentration of PbTx-1 needed in this axonal
preparation is attributed to the presence of 2-3 layers of glial
cells surrounding the isolated axon and limiting the access of the
toxin molecules to reach their receptor sites on the axonal membrane
(Pichon et al., 1983). Future studies on cultured isolated
neurons will be carried out to establish the efficacy of PbTx-1 in
cockroach neuronal membranes.
The dramatic differences in allosteric
modulation observed between the locust and cockroach sodium channels
are further emphasized by the previously suggested pharmacological
similarity between these two insect neuronal preparations. This
similarity has been demonstrated by comparable binding characteristics
and similar mutual competitive inhibition of an excitatory (AaIT, from
the scorpion Androctonus australis Hector) and depressant
(LqhIT
Hence, the lack of allosteric interaction
between brevetoxin and Lqh
The above assumption is in
concert with previous biochemical examinations of insect neuronal
sodium channel polypeptides, which have indicated that some structural
differences exist among sodium channel proteins expressed in CNS from
various insect orders, including locust and cockroach (Gordon et
al., 1990, 1993; Moskowitz et al., 1994). Moreover, our
recent binding experiments further suggest the presence of differences
between these two insect sodium channels. The K
The previous observations that alterations
of some hydrophobic amino acids of sodium channels cause a dramatic
change in gating behavior (Auld et al., 1990) suggest that
they are involved in critical interaction with one or more adjacent
transmembrane helices. It is possible that brevetoxin, when it occupies
its receptor site, interacts differently with its surrounding
hydrophobic recognition sites associated to the transmembrane
Thus, detailed comparative pharmacological studies may yield
some insight into the dynamics and mechanism of action of sodium
channels. The combination of pharmacology and molecular biology
techniques may lead to the identification of the precise peptide
sequences to which the brevetoxins as well as the
The IC
Our sincere thanks to Prof. Eliahu Zlotkin from the
Hebrew University of Jerusalem for the kind gift of the Lqh
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-scorpion
toxins at receptor site 3, on three different neuronal sodium channels:
rat brain, locust, and cockroach synaptosomes. We used
I-AaH II, the most active
-scorpion toxin on
vertebrates, and
I-Lqh
IT, shown to have high
activity on insects, as specific probes for receptor site 3 in rat
brain and insect sodium channels. Our results reveal that brevetoxin
PbTx-1 generates three types of effects at receptor site 3: 1) negative
allosteric modulation in rat brain sodium channels, 2) positive
modulation in locust sodium channels, and 3) no effect on cockroach
sodium channel. However, PbTx-1 activates sodium channels in cockroach
axon similarly to its activity in other preparation. Veratridine
positively modulates both rat brain and locust sodium channels but had
no effect on
-toxin binding in cockroach. The dramatic differences
in allosteric modulations in each sodium channel subtype suggest
structural differences in receptor sites for PbTx-1 and/or at the
coupling regions with
-scorpion toxin receptor sites in the
different sodium channels, which can be detected by combined
application of specific channel modifiers and may elucidate the dynamic
gating activity and the mechanism of allosteric interactions among
various neurotoxin receptors.
(
)cause persistent activation of the channel at the
resting membrane potential by blocking sodium channel inactivation and
shifting the voltage dependence of channel activation to more negative
membrane potentials. Receptor site 3 binds
-scorpion toxins and
sea anemone toxins that inhibit sodium channel inactivation. They also
enhance persistent activation of sodium channels by lipid-soluble
toxins acting at receptor site 2, and their affinity to receptor site 3
is reduced by depolarization.
-Scorpion toxins bind to receptor
site 4 and shift the voltage dependence of sodium channel activation.
The hydrophobic polyether toxins brevetoxin (PbTx) and ciguatoxin bind
to receptor site 5 and shift the activation to more negative membrane
potentials (reviewed in Catterall(1986); Strichartz et al. (1987); Baden(1989)). Receptor site 6 binds the
-conotoxin
TxVI that inhibits sodium channel inactivation in mollusc neurons but
binds with high affinity to both mollusc and rat brain sodium channels
(Fainzilber et al., 1994, 1995).
H-saxitoxin and tetrodotoxin (receptor site 1,
Gordon et al., 1985), tritiated derivative of batrachotoxin (
H-BTX-B) and veratridine (receptor site 2, Soderlund et al., 1989; Dong et al., 1993; Church and Knowles,
1993),
I-
-scorpion (Lqh
IT) and
I-ATX II sea anemone toxins (receptor site 3) (Gordon and
Zlotkin, 1993; Pauron et al., 1985), and
I-
-scorpion toxins (Ts VII (from the scorpion Tityus serrulatus, called also
-Tityus toxin),
Css VI (
-scorpion toxin VI from the venom of the scorpion C.
suffusus suffusus), receptor site 4) (Lima et al., 1986,
1989) on locust, cockroach, and other insect neuronal membranes. The
presence of receptor site 5 has not yet been examined in insects.
-subunit of about 240-280 kDa (for a review, see
Catterall (1992); Gordon et al.(1988, 1990, 1993)). The
primary structure of voltage-gated sodium channel
-subunits
contains four homologous internal repeats (domains I-IV), each having
six putative transmembrane segments designated S1-S6 (Noda et
al., 1986). Insect sodium channels were shown to resemble their
vertebrate counterparts by their primary structure (Loughney et
al., 1989), topological organization (Gordon et al.,
1992; Moskowitz et al., 1994), and basic biochemical (Gordon et al., 1988, 1990, 1992, 1993; Moskowitz et al.,
1991, 1994) and pharmacological (Pelhate and Sattelle, 1982)
properties. On the other hand, a possible uniqueness of the insect
sodium channels was suggested by the selective activity of the
excitatory and depressant insect-selective toxins. Thus, a comparative
study of mammalian and insect neurotoxin receptor sites on the
respective sodium channels may elucidate the structural features
involved in the binding and activity of the various neurotoxins and may
contribute to the clarification of a structure-function relationship in
sodium channels.
-scorpion toxins at
receptor site 3 on three different neuronal sodium channels: rat,
locust, and cockroach CNS. These three sodium channel subtypes have
been chosen since the rat brain channel is the most studied one, and we
initially intended to use it as a known reference in comparison to the
others; locust neuronal membranes served as the main sodium channel
source for neurotoxin binding studies in insects, and cockroach axons
have been used as the main preparation for physiological effects of
neurotoxins.
-scorpion
toxin on vertebrates (Jover et al., 1980b), and Lqh
IT,
the recently characterized
-scorpion toxin that reveals
significantly higher activity to insects as compared with vertebrates
(Eitan et al., 1990; Gordon and Zlotkin, 1993) as specific
probes for receptor site 3 in rat brain and insect sodium channels,
respectively. Lqh
IT binding characteristics have been shown to be
similar to those described for
-scorpion toxins in vertebrate
sodium channels, except that its binding is not dependent on membrane
potential (Gordon and Zlotkin, 1993). Thus, the receptor site for
Lqh
IT on insect sodium channels is considered to be homologous to
receptor site 3 in mammalian sodium channels (Eitan et al.,
1990; Gordon and Zlotkin, 1993; Zlotkin et al., 1994).
-scorpion toxin in the various sodium channels in a
significantly different manner. These differences may indicate the
presence of important structural and/or functional differences that may
be present in these channel subtypes. This study may help to elucidate
the structural elements responsible for the conformational changes
induced on the sodium channels upon neurotoxin binding and gating.
Materials
Scorpion toxin AaH II was
purified as previously described (Miranda et al., 1970).
LqhIT, used for radioiodination and saturation curves (Fig. 3) was a generous gift of Prof. E. Zlotkin (The Hebrew
University, Jerusalem, Israel). Lqh
IT used for nonspecific binding
determinations and brevetoxin PbTx-1 were from Latoxan (A.P. 1724,
05150 Rosans, France). Veratridine was from Sigma. Carrier-free
Na
I was from Amersham Corp. All other chemicals were of
analytical grade. Filters for binding assays were glass fiber GF/C
(Whatman, United Kingdom) preincubated in 0.3% polyethylenimine
(Sigma).
Figure 3:
Brevetoxin PbTx-1 enhances the binding of I-Lqh
IT to locust neuronal membranes. A,
locust neuronal membranes (15 µg of protein) were incubated with
0.1 nM
I-Lqh
IT and increasing
concentrations of PbTx-1 for 60 min at 22 °C, as described under
``Experimental Procedures.'' Nonspecific binding, determined
in the presence of 1 µM of unlabeled Lqh
IT, was
subtracted. The amount of
I-Lqh
IT bound at each data
point represents the mean ± S.E. of three to six experiments,
expressed as a percentage of the maximal specific binding without
additions (indicated by the brokenline). B,
Scatchard analysis of saturation curves of Lqh
IT binding to locust
neuronal membranes (see ``Experimental Procedures'') in the
presence or absence of 200 nM PbTx-1. Scatchard plots were
analyzed with the program LIGAND.
, control, no brevetoxin.
, + 200 nM PbTx-1 (Brev). Equilibrium
binding constants determined (n = 3) were as follows: K = 0.46 ± 0.14 nM, B
= 0.33 ± 0.05 pmol/mg (control Lqh
IT, no
additions); K = 0.23 ± 0.11 nM, B
= 0.37 ± 0.04 pmol/mg (+, 200 nM PbTx-1).
Neuronal Membrane Preparations
Rat brain
synaptosomes were prepared from adult albino Wistar rats (about 300 g,
laboratory bred), according to the procedure of Dodd et al. (1981). Insect synaptosomes (PL preparation) were
prepared from the CNS of adult locusts (Locusta migratoria)
and cockroach (Periplaneta americana) according to established
methods (Gordon et al., 1990, 1992; Moskowitz et al.,
1994). All buffers contained a mixture of proteinase inhibitors
composed of phenylmethylsulfonyl fluoride (50 µg/ml), pepstatin A
(1 mM), iodoacetamide (1 mM), and 1 mM
1,10-phenantroline. Membrane protein concentration was determined using
a Bio-Rad protein assay, with bovine serum albumin as standard.
Radioiodination
AaH II was radioiodinated
by lactoperoxidase as previously described (Rochat et al.,
1977) using 1 nmol of toxin and 1 mCi of carrier free
NaI. Lqh
IT was iodinated by Iodogen (Pierce) using 5
µg of toxin and 0.5 mCi carrier free Na
I as
previously described (Gordon and Zlotkin, 1993). The monoiodotoxins
were purified according to Lima et al.(1989) using a Merck RP
C
column and a gradient of 5-90% B (A = 0.1%
trifluoroacetic acid, B = acetonitrile, 0.1% trifluoroacetic
acid) at a flow rate of 1 ml/min. The concentration of the radiolabeled
toxins was determined according to the specific activity of the
I corresponding to 2424 dpm/fmol monoiodotoxin.
Binding Assay
Equilibrium saturation
assays were performed using increasing concentrations of the unlabeled
toxin in the presence of a constant low concentration of the
radioactive toxin. To obtain saturation curves, the specific
radioactivity and the amount of bound toxin were calculated and
determined for each toxin concentration. Standard binding medium
composition was (in mM): choline Cl, 140; CaCl,
1.8; KCl, 5.4; MgSO
, 0.8; HEPES, 25, pH 7.4; glucose, 10;
bovine serum albumin, 2 mg/ml. Wash buffer composition was (in
mM): choline Cl, 140; CaCl
, 1.8; KCl, 5.4;
MgSO
, 0.8; HEPES, 25, pH 7.4; bovine serum albumin, 5
mg/ml.
L, 50 and 3.3 µg/ml for locust and
cockroach, respectively) were suspended in 0.15 or 0.3 ml binding
buffer, containing
I-AaH II or
I-Lqh
IT, respectively. After incubation for the
designated time periods, the reaction mixture was diluted with 2 ml of
ice-cold wash buffer and filtered through GF/C under vacuum. Filters
were rapidly washed with an additional 2
2-ml buffer.
Nonspecific toxin binding was determined in the presence of 0.2
µM unlabeled AaH II or 1 µM Lqh
IT,
respectively, and consists typically of 15-20% of total binding
for
I-AaH II or
I-Lqh
IT, using rat
brain or locust membranes, respectively, and about 1% using cockroach
membranes. The experiments with the rat brain preparation were carried
out at 37 °C, and those with insect membranes were carried out at
22 °C. Equilibrium saturation or competition experiments were
analyzed by the iterative computer program LIGAND (Elsevier Biosoft,
UK). Kinetic experiments were analyzed according to Weiland and
Molinoff(1981). Each experiment was performed at least three times.
Electrophysiology
Adult male cockroaches (P. americana) were used throughout these experiments. A
segment (1.5-2.5 mm) of one giant axon was isolated from a
connective linking the 4th and 5th abdominal ganglia and cleaned of
adhering fibers. The preparation was transferred to an experimental
chamber in which two lateral Ag-AgCl electrodes were in contact with
the severed ends of the axon and a central Ag-AgCl electrode was in
contact through the external bathing solution with a
100-150-µm segment of the dissected axon. The preparation was
immersed in paraffin oil, and the ``artificial node'' created
by the non-electrolyte (Pichon and Boistel, 1967) was voltage clamped
as described in detail earlier (Pelhate and Sattelle, 1982).
, 5.4; MgCl
, 5.0;
HEPES buffer, 1; pH 7.2. Experiments were performed at 19-21
°C. When necessary, potassium current was suppressed largely by 0.5
mM 3,4-diaminopyridine. Brevetoxin PbTx-1 (from Latoxan, A.P.
1724, 05150 Rosans, France) was dissolved in acetonitrile before
addition to the saline to a final test concentration of 1.15-4.6
µM PbTx-1 and or (v/v) acetonitrile and was externally
applied on the axonal preparation. A preliminary test demonstrated no
detectable effects of the (v/v) acetonitrile saline solution.
Inhibition of
-Scorpion Toxin Binding by
Brevetoxin PbTx-1 on Rat Brain Sodium Channels
I-AaH II to rat brain synaptosomes is remarkably reduced
in the presence of brevetoxin PbTx-1, the most active brevetoxin analog
(Baden, 1989). This result was rather surprising since brevetoxin
PbTx-2 was reported to have no effect on
-scorpion toxin (Lqq V)
binding to rat brain synaptosomes (Sharkey et al., 1987), and
PbTx-1 did not alter the binding of
I-Lqq V to
neuroblastoma cells (Catterall and Risk, 1980). In addition, Lqq V had
no effect on the specific binding of
H-PbTx-9 (Trainer et al., 1993). The different brevetoxins are known to bind
competitively to the same receptor site 5 on sodium channels with
similar binding affinities (Poli et al., 1986; Baden, 1989).
) is 31.6 ± 12 nM. This value is in
accordance with the previously published PbTx-3 concentration that
cooperatively enhanced aconitine-stimulated
Na influx
(Poli et al., 1986) and the effective dose (ED
)
for PbTx-2 that enhanced
H-BTX-B binding (Sharkey et
al., 1987) in rat brain synaptosomes.
Figure 1:
Negative allosteric effect of
brevetoxin PbTx-1 on I-AaH II binding in rat brain
synaptosomes. A, inhibition of
I-AaH II binding.
Rat brain synaptosomal membranes were incubated 30 min at 37 °C (as
described under ``Experimental Procedures'') with 0.12 nM
I-labeled AaH II and increasing concentrations of
brevetoxin PbTx-1. Nonspecific binding, determined in the presence of
0.2 µM native AaH II, was subtracted from all data points.
Data are shown as percent inhibition of specific AaH II binding. Each
data point represents the mean ± S.E. of three to six
experiments. IC
values were calculated using DRUG analysis
in the LIGAND program and determined as (mean ± S.E.) 31.6
± 12 nM (n = 6). B, Scatchard
analyses of AaH II specific binding to rat synaptosomal membranes, in
the presence and absence of 100 nM brevetoxin PbTx-1.
Membranes were incubated with 0.12 nM
I-labeled
AaH II and the indicated concentrations of unlabeled AaH II in the
presence and absence of 100 nM brevetoxin PbTx-1 for 30 min at
37 °C, and specific binding was determined as described. Scatchard
plots were analyzed with the program LIGAND. Equilibrium binding
constants determined in this experiment were as follows: K = 0.3 nM, B
= 0.32
pmol/mg (
, AaH II control, no brevetoxin); and K = 1.1 nM, B
= 0.3
pmol/mg (
, + 100 nM brevetoxin (BREV)
PbTx-1). C, dissociation rates of
I-labeled AaH
II from rat synaptosomal membranes in the presence or absence of
brevetoxin PbTx-1. Membranes were pre-equilibrated for 30 min at 37
°C with 0.16 nM
I-labeled AaH II, and
dissociation was initiated by the addition of 0.2 µM
unlabeled AaH II with or without 0.5 µM brevetoxin PbTx-1.
Specific binding at each point was determined as described.
,
control AaH II only;
, + 0.5 µM brevetoxin (BREV) PbTx-1. Data analysis was according to Weiland and
Molinoff (1981); k
= 1.23
10
± 1.67
10
s
(no brevetoxin); k
= 2.8
10
± 2.5
10
s
(+ 0.5 µM
brevetoxin PbTx-1).
Scatchard analysis of AaH
II binding in the presence of 100 nM brevetoxin shows a
3.6-fold reduction in affinity (K of 1.1
± 0.3 nMversus 0.3 nM with and
without PbTx-1), with no significant change in the binding capacity (Fig. 1B). Since brevetoxin is known to bind to receptor
site 5 and AaH II to receptor site 3 on rat brain sodium channels, we
examined the effects of brevetoxin on the dissociation kinetics of AaH
II from its receptor site. As can be seen from Fig. 1C,
PbTx-1 increased the rate of AaH II dissociation. The t
for AaH II dissociation is reduced from 9.4
± 1.5 min in the absence of PbTx-1 to 4.1 ± 0.4 min in
the presence of 0.5 µM brevetoxin. These data are
consistent with a negative allosteric interaction (Willow and
Catterall, 1982; Fainzilber et al., 1994) between receptor
sites 5 and 3 on rat brain sodium channels, whereby occupation of site
5 by PbTx-1 causes a decrease in the stability of the
-toxin-receptor complex at site 3. The latter is responsible for
the reduction in affinity of AaH II to its receptor site and accounts
for the apparent binding inhibition (Fig. 1).
Veratridine Reverses the Inhibition of AaH II Binding
Induced by Brevetoxin in Rat Brain Synaptosomes
-scorpion toxins to receptor site 3 in
rat brain synaptosomes up to 2-fold (Ray et al., 1978) or less
(Jover et al., 1980b). Additional synergistic binding
interactions have been described to occur between receptor sites 2, 3,
and 5 on rat brain sodium channels (Catterall and Gainer, 1985;
Catterall et al., 1981; Sharkey et al., 1987; Poli et al., 1986).
I-AaH II up to
1.3-fold above the control level, with an ED
of about 200
µM (Fig. 2A, opensymbols). As demonstrated in Fig. 2A, the
inhibition of AaH II binding induced by 100 nM brevetoxin
(about 65% inhibition, indicated by the brokenline in Fig. 2A) is reversed or antagonized in a
dose-dependent manner by veratridine. The recovery in AaH II binding is
observed at the same range of veratridine concentrations that induced
the increase in AaH II binding (Fig. 2A, filledsymbols).
Figure 2:
Effects of the concurrent presence of
brevetoxin PbTx-1 and veratridine on the binding of I-AaH
II to rat brain synaptosomes. A, reversal of
brevetoxin's inhibition by veratridine on
I-AaH II
binding. Rat synaptosomal membranes were incubated in the presence of
0.15 nM
I-labeled AaH II and increasing
concentrations of veratridine in the absence (
, VER) or
the presence of 100 nM brevetoxin PbTx1 (
, +BREV). Specific binding was determined as described in
Fig. 1 and under ``Experimental Procedures.'' Results are
shown as percentage of maximal
I-AaH II bound with no
additions (100%). Each data point represents the mean ± S.E. of
three experiments. The percentage of
I-AaH II bound in
the presence of 100 nM PbTx-1 alone is indicated by the brokenline. B, rat synaptosomal membranes
were incubated 30 min at 37 °C with increasing concentrations of
brevetoxin PbTx-1 in the absence or the presence of indicated
concentrations of veratridine. Results are shown as percentage of
maximal
I-AaH II bound with no additions (indicated by
the brokenline). For clarity of presentation, only
the curves in the presence of 20 µM veratridine
(that is similar to brevetoxin alone) and 200 µM veratridine are presented. The IC
values determined
using DRUG analysis in the LIGAND program are presented in Table
II.
To analyze the allosteric interactions
observed between veratridine and brevetoxin on the -scorpion toxin
receptor site, we tested the effect of increasing concentrations of
PbTx-1 in the presence of several veratridine concentrations (Fig. 2B and ). Brevetoxin-induced
inhibition of AaH II binding is not affected by the presence of low
concentrations of veratridine (10-25 µM). On the
other hand, increasing concentrations of brevetoxin are able to prevent
or decrease the enhancement on AaH II binding induced by 200
µM veratridine (Fig. 2B), reducing the
specific binding back to the control level. In the presence of
saturating concentrations of both allosteric modifiers, the specific
binding of AaH II is restored (Fig. 2B).
values for brevetoxin
inhibitory effect on AaH II binding. A 10-fold increase in the
IC
of brevetoxin has been obtained in the presence of 100
µM veratridine, and higher concentration resulted in a
100-fold increase in the IC
values ().
Effect of Brevetoxin PbTx-1 on Insect Sodium Channels
-scorpion toxin receptor site on insect sodium channels, we have
used synaptosomes prepared from CNS of both locust and cockroach for
comparative purposes.
I-Lqh
IT as specific
probe for receptor 3 in insect sodium channels. Lqh
IT binds to a
single class of high affinity receptor sites in locust (Gordon and
Zlotkin, 1993) and cockroach
(
)sodium channels.
The binding of Lqh
IT to locust neuronal membrane has been
demonstrated to be cooperatively increased by veratridine, whereby 100
µM veratridine increases both the affinity and capacity of
Lqh
IT receptor sites (Gordon and Zlotkin, 1993). Veratridine has
been shown to be as active on cockroach axon (Pelhate and Sattelle,
1982) and cultured cockroach neuronal cells
(
)as
in vertebrate electrophysiological preparations (reviewed in
Catterall(1980)).
Brevetoxin Enhances the Binding of Lqh
Brevetoxin PbTx-1 increases 1.8-fold the binding of
LqhIT to Locust Sodium
Channels
IT to locust synaptosomes, with an apparent ED
of
24.4 ± 4.1 nM (Fig. 3A). Scatchard
analysis in the presence of brevetoxin reveals that saturating
concentrations of PbTx-1 increase the affinity of Lqh
IT (K
= 0.46 ± 0.14 nMversus 0.23 ± 0.11 nM without and with
200 nM brevetoxin) to its receptor site in locust neuronal
sodium channels, with no effect on the number of its receptor sites (Fig. 3B). The effects of veratridine and brevetoxin on
Lqh
IT binding in cockroach neuronal membranes have been studied in
parallel under similar conditions. Since most recently we have shown
that Lqh
IT binds with a 10-fold higher affinity to cockroach
sodium channels (K
about 20-30
pM)
than to locust,
I-Lqh
IT was
used at lower concentrations, between 20 and 60 pM, a range in
which the specific binding to the cockroach membranes markedly
increased with the toxin concentration (data not shown). Surprisingly,
no significant effect of brevetoxin or veratridine has been detected on
Lqh
IT binding on cockroach sodium channels (see Fig. 5).
Figure 5:
Comparison between the effects of
brevetoxin (BREV) PbTx-1 and veratridine (VER) on
-scorpion toxin binding in rat brain, locust, and cockroach
neuronal membranes. Rat brain synaptosomes were incubated with
I-AaH II (as described in Figs. 1 and 2) in the absence
or presence of PbTx-1 or veratridine, as indicated under the columns. Locust or cockroach neuronal membrane were incubated
with
I-Lqh
IT (as described in Fig. 3) without or
with the indicated concentrations of PbTx-1 or veratridine. All data
are shown as percentage of maximal
I-
-scorpion toxin
bound in control with no additions (control, 100%). Each column represents a mean ± S.E. of at least three
experiments.
Activity of Brevetoxin on Cockroach
Axons
The lack of any effect of brevetoxin PbTx-1 on the
binding of the -scorpion toxin in cockroach neuronal membranes, in
contrast to its significant allosteric enhancement of Lqh
IT
binding in locust sodium channels, could result either from inherent
differences at the brevetoxin receptor site between locust and
cockroach sodium channels or from lack of activity of PbTx-1 on the
cockroach neuronal membrane. Since no data are available in the
literature whether brevetoxin is active in insect neurons, we have
studied the effects of PbTx-1 on an isolated axon of the cockroach (Fig. 4).
Figure 4:
Action of PbTx-1 on an isolated cockroach
axon under current and voltage clamp. A1, electrical activity
of PbTx-1 recorded during the first 10 min of superfusion of the axon
with 4.6 µM PbTx-1. The membrane was depolarized by the
action of the toxin by 15 mV after 10 min (uppertrace), accompanied by the appearance of repetitive
activity. Lowertrace, control; middletrace, after 6 min. A2, after the first 10 min
of action of PbTx-1, normal shaped action potential bursts alternate
with under threshold oscillations at a frequency of 180-200 Hz
when the axonal membrane was artificially repolarized to the normal
resting potential by injection of a constant hyperpolarizing current of
8-10 nA. B, sodium currents in a step depolarization
from a holding potential of -60 to -10 mV. B1,
control. B2, after 10 min external application of 2.3
µM PbTx-1. Na currents at E
= -10 mV during a pulse of 6 ms are
shown. The K
outward current was suppressed with
5.10
M of 3,4-diaminopyridine. Sodium
current activation and inactivation developed normally and completely.
The inward Na
peak value decreased by 15-25%. At
this scale, no maintained Na
current was observed. C, Na
current recorded at high vertical
magnification during 6-ms pulses from holding potential of -70 mV
to E
= -55 mV (C1a to C3a) or 35-ms
pulses (C1b to C3b). Slow and maintained inward Na
currents were developed after 10 min (C2a and C2b) and 20 min
(C3a and C3b) external application of 2.3 µM PbTx-1, as
compared to the control (C1a and C1b). There was a shift of about 15 mV
in sodium conductance (gNa) increase toward negative potentials,
developing a Na
current that corresponds to 6% of the
maximum peak current at E
= -10
mV.
Application of 2.3 µM PbTx-1 caused a
small (5 mV) depolarization of the axonal membrane, but higher
concentration (4.6 µM) resulted in a 15-mV depolarization (Fig. 4A, panel1), which immediately
induced a sustained repetitive activity (even in the absence of
electrical stimulation), consisting of normal-shaped action potentials
with inserted small ones of 20-25 mV in amplitude (Fig. 4A, panel2). Similar type of
repetitive activity has been observed on this preparation in the first
stages of action of aconitine (see ; Pelhate and
Sattelle(1982)) and of insect-selective excitatory toxin activity
(Pelhate and Zlotkin, 1982; Lester et al., 1982). The
repetitive firing established itself at a very regular frequency of
180-200/s (which is the maximum frequency possible to record
using this preparation). Artificial repolarization to the resting
potential of -60 to -50 mV, or even hyperpolarization to a
level 10-20 mV more negative than the resting potential (to
-70 mV), suppressed the repetitive firing, but a later return to
normal resting potential (-60 mV or to -50 mV) immediately
induced a sustained repetitive activity (Fig. 4A, panel2).
current when a large depolarizing pulse (from -60 to
-10 mV) was applied (Fig. 4B). The increase in
Na
conductance reaches its maximum at E
= -10 mV and inactivates completely in 3-4 ms,
as under control (normal) conditions. At this potential, the main
modification observed in the presence of PbTx-1 was a decrease in the
peak Na
current by 15-25% (Fig. 4B). Only a part of this decrease may be
attributed to a run-down of the preparation when experiments were
carried on for more than 30-45 min (Fig. 4B, panels1 and 2). However, PbTx-1 has no
effect on the K
conductance of the axon.
inward current at potentials -55 to -50
mV (during a voltage pulse from holding potential = -70 to E
= -55 or -50 mV), potentials
at which normally no Na
current exists (Fig. 4C). These sodium currents, developed near the
resting potential of the axon, may rationally explain the
depolarization and repetitive activity observed in the presence of
brevetoxin.
-scorpion toxin receptor site
caused by the single and concurrent occupancy of receptor sites 2 and 5
by the two hydrophobic activators of sodium channels, brevetoxin PbTx-1
and veratridine, revealed the occurrence of dramatic differences in the
allosteric modulations in each sodium channel subtype studied. Our
results may contribute to the elucidation of a structure-function
relationship of sodium channels and may suggest new possibilities to
tackle the problem of studying the cooperative, dynamic gating activity
of sodium channel as well as the mechanism of action of the various
neurotoxins on the molecular level.
Comparison among the Allosteric Interactions Revealed in
Rat, Locust, and Cockroach Sodium Channels
Our results show
that allosteric interactions induced by brevetoxin at the
-scorpion toxin receptor site may reveal structural and functional
differences in sodium channel subtypes. Brevetoxin PbTx-1 generates
three types of allosteric effects on
-scorpion toxins receptor
sites: 1) negative modulation in rat brain sodium channels ( Fig. 1and Fig. 2), 2) positive modulation in locust sodium
channels (Fig. 3), and 3) no effect in cockroach sodium channels.
However, the lack of allosteric modulation does not result from a lack
of binding or activity, since brevetoxin is shown, for the first time
to activate the cockroach axonal sodium channels in a comparable manner
to its effect in other systems (Fig. 4). Veratridine revealed a
positive allosteric modulation in both rat brain and locust sodium
channels but had no effect in the cockroach on the
-scorpion toxin
receptor site (see Fig. 5). Thus, the differential allosteric
interactions may indicate the existence of structural/functional
differences in the receptor site for brevetoxin and/or at the regions
that produce the coupling (the conformational change) with the
-scorpion toxin receptor site in the three sodium channels tested.
Negative Allosteric Modulation by Brevetoxin at
Receptor Site 3 on Rat Brain Sodium Channels
Despite the
known activity of brevetoxin to shift the voltage dependence of
activation of sodium channels to more negative potentials, brevetoxin
PbTx-1 has been shown to have no effect on -scorpion toxin (Lqq V)
binding to neuroblastoma cells (Catterall and Risk, 1980), and PbTx-2
revealed no effect on rat brain synaptosomes (Sharkey et al.,
1987). Our results, on the other hand, demonstrate a remarkable
negative allosteric modulation at receptor site 3 by PbTx-1 in rat
brain synaptosomes.
-scorpion toxins employed, AaH II in our
case and Lqq V in the others, which suggests that the interaction
between brevetoxin and
-scorpion toxin sites may be, at least
somewhat, toxin specific; and 3) the inherent structural differences in
brevetoxins. The different brevetoxins are known to bind competitively
to the same receptor site with similar binding affinities (Poli et
al., 1986; Baden, 1989). However, PbTx-1 has been shown to be the
most effective analog to enhance
H-BTX-B binding to rat
brain sodium channels (Trainer et al., 1993). The greater
conformational flexibility in the backbone structure of PbTx-1 (Gawley et al., 1992) as compared to that of PbTx-2 may be related to
its ability to allosterically modulate the
H-BTX-B binding
(Trainer et al., 1993) as well as the
-scorpion toxin
binding ( Fig. 1and Fig. 2).
The Conformational Changes Induced by Brevetoxin
Reveal Similarity to the Effect of Depolarization at Receptor Site 3 on
Rat Brain Sodium Channels
The voltage dependence of
-scorpion toxin binding has been suggested to arise from the
voltage dependence of activation of sodium channels, whereby the state
leading to activation results in a conformational change at the
-scorpion toxin receptor site, leading to reduced affinity for the
toxin (Catterall, 1977, 1979; Ray et al., 1978). We suggest
that PbTx-1 induces a conformational change in receptor site 3 on the
sodium channel that resembles the one caused by depolarization,
resulting in both cases in a decreased affinity of an
-scorpion
toxin to its receptor site.
-scorpion toxin binding, may
induce different conformational changes by binding to receptor site 5,
sufficient to reduce the activation energy but not to produce the
coupling with the
-scorpion toxin receptor. The same reasoning may
apply to the lack of effect of PbTx-1 on
-scorpion toxin receptor
site on cockroach sodium channels. Thus, the use of different
structural analogs of brevetoxins and/or different sodium channel
subtypes may differentiate between the conformational changes that lead
to activation of the channel and those that result in modification of
-scorpion toxin receptor site. This hypothesis, however, deserves
further study.
-scorpion toxin more remarkably from
the depolarized membrane conformation of the sodium channel than from
the one at resting membrane potential (Ray et al., 1978; Jover et al., 1980b). In analogy, veratridine enhances the
-scorpion toxin binding by almost 3-fold at the
brevetoxin-modified receptor, as demonstrated in Fig. 2A, but only by about 1.3-fold at the normal (at
resting potential) receptor state (Fig. 2A, opensymbols). The recent partial localization of the receptor
site for brevetoxin in rat brain sodium channels (Trainer et
al., 1994) is consistent with our hypothesis (see below).
IT to locust sodium channels (Fig. 3) (Gordon and Zlotkin, 1993) and that of
-scorpion
toxin (Css II, from the venom of the scorpion Centruroides suffusus
suffusus) to rat brain sodium channels (Jover et al.,
1980a; Sharkey et al., 1987).
Allosteric Modulation at Receptor Site 3 by
Concurrent Occupancy by Brevetoxin and Veratridine on Rat Brain Sodium
Channels
Our results concerning the combinations of
concomitant effects of brevetoxin and veratridine at the -scorpion
toxin receptor site may be explained in terms of previous studies. Our
data demonstrating that veratridine 1) reverses the inhibition induced
by brevetoxin on AaH II binding (Fig. 1A) and 2) causes
an increase in IC
values for brevetoxin (in the presence
of increasing concentrations of veratridine, Fig. 2B and ) may be explained in terms of increased binding affinity
of veratridine to receptor site 2 in the presence of PbTx-1 (Sharkey et al., 1987; Trainer et al., 1993). The increase in
veratridine binding in turn cooperatively increases the binding of AaH
II to receptor site 3 (Ray et al., 1978) and
``overcomes'' the inhibitory effect of brevetoxin, resulting
in decreased levels of inhibition and the need for higher
concentrations of brevetoxin to induce its effect.
Comparison between Locust and Cockroach Sodium
Channels
In contrast to rat brain, brevetoxin PbTx-1
reveals a positive allosteric modulation at the -scorpion toxin
receptor site in locust sodium channels. The allosteric enhancement in
Lqh
IT binding produced by brevetoxin is similar to that impelled
by veratridine (1.4-1.8-fold, Fig. 3) (Gordon and Zlotkin,
1993). Veratridine has been suggested to increase the affinity of
Lqh
IT only slightly (1.2-fold) and to increase the receptor
capacity by 1.4-1.5-fold (Gordon and Zlotkin, 1993). Brevetoxin,
on the other hand, is suggested to enhance the Lqh
IT binding by
increasing its affinity 1.8-fold, which may fully account for the
binding enhancement (Fig. 3B). These results demonstrate
for the first time the occurrence of allosteric modulation of insect
sodium channels by brevetoxin. However, the allosteric interactions
observed on the locust sodium channels dramatically contrast the lack
of any modulation at the Lqh
IT receptor site observed on the
cockroach sodium channels (Fig. 5).
IT in cockroach CNS. Although we cannot disregard
this possibility, it seems less likely to account for our results.
Veratridine and brevetoxin have been shown to bind and affect a very
large range of sodium channel subtypes in CNS and periphery of various
animal species from vertebrate and invertebrate phyla (Hille, 1992;
Baden, 1989). The similarity between the binding capacity of Lqh
IT
and those previously reported for the excitatory and depressant
insect-selective scorpion toxins (Moskowitz et al., 1994)
suggest that it may represent the majority of sodium channel
population(s) present in cockroach CNS. No information is presently
available on the existence of sodium channel subtypes resistant to
veratridine or scorpion toxins in cockroach CNS, and further study is
required to enable the examination of this possibility. Thus, we
discuss below the alternative explanation that seems to accommodate
more likely with our present results on the differences in allosteric
modulation of Lqh
IT binding in locust and cockroach.
) insect-selective toxins on both locust and
cockroach sodium channels, which markedly differed from the competitive
interactions revealed on other insect neuronal membranes (Moskowitz et al., 1994).
IT receptor sites on cockroach sodium
channels may indicate some structural/functional differences between
cockroach and locust sodium channels.
obtained for Lqh
IT in cockroach neuronal membrane is
lower by at least 10-fold than that obtained in locust sodium
channels.
It may be assumed that the receptor site for
-scorpion toxins in cockroach sodium channels is at its most
favorable high affinity conformational state for the toxin binding, and
therefore it cannot be further positively modified by the allosteric
interactions induced on the channel by brevetoxin and/or veratridine
binding.
Implications for Brevetoxin and
Brevetoxin is proposed to bind at the
transmembrane interface between domains I and IV, near the S5
transmembrane segment of domain IV and the S6 segment of domain I
(Trainer et al., 1994). The binding-induced conformational
changes might induce opening of the channel (Gawley et al.,
1992). Localization of the brevetoxin binding site between domains I
and IV (Trainer et al., 1994) is associated to the
localization of the -Scorpion Toxin
Receptor Sites
-scorpion toxin receptor site to the
extracellular amino acid loops (between transmembrane segments S5 and
S6) in domains I and IV of the rat brain sodium channels (Thomsen and
Catterall, 1989). The proximity in localization of receptor sites for
these two different sodium channel modifiers further rationalizes our
results, which indicate a strong allosteric modulation of the
-scorpion toxin receptor site by brevetoxin in both rat brain and
locust sodium channels.
-helices of the rat and insect sodium channels. The different
perturbation of the intact sodium channel structure induces
differential conformational changes expressed in alteration of the
channel activation function as well as modification of the adjacent
receptor site for
-scorpion toxins and/or receptor site 2.
Disruption or alteration of the structure by substitution with other
hydrophobic residues, as may naturally occur in insect sodium channels,
or by hydrophobic toxin binding (such as brevetoxin and veratridine)
could alter the conformational changes or interactions required for
normal channel gating. The amino acid substitutions, present in sodium
channel transmembrane segments IVS5 and IS6 in rat brain sodium
channels as compared to the Drosophila sodium channels (Noda et al., 1986; Loughney et al., 1989), may contribute
to the differential allosteric modulations detected in our study.
Future site-directed mutagenesis of the rat brain sodium channel may be
guided, in part, by the known differences in Drosophila channels and emphasize the need to reveal the primary structure of
different insect sodium channels, which may lead and suggest new
possible functional differences that are revealed, in part, by our
study.
-scorpion toxins
bind, both in rat brain and insect sodium channels. These may lead to
the location of the amino acids that control gating processes and will
help to clarify the role of these toxins in the alteration of normal
sodium channel function and the mechanism of their allosteric
interactions with other neurotoxins.
Table: Toxins bound by identified neurotoxin receptor
sites on sodium channels
Table: Veratridine increases the IC for
brevetoxin inhibition of
I-AaH II binding in rat brain
synaptosomes
values were determined using the
DRUG analysis in the LIGAND program. The maximal
I-AaH II
binding was taken in the presence of the indicated veratridine
concentration, and the inhibition curves were extrapolated to the
binding determined in the presence of PbTx-1 alone.
-toxin II from the venom of the
scorpion A. australis Hector; [
H]BTX-B,
[
H]batrachotoxin A 20-
-benzoate; CNS,
central nervous system; Lqh
IT,
-toxin specific to insects,
from the venom of the scorpion Leiurus quinquestriatus
hebreus; LqhIT
, depressant insect-selective toxin from
the scorpion L. quinquestriatus hebreus; Lqq V,
-toxin V
from the venom of the scorpion Leiurus quinquestriatus
quinquestriatus, also called LqTx or ScTx; PbTx, brevetoxin from
the marine dinoflagellate Ptychodiscus brevis.
IT
toxin. We are grateful to Procida Co. (Marseille, France) for the
generous gift of the P. americana.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.