(Received for publication, July 5, 1995; and in revised form, January 22, 1996)
From the
Sodium channels posses receptor sites for many neurotoxins, of
which several groups were shown to inhibit sodium current inactivation.
Receptor sites that bind - and
-like scorpion toxins are of
particular interest since neurotoxin binding at these extracellular
regions can affect the inactivation process at intramembranal segments
of the channel. We examined, for the first time, the interaction of
different scorpion neurotoxins, all affecting sodium current
inactivation and toxic to mammals, with
-scorpion toxin receptor
sites on both mammalian and insect sodium channels. As specific probes
for rat and insect sodium channels, we used the radiolabeled
-scorpion toxins AaH II and Lqh
IT, the most active
-toxins on mammals and insect, respectively. We demonstrate that
the different scorpion toxins may be classified to several groups,
according to their in vivo and in vitro activity on
mammalian and insect sodium channels. Analysis of competitive binding
interaction reveal that each group may occupy a distinct receptor site
on sodium channels. The
-mammal scorpion toxins and the
anti-insect Lqh
IT bind to homologous but not identical receptor
sites on both rat brain and insect sodium channels. Sea anemone toxin
ATX II, previously considered to share receptor site 3 with
-scorpion toxins, is suggested to bind to a partially overlapping
receptor site with both AaH II and Lqh
IT. Competitive binding
interactions with other scorpion toxins suggest the presence of a
putative additional receptor site on sodium channels, which may bind a
unique group of these scorpion toxins (Bom III and IV), active on both
mammals and insects. We suggest the presence of a cluster of receptor
sites for scorpion toxins that inhibit sodium current inactivation,
which is very similar on insect and rat brain sodium channels, in spite
of the structural and pharmacological differences between them. The sea
anemone toxin ATX II is also suggested to bind within this cluster.
Scorpion venom toxicity to humans has mainly been attributed to
the pharmacological properties of toxic polypeptides that interfere
with the sodium conductance in mammalian excitable tissues. The
principal toxic compounds in scorpion venoms belong to a clearly
defined family of homologous proteins, composed of single chain of
63-70 amino acid polypeptides cross-linked by four disulfide
bridges (Miranda et al., 1970; Kopeyan et al., 1974;
Darbon et al., 1982; Gregoire and Rochat, 1983). These sodium
channel neurotoxins have been classified into several structural groups
on the basis of primary structure (Rochat et al., 1979; Dufton
and Rochat, 1984; Posanni, 1985; Watt and Simard, 1984) and
immunological (Delori et al., 1981) criteria. The four first
groups (I-IV) reveal a good correlation between amino acid
sequences and pharmacological properties and contain the -scorpion
toxins active on vertebrates. The other groups contain the
-scorpion toxins and the toxins active on insect sodium channels
(excitatory and depressant insect-selective toxins) (reviewed by
MartinEauclaire and Couraud(1995)). The specificity of these toxins
vary considerably (Zlotkin et al., 1978). Thus toxins
specifically active on mammals (Miranda et al., 1970),
insects, or crustaceans have been already described (Zlotkin, 1987).
All these different toxins affect sodium conductance in various
excitable tissues, thus serving as important pharmacological tools for
the study of excitability and sodium channel structure.
Voltage-dependent sodium channels are integral plasma membrane proteins responsible for the generation and propagation of action potentials in most excitable tissues. Being a critical element in excitability, sodium channels serve as specific targets for many neurotoxins. These toxins occupy different receptor sites on a sodium channel and have been used as tools for functional mapping and characterization of the channel (reviewed by Catterall(1986, 1992)).
At least six neurotoxin receptor sites have been identified by
direct radiotoxin binding on the mammalian sodium channels and
additional, as yet unidentified receptor sites have been noticed (Table 1). 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:
[H]saxitoxin and TTX (
)(receptor site
1, Gordon et al., 1985); tritiated derivative of batrachotoxin
([
H]batrachotoxin A 20-
-benzoate) 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, Css VI, receptor site 4; Lima et al., 1986, 1989), on
locust, cockroach and other insect neuronal membranes. The presence of
receptor site 5 has been most recently demonstrated by the
electrophysiological activity of brevetoxin on cockroach axons and its
allosteric modulation on Lqh
IT binding on locust sodium channels
(Cestele et al., 1995). The presence of receptor site 6, which
binds the
-conotoxin TxVI, has also been suggested on insect
sodium channels. (
)
Sodium channels from various excitable
tissues and animal phyla contain a major -subunit of about
240-280 kDa (Catterall, 1992; Gordon et al., 1988, 1990,
1993), composed of about 2000 amino acids comprising four homologous
repeated domains (I-IV), each containing six putative
transmembrane
-helices (for a review, see Gordon(1990) and
Catterall(1992)). 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; Pelhate
and Zlotkin, 1982; Cestele et al., 1995) properties. On the
other hand, a possible uniqueness of the insect sodium channels was
suggested by the description of two groups of scorpion toxins that
modify sodium conductance exclusively in insect neuronal preparations,
the excitatory and depressant insect-selective toxins (Pelhate and
Zlotkin, 1982; Zlotkin et al., 1985, 1991). These toxins bind
selectively to insect sodium channels at two distinct receptor sites
(Gordon et al., 1992; Moskowitz et al., 1994) and
therefore indicate the existence of unique features in the structure of
insect channels, as compared to their mammalian counterparts (Gordon et al., 1984, 1992, 1993). 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 structure-function relationship in sodium channels.
Receptor sites for peptide neurotoxins that inhibit sodium current
inactivation in neurons (the classical effect induced by -scorpion
and sea anemone toxins; see Table 1) are of particular interest
for the study of the dynamics of channel gating, since neurotoxin
binding at these extracellular regions can affect the inactivation
process at intramembranal segments of the channel (Catterall, 1992).
The most studied neurotoxins that induce inhibition of sodium current
inactivation are the
-scorpion toxins and sea anemone toxins,
which are believed to share receptor site 3 on sodium channels (Couraud et al., 1978; Catterall and Beress, 1978; Catterall, 1980).
Several
-scorpion toxins have been identified by their high
toxicity to mammals and by a high homology in their amino acid sequence
(reviewed by Martin-Eauclaire and Couraud(1995)).
In the present
study we have used AaH II, the -scorpion toxin that reveals the
highest affinity to rat brain synaptosomes (Jover et al.,
1978), and Lqh
IT, the
-scorpion toxin that reveals
significantly higher activity to insects as compared to 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 to locust neuronal
membranes have been shown to be similar to those described for the
-scorpion toxins Lqq V (Ray et al., 1978) and AaH II
(Jover et al., 1978) on rat brain 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
has been considered to be homologous to receptor site 3 in vertebrate
sodium channels (Eitan et al., 1990; Gordon and Zlotkin, 1993;
Zlotkin et al., 1994).
We have compared the toxic activity and binding interactions of various scorpion toxins on mammals and insects. Three different neuronal sodium channel preparations have been chosen: rat brain synaptosomes, which are the most studied; and two different insect central nervous system membranes, locust and cockroach neuronal membranes, which served for neurotoxin binding studies in insects. Cockroach axons have been used as the main preparation for physiological effects of neurotoxins in insects. We have tested binding interactions of several different scorpion toxins, which reveal peculiarity in their toxic and pharmacological behavior, to get some insight into their possible receptor sites on sodium channels.
The
results of our comparative study suggest that scorpion toxins affecting
inactivation of sodium current may be divided into several different
groups according to their mammal versus insect activities,
each possessing its distinct receptor site on sodium channels. The
-toxin receptor site on sodium channels is suggested to be a
macrosite, which includes the Lqh
IT/Lqq III receptor site that
partially overlaps with both ATX II and AaH II receptor sites. The
other groups of
-like scorpion toxins are suggested to bind to
distinct receptor sites on both rat brain and insect sodium channels,
which interact with receptor site 3. A cluster of receptor sites that
preferentially bind scorpion toxins affecting current inactivation is
suggested to be present on both rat brain and insect sodium channels.
ATX II receptor site is suggested to be included in this cluster.
Rat brain
synaptosomes (100 µg of protein/ml) or insect synaptosomes
(PL, 50 µg/ml and 3.3 µg/ml, for locust and
cockroach, respectively) were suspended in 0.15 or 0.3 ml of 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 filters under vacuum.
Filters were rapidly washed with an additional 2
2 ml of
buffer. Nonspecific toxin binding was determined in the presence of 0.2
µM unlabeled AaH II or 1 µM Lqh
IT,
respectively, and consist 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 for 30 min at 37 °C and those with insect membranes, for 60 min
at 22 °C. Equilibrium saturation or competition experiments were
analyzed by the iterative computer program LIGAND (Elsevier Biosoft).
Each experiment was performed at least three times.
Figure 1: Comparison of scorpion toxin amino acid sequences classified according to their structural homology. A, the structural group is marked on the left (I-IV). The sequences were aligned for maximum similarity by eye inspection. B, a table presenting the percentage of identical and conserved (in brackets) residues calculated for maximum homology between each pair of protein sequences.
Figure 2:
Correlation between the toxicity to mice
(intracerebroventricular) of different scorpion toxins and the
concentration required to inhibit the binding of I-AaH II
to rat brain synaptosomes (IC
), relative to the toxicity
and IC
of AaH II. The data are from Table 2. Abcissa, LD
values of each toxin divided by the
LD
of AaH II; ordinate, IC
values of
each toxin divided by IC
of AaH II. Upper inset,
competitive inhibition curves of several toxins for
I-AaH
II binding to rat brain synaptosomes. The results are presented as
percent of AaH II maximal specific binding with no competitor.
Nonspecific binding, measured in the presence of 200 nM AaH
II, was subtracted from all data points. Lower inset,
enlargement of the correlation curve (main panel, lower
left corner), presenting the correlation between toxicity and
binding inhibition of some classical
-scorpion
toxins.
Examination of the correlation between toxicity to mice
and the toxins' potency in competing for binding of AaH II on rat
brain sodium channels reveals a certain peculiarity ( Table 2and Fig. 2). The graphic presentation of this correlation (Fig. 2) suggests that toxins related to -scorpion toxins
comprise at least four groups: 1) the ``classical''
-toxins, such as AaH I-III and Lqq V (belonging to
structural groups I and II), which reveal a perfect correlation between
their toxicity and binding inhibition properties in rat brain (Fig. 2, lower inset); 2) Lqq IV, which exhibits a
lower toxicity (54-fold less toxic than AaH II) and inhibits AaH II at
significantly higher concentrations than other
-toxins (Table 2) (this toxin holds an intermediate position on the
correlation curve; Fig. 2); 3) Lqq III, which is 2200-fold less
toxic to mice than AaH II, and inhibits the binding of AaH II at very
high concentration ( Fig. 2and Table 2) (Lqq III is highly
homologous to the anti-insect
-toxin Lqh
IT (see Fig. 1) and holds a unique place in this correlation curve); 4)
toxins belonging to structural group III, represented by Bom III and
Bom IV, which are toxic to mice but do not compete for AaH II binding
and consequently do not reveal any correlation between these parameters (Table 2). The peculiarity of these toxins prompt us to
re-examine their toxicity and pharmacology by a comparative approach,
using sodium channels from rat and insect central nervous system.
Figure 3:
Action of AaH II, Bom III, and Bom IV on
isolated rat cerebellar granule cells in culture, under voltage clamp
conditions. Outward Na currents from cerebellar
granule cells were detected before and 3 min after addition of 0.5
nM AaH II (A), 2.5 or 5 nM Bom III (B and C), and 10 or 25 nM Bom IV (D and E). The cells were held at -90 mV, and depolarization
was induced by a 50-ms test pulse to -20 mV. Superimposed traces
before and after addition (arrow) of toxins are shown. Note
the evident toxin effect on slowing the current inactivation and the
slight decrease on Na
peak current (A, C, and E). F and G, I-V
activation curves obtained by 8 mV voltage steps from -60 mV to
+60 mV, before (black circles) and after (open
circles) addition of 0.5 nM AaH II (F) or 25
nM Bom IV (G). No difference in the activation
threshold was observed, but the slope of the curve was decreased after
toxin action (G). Steady-state inactivation curves were
determined using a 200-ms prepulse from -110 mV to +20 mV in
10-mV steps, followed by a test pulse to +40 mV, before (black
squares) and after (open squares) addition of 0.5 nM AaH II (F) or 25 nM Bom IV (G). Note
the left shift of the curves.
Figure 4:
The effects of Bom III on an isolated
cockroach axon under current and voltage clamp. A,
superimposed records of action potentials evoked by a short current
pulse (0.5 ms, 10 nA) during a Bom III (5 µg/ml, 0.625
µM) superfusion. The short control action potential is
progressively transformed into a ``plateau'' potential seen
also in B. B, after 12 min of Bom III application. C, control Na current associated to a 5 ms in
duration voltage pulse to E
= -20 mV
from a holding potential E
= -60 mV
after blockage of I
by 10 mM 3-4
diaminopyridine. Note the complete inactivation of I
after less than 2 ms. D, superimposed recordings every
15 s during the application of 0.5 µg/ml (62.5 nM) Bom
III; note the progressive slowing of the current tracks accompanied
here by a slight increase in the peak current. At the end of the
voltage pulse, the maintained Na
current turns off
rapidly. E, the peak as well as the maintained Na
current are blocked by a 60-s application of TTX (1
µM). Near each trace the time of TTX application is marked
in seconds. F, potassium current associated to a voltage pulse
to E
= +20 mV (E
= -60 mV), after blockage of I
by 1 µM TTX: after a 10-min application of Bom III
(62.5 nM), no significant change is detected in the magnitude
as well as in the kinetics of I
.
In cerebellar granule cells under
voltage-clamp conditions, extracellular addition of 0.5 nM AaH
II induced a classical -scorpion toxin effect, namely a slight,
progressive decrease of the Na
peak current
accompanied by an evident slowing of inactivation time course (Fig. 3A). In the same experimental conditions, the
main effect induced by Bom III and Bom IV was slowing down the decline
of Na
currents (Fig. 3, B-E),
similarly to the one observed with AaH II (Fig. 3A),
but Bom IV affects the sodium conductance at higher concentration (Fig. 3, D and E). The higher concentration of
Bom III and IV needed for maximal effects are in concert with the lower
activity of these toxins on mice (see Table 2). Steady-state
inactivation curves obtained before and after addition of 0.5 nM AaH II or 25 nM Bom IV showed a notable shift to the
left, to more hyperpolarized potentials for both AaH II and Bom IV (Fig. 3, F and G). However, examination of the
current changes induced by AaH II compared to Bom toxins reveals that
the latter affect the Na
conductance in an additional
manner, namely slowing the activation kinetics. Although we did not
quantitatively analyzed the activation kinetics of the sodium currents,
they appear to be slowed by both Bom toxins (Fig. 3, C and E) but not by AaH II (Fig. 3A), as
indicated by the rising phase and time-to-peak current. Unlike AaH II,
Bom IV reduced the slope of the activation curve (Fig. 3G). These discrepancies between the two groups
of toxins could indicate that Bon IV may modify additional properties
of the channel. Since plural mechanisms may account for slowing the
decline of sodium current, including reopening of channels that are
closed along the inactivation pathway as well as those with slowed or
modified activation, further experimentation would be necessary to
determine the exact nature of the mechanism involved. Thus, both Bom
III and IV induce an apparent phenomenologically similar effect to that
of the
-scorpion toxin AaH II on the slowed decline of sodium
currents in mammalian neurons, but reveal difference on the activation
kinetics. The latter may suggest that the Bom toxins exert their
effects by binding to distinct receptor site on the sodium channels.
The similarity in macroscopic effects on the decline of sodium
currents has been further exemplified on cockroach axonal preparation (Fig. 4). AaH II and LqhIT were demonstrated to induce
prolongation of action potentials in an isolated giant axon of the
cockroach due to inhibition of the sodium current turning off (Pelhate
and Zlotkin, 1982; Eitan et al., 1990). Bom III affects the
cockroach axonal membrane in a similar way (Fig. 4A) at
concentrations similar to those needed for insect-selective toxins
activity in this preparation (Eitan et al., 1990; Pelhate and
Zlotkin, 1982). In voltage clamp conditions, 10-fold lower
concentration of Bom III (62.5 nM) inhibits the inactivation
of the sodium current, with no effect on the potassium conductance (Fig. 4B), similar to the effect of
-scorpion
toxins in vertebrate and insect preparation (Duval et al.,
1989; Wang and Strichartz, 1983; Eitan et al., 1990; Pelhate
and Zlotkin, 1982).
Thus, the scorpion toxins listed in Table 3reveal some similar electrophysiological phenomenology on
sodium conductance (inhibition of sodium current inactivation) in both
mammal and insect excitable membranes, as described previously for ATX
II and other polypeptide neurotoxins derived from Conus snail
and coral venom (Catterall and Beress, 1978; Gonoi et al.,
1986, 1987; Hasson et al., 1993; Fainzilber et al.,
1995). Such effects may be a result of many different kinetic
modifications produced by different specific action, following binding
of the chemically different toxins to distinct receptor sites on sodium
channels (see Gonoi et al.(1986, 1987) and Fainzilber et
al.(1994, 1995)). Moreover, the Bom toxins have been shown to
alter, in addition, the activation kinetics (Fig. 3, C and E). Accordingly, Bom III and IV do not interact with
receptor site 3 on vertebrate sodium channels, as indicated by their
inability to inhibit the binding of AaH II in rat brain synaptosomes ( Table 2and Fig. 2, upper inset). For the
convenience of discussion and to be consistent with previous
classification (Vargas et al., 1987; Maritn-Eauclaire et
al., 1992), we suggest to term them as -like scorpion toxins.
-Like toxins include neurotoxins that are toxic to vertebrates,
and induce inhibition of sodium current inactivation by occupying a
different receptor site from that of
-scorpion toxins.
The activity of
these -like toxins on both mammals and insects allowed the
examination of their interaction with Lqh
IT binding on insect
sodium channels. Lqh
IT shares 53-77% identity with other
-scorpion toxins affecting mammals (Fig. 1B), but
it reveals high toxicity to insects (Eitan et al., 1990; Table 3).
Comparative binding study of LqhIT in the two
insect neuronal membrane preparations, from locust and cockroach
central nervous system (Fig. 5) revealed that the affinity of
I-Lqh
IT to cockroach synaptosomes is about
10-15-fold higher than its binding affinity to locust neuronal
membranes (K
= 0.03 ± 0.01 nM in cockroach and 0.46 ± 0.14 in locust; Fig. 5, panels A and B (insets) and panel
D). This is the highest affinity described so far for an
-scorpion toxin to any sodium channel preparation (see Table 3). Lqq III, which possess only three amino acid
substitutions as compared to Lqh
IT (Kopeyan et al., 1993; Fig. 1), reveals similar IC
to that of Lqh
IT
on cockroach sodium channels (Fig. 5C and Table 3). Thus, these two homologous toxins are suggested to
share the same receptor site on insect sodium channels. Depolarization
of the membrane by osmotic lysis does not affect Lqh
IT binding to
cockroach (data not shown), conforming the independence of the binding
on membrane polarization, as described previously in locust (Gordon and
Zlotkin, 1993).
Figure 5:
Competitive inhibition curves for I-Lqh
IT binding by
- and
-like scorpion
toxins. Insect neuronal membranes were incubated with
I-Lqh
IT and increasing concentrations of the other
toxins (as described under ``Experimental Procedures''). The
amount of
I-Lqh
IT bound is expressed as the
percentage of the maximal specific binding in the system without
additional toxins. All curves were analyzed by LIGAND program, and
IC
values were calculated using DRUG analysis. The lines
are drawn by hand. A, cockroach neuronal membranes (1 µg
of protein) were incubated with 30-60 pM of the labeled
toxin. Inset, Scatchard analysis of a saturation binding
curve. The membranes were incubated for 1 h at 22 °C with
increasing concentrations of
I-Lqh
IT
(``hot'' saturation), as described under ``Experimental
Procedures.'' Equilibrium binding constants, obtained by the
computer program analysis (LIGAND) were as follows: K
= 32.9 ± 8.2 pM; B
= 1.85 ± 0.62 pmol/mg protein.
There was a very good accordance between the binding constants obtained
by ``cold'' and ``hot'' saturation curves (0.03
± 0.01 nM, n = 4). B. Locust
neuronal membranes (15 µg of protein) were incubated with 0.1
nM of
I-Lqh
IT. Inset, Scatchard
analysis of a ``cold'' saturation binding curve (see
``Experimental Procedures''). The equilibrium binding
constants, obtained as in A, were: K
= 0.46 ± 0.14 nM; B
= 0.33 ± 0.05 pmol/mg. The IC
values
are presented in Table 3. C-E, comparison between
I-Lqh
IT binding inhibition by various neurotoxins on
cockroach (black symbols) and locust (empty symbols)
neuronal membranes. Note the shifts in the competition curves obtained
by the different inhibitors in locust versus cockroach
membranes (see text). The IC
values are presented in Table 3.
The -toxins highly active on mammals (see Table 2) are able to inhibit Lqh
IT binding on both cockroach
and locust membranes, but at concentrations higher by about 3-4
orders of magnitude than Lqh
IT (Fig. 5, A and B, and Table 3). In accordance, the toxicity of the
classical
-toxins to insects is very low (Table 3). The
inhibitory potency of the classical
-toxins in each insect
neuronal preparation is comparable (IC
around 1 µM in locust and in the range of 60-325 nM in
cockroach; Fig. 5, A and B, and Table 3), supporting the notion that the
-mammal toxins bind
to a homologous, perhaps overlapping receptor site on insect sodium
channels, but with a much weaker affinity, as compared to Lqh
IT.
The toxins that reveal no inhibition on AaH II binding in rat brain
sodium channels, Bom III and Bom IV, but were shown to be active on
mice (Bom III and Bom IV are 12.5 and 3.5 times less active on mice
than AaH II by subcutaneous injection, respectively; Table 3),
are able to compete for LqhIT binding at nanomolar concentrations (Fig. 5, A and B, and Table 3). The
relative higher toxicity of Bom IV as compared to Bom III in insects is
accompanied by lower IC
values in both cockroach and
locust (Fig. 5E and Table 3).
The intermediate
position of Lqq IV, suggested by the correlation of toxicity and
binding in mammals ( Fig. 2and Table 2), is supported by
its very low toxicity to insect (LD in the range of the
classical
-mammal scorpion toxins; see Table 3). However,
Lqq IV competitively inhibits the binding of Lqh
IT in both locust
and cockroach at moderate concentrations (Fig. 5D).
Unlike the increase in IC
detected between cockroach and
locust for Lqh
IT and ATX II inhibition (Fig. 5, C and D), the IC
of Lqq IV is lower in locust (Fig. 5D and Table 3), in contrast to all the
other toxins (Table 3), suggesting that this toxin binds to a
different receptor site than Lqh
IT.
ATX II has been shown to
compete for -scorpion toxins on binding to both rat brain (Couraud et al., 1978; Catterall and Beress, 1978) and locust (Gordon
and Zlotkin, 1993) sodium channels. Accordingly, ATX II inhibits at low
concentration (IC
= 0.53 ± 0.03 nM)
the binding of Lqh
IT to cockroach sodium channels (Fig. 5C), suggesting similarity between their receptor
sites.
Figure 6:
Effects of concurrent presence of
brevetoxin PbTx-1 and veratridine on the binding of I-Lqh
IT to locust and cockroach neuronal membranes. A, effect of veratridine in the presence of brevetoxin on
I-Lqh
IT binding. Locust neuronal membranes were
incubated with 0.1 nM
I-Lqh
IT, in the
presence (full symbols) or absence (empty symbols) of
20 nM brevetoxin PbTx-1, with increasing concentrations of
veratridine. Results are shown as percentage of
I-Lqh
IT bound in the presence of brevetoxin alone.
The increase in
I-Lqh
IT binding by veratridine alone (empty symbols) is shown as percentage of the maximal binding
with no addition. The difference between the two curves (with locust
membranes) indicates the synergic increase in
I-Lqh
IT binding induced by veratridine in the
presence of 20 nM PbTx-1, over the combined effect of both.
The increase in binding by brevetoxin alone equals 121.2 ± 10.4%
(in locust, n = 3). Cockroach membranes were incubated
in the presence of 30 -60 pM
I-Lqh
IT and
increasing concentrations of the two effectors. No significant effect
of veratridine and PbTx-1 was detected in cockroach membranes, under
any experimental conditions. B, effect of brevetoxin PbTx-1 on
the veratridine-increased
I-Lqh
IT binding. Locust
neuronal membranes were incubated with 0.1 nM
I-Lqh
IT in the presence (full symbols)
or absence (empty symbols) of 100 µM veratridine,
with increasing concentrations of brevetoxin PbTx-1. Results are shown
as percentage of maximal
I-Lqh
IT bound with no
additions. Cockroach neuronal membranes were incubated as in A, and no effect of brevetoxin was
detected.
In contrast to the situation in locust, neither veratridine nor
brevetoxin reveals any significant effect on LqhIT binding on
cockroach sodium channels (Fig. 6). To further examine this
discrepancy, we tested the effects of concurrent presence of both
lipid-soluble sodium channel activators on the binding of Lqh
IT in
the two insect neuronal membranes. The effect of veratridine is further
enhanced by 2-fold in the presence of 20 nM brevetoxin (over
the combined effects of veratridine and brevetoxin; Fig. 6A). Brevetoxin (at 20 nM) alone induces
121 ± 10% increase in Lqh
IT binding (see Fig. 6B). Thus, veratridine enhances in a synergic
manner the binding of Lqh
IT at the brevetoxin-modified receptor
site in locust sodium channels (Fig. 6A). The synergic
effect of veratridine in the presence of 20 nM brevetoxin on
Lqh
IT binding may be explained by the increase in concentration of
Lqh
IT receptor sites previously observed in the presence of 100
µM veratridine (Gordon and Zlotkin, 1993). All the
available receptor sites for Lqh
IT are, in turn, modified to a
higher affinity state by PbTx-1, resulting in an apparent cooperative
increase in Lqh
IT binding (see Cestele et al.(1995) and Fig. 6B). The effect of brevetoxin on the binding of
Lqh
IT has been measured in the presence of saturating
concentration (100 µM) of veratridine. As is demonstrated
in Fig. 6B, the effect of PbTx-1 on the veratridine
increase in Lqh
IT binding is additive (Fig. 6B).
Brevetoxin was shown to increase the affinity of Lqh
IT with no
effect on the receptor concentration (Cestele et al., 1995).
No effect is detected on the binding of Lqh
IT on cockroach sodium
channels under any conditions or combinations tested (Fig. 6).
The differences in allosteric modulation of Lqh
IT binding indicate
the presence of structural differences between locust and cockroach
sodium channels.
The present study examines, for the first time, the
interaction of different scorpion neurotoxins, all affecting sodium
current inactivation and toxic to mammals, with -scorpion toxin
receptor sites on sodium channels in mammals versus insects.
Our results suggest that
- and
-like (see
``Results'') scorpion toxins may be divided into several
groups, according to their activity on mammalian and insect sodium
channels. Each group may occupy a distinct receptor site on sodium
channels and form together a putative macrosite (see below and
Fainzilber et al. (1995)). This macrosite, which is composed
of receptor sites for scorpion toxins that inhibit sodium current
inactivation, is very similar on insect and rat brain sodium channels,
in spite of the structural and pharmacological differences between
them. The sea anemone toxin ATX II is also suggested to bind within
this macrosite.
The third group
consists of Bom III and IV, which are shown to be active on both insect
and mice and compete at nanomolar concentrations for the binding of
LqhIT to insect sodium channels, but do not inhibit at all the
binding of AaH II to rat brain synaptosomes. Bom III and IV are
similarly active on mice and on insects (Table 3) and inhibit
sodium current inactivation in both rat neuronal cells (Fig. 3)
and in cockroach axon (Fig. 4). The fourth group consists of Lqq
III and Lqh
IT. These two homologous toxins demonstrate the highest
affinity to insects, as opposed to the very low affinity to rat brain
sodium channels (Table 3). The activity of Lqh
IT is very
similar to that of Lqq III, but it reveals slightly higher specificity
to insects versus mammals, which is also reflected by its
lower ability to inhibit the binding of AaH II in rat brain membranes
(as compared to Lqq III, Table 3). Thus, Lqh
IT and Lqq III
are considered anti-insect
-scorpion toxins.
The
positive cooperative interaction observed between veratridine and
-scorpion toxins (Lqq V and AaH II) on rat brain sodium channels
(Ray et al., 1978; Jover et al., 1980b; Cestele et al., 1995), comparable to the cooperativity detected
between veratridine and Lqh
IT binding on locust sodium channel (Fig. 6) (Gordon and Zlotkin, 1993; Cestele et al.,
1995), further support the similarity in the
-scorpion toxins
receptor sites on insect and rat brain sodium channels.
The low
affinity revealed by the -mammal toxins on insects is in contrast
to the high affinity observed on rat brain sodium channels, indicating
differences in receptor site structures on mammal versus insect sodium channels. However, the complete inhibition of
Lqh
IT binding, especially on cockroach sodium channels and the
shift in affinity detected in cockroach versus locust (which
correspond to a concentration change of about 1 order of magnitude
between Lqh
IT binding inhibition in cockroach as compared to
locust neuronal membranes; Fig. 5and Table 3), which
conforms with the shift in affinity of Lqh
IT on these insect
sodium channels (Fig. 5D), supports that the
competition may result from binding to homologous, similar or
overlapping receptor sites.
The sea anemone toxin ATX II and the
-scorpion toxins AaH II and Lqq V have been shown to compete on
binding to vertebrate excitable cells and to have similar
pharmacological and electrophysiological activities (Couraud et
al., 1978; Jover et al., 1978; Catterall and Beress,
1978; Salgado and Kem, 1992). On this basis they were considered to
bind to a common receptor site on mammalian sodium channels. The
competition of ATX II for
-mammal toxins binding on rat brain as
well as for Lqh
IT binding on insect sodium channels (Gordon and
Zlotkin, 1993) (Fig. 5C and Table 3) strongly
suggests that these
-scorpion toxins, having different specificity
to mammal versus insect sodium channels, may bind to closely
related receptor sites, which might also (at least partially) overlap
with ATX II in the different sodium channel subtypes.
Our results
demonstrate that ATX II and AaH II reveal inverse affinities toward
insect and mammal sodium channels, as detected by their competitive
inhibition on LqhIT binding; the IC
of AaH II on
insect sodium channels is increased by about 2 orders of magnitude, in
contrast to a similar decrease in IC
of ATX II (Table 3). These contrary affinities may indicate that at least
some of the recognition sites that are involved in the high affinity
binding of these two different toxins might be chemically different on
mammal and insect sodium channels. The comparable shift in IC
values between ATX II and Lqh
IT in cockroach versus locust (Fig. 5C) conforms that the receptor site
for ATX II is highly similar to that of Lqh
IT on the two insect
sodium channels, but different (at least in part) from the one of AaH
II. The membrane potential-independent binding of Lqh
IT is
comparable to the ability of ATX II to compete in a
potential-independent manner with Lqh
IT for binding in locust
neuronal membranes (Gordon and Zlotkin, 1993), further supporting the
notion that ATX II receptor site might be very similar to that of
Lqh
IT on insect sodium channels. These and previous (Catterall and
Beress, 1978; Catterall and Coppersmith, 1981; Frelin et al.,
1984; Renaud et al., 1986) results suggest that ATX II and
-scorpion toxins may not bind to identical receptor site on
mammalian sodium channels, but rather to overlapping (at least in part)
sites.
The specificity and differences in the insect versus mammal activity of the - and
-like scorpion toxins may
be attributed, in part, to structural differences among both the toxins
and the homologous receptor sites on insect and mammalian sodium
channels. Clarification of the structural basis for selectivity in
action of toxins will require three-dimensional structural knowledge of
the toxins coupled with molecular localization of the amino acids
directly interacting with the recognition points within the receptor
site structure and are important areas of future studies.
The lack of correlation between toxicity and IC of Lqq
IV in mammals ( Table 2and Table 3) suggest that this
structurally different toxin (Fig. 1) may bind to a distinct
receptor site also on rat brain sodium channels. The relatively lower
toxicity ratio as compared to the IC
ratio (Table 2)
suggest that Lqq IV's relatively weak competitive inhibition on
AaH II binding is due to a steric interference between their binding
areas, suggesting the presence of distinct receptor site for each.
Presently, no direct binding data are available on Lqq IV, making it
difficult to relatively localize its binding area. It is suggested to
occupy a closely related area to those of AaH II and Lqh
IT.
In contrast to the lack of
interaction between AaH II and Bom III and IV on rat brain
synaptosomes, the binding of LqhIT to insect sodium channels is
inhibited by nanomolar concentrations of these toxins (Fig. 5).
The two toxins reveal similar IC
values in locust and
cockroach, in contrast to the marked shift in IC
detected
with other toxins (Fig. 5E and Table 3). These
results suggest that Bom III and IV may bind to a separate receptor
site than Lqh
IT on insect sodium channels. Unlike the situation in
rat brain synaptosomes, the receptor sites for
-scorpion toxins
and Bom III and IV must be present on the same insect sodium channel
population. Bom III receptor site (or binding areas) may partially
overlap or be in a close proximity to that of Lqh
IT.
These
results may suggest that each -like toxin group binds to a
different receptor site on the sodium channel extracellular surface.
The competitive binding interactions observed among the most specific
scorpion toxins to mammal and insect sodium channels, AaH II and
Lqh
IT, respectively, suggest that all the
-like scorpion
toxins may bind to a common area, or a macrosite, present on sodium
channels in the different animal phyla, and shared also by the sea
anemone toxin ATX II. Interestingly, the
-conotoxins (Fainzilber et al., 1994, 1995) may occupy a different area, or macrosite
on the sodium channel surface (see Fainzilber et al.(1995) for
a tentative model). All these peptide toxins reveal similar apparent
electrophysiological effect, namely inhibition of sodium current
inactivation, with different specificity to various animal groups
(
TxVIA is active only on mollusk sodium channels; Lqh
IT and
AaH II are preferably active on insect and mammalian sodium channels,
respectively).
The affinity of LqhIT is 10-fold higher in
cockroach as compared to locust sodium channels (Table 3, Fig. 5). Similar change in affinity is revealed by ATX II and
some
-mammal scorpion toxins (Table 3, Fig. 5). These
differences in binding interactions observed with the various toxins
indicate that the receptor sites for Lqh
IT, that may be shared (or
partially overlap) also by these other toxins, may differ in structure
on the two insect sodium channels. The cockroach sodium channels form
receptor sites with the highest affinity.
Allosteric interactions
between brevetoxin, veratridine, and LqhIT receptor sites provide
further evidence for the structural differences between sodium channels
in locust and cockroach central nervous system. Both lipophilic sodium
channel activators (brevetoxin and veratridine) cooperatively enhance
the binding of Lqh
IT to locust sodium channels (Cestele et
al., 1995) (Fig. 6), but reveal no effect on Lqh
IT
binding to cockroach sodium channels, not even under concurrent
presence of both allosteric modulators (Fig. 6). 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. Hence, the lack of
allosteric interaction between these receptor sites on cockroach sodium
channels may indicate some structural/functional difference between
cockroach and locust sodium channels, perhaps also in the coupling
between receptor sites of Lqh
IT and brevetoxin and veratridine.
The differences revealed by -scorpion toxin binding between
locust and cockroach sodium channels are in accordance with previous
biochemical examination of various insect neuronal sodium channel
polypeptides. Sodium channel proteins immunoprecipitated from various
insect central nervous systems revealed variations in their molecular
mass and partial proteolytic peptide maps, indicating the presence of
structural differences among them (Gordon et al., 1990, 1993;
Moskowitz et al., 1994).
Our results suggest that the
structurally related -like scorpion toxins may be classified
according to their relative specificity in action and binding to
mammals and insect sodium channels. Despite the competitive binding
interaction, each toxin group is suggested to bind to a distinct,
different receptor site, which together may confine a large macrosite
on the extracellular surface on sodium channels. Such a macrosite,
which preferentially bind scorpion toxins affecting current
inactivation and is shared also by ATX II, is suggested to be present
on both rat brain and insect sodium channels, despite the structural
and pharmacological differences among them.
Our study emphasizes the lack of structural information on the molecular level on these receptor sites. Localization of the attachment points comprising these receptor sites may shed light on the mechanism of action of toxins that modify sodium channel gating. Use of known selective sodium channel neurotoxins as pharmacological sensors for minor, subtle differences in their receptor sites on sodium channels in different animal phyla may provide a rational approach to this complex problem, and contribute to the elucidation of the structural basis for their selectivity and to structure-function relationship in sodium channels.