©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Scorpion Toxins Binding on Rat Brain and Insect Sodium Channels Reveal Divergent Allosteric Modulations by Brevetoxin and Veratridine (*)

Sandrine Cestèle (1)(§), Rym Ben Khalifa (2), Marcel Pelhate (2), Hervé Rochat (1), Dalia Gordon (1)(¶)

From the (1)Faculty of Medicine Nord, Institut Féderatif de Recherche Jean Roche, Laboratory of Biochemistry, URA CNRS 1455, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France and the (2)Laboratory of Neurophysiology, URA CNRS 611, University of Angers, 49045 Angers Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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-LqhIT, 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.


INTRODUCTION

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),()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).

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 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 (LqhIT) 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.

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 -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.

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 -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.

We have used AaH II, the most active -scorpion toxin on vertebrates (Jover et al., 1980b), and LqhIT, 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. LqhIT 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 LqhIT 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).

Our comparative approach reveals that the two groups of lipid-soluble activators of sodium channels, brevetoxin and veratridine, modulate the binding of an -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.


EXPERIMENTAL PROCEDURES

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). LqhIT used for nonspecific binding determinations and brevetoxin PbTx-1 were from Latoxan (A.P. 1724, 05150 Rosans, France). Veratridine was from Sigma. Carrier-free NaI 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-LqhIT to locust neuronal membranes. A, locust neuronal membranes (15 µg of protein) were incubated with 0.1 nMI-LqhIT 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 LqhIT, was subtracted. The amount of I-LqhIT 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 LqhIT 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 LqhIT, 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. LqhIT was iodinated by Iodogen (Pierce) using 5 µg of toxin and 0.5 mCi carrier free NaI 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.

Rat brain synaptosomes (100 µg of protein/ml) or insect synaptosomes (PL, 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-LqhIT, 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 LqhIT, respectively, and consists typically of 15-20% of total binding for I-AaH II or I-LqhIT, 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).

Normal physiological saline had the following composition (in mM): NaCl, 200; KCl, 3.1; CaCl, 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.


RESULTS

Inhibition of -Scorpion Toxin Binding by Brevetoxin PbTx-1 on Rat Brain Sodium Channels

Preliminary experiments revealed that the specific binding of 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).

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) 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 nMI-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 nMI-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 nMI-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

Previous studies have demonstrated that veratridine enhanced the binding of radiolabeled -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).

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 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 nMI-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).

As can be seen in , veratridine at higher concentrations (100 µM and above, see Fig. 2A) induces a significant shift in the IC 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 ().

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).

Effect of Brevetoxin PbTx-1 on Insect Sodium Channels

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 -scorpion toxin receptor site on insect sodium channels, we have used synaptosomes prepared from CNS of both locust and cockroach for comparative purposes.

We used I-LqhIT as specific probe for receptor 3 in insect sodium channels. LqhIT binds to a single class of high affinity receptor sites in locust (Gordon and Zlotkin, 1993) and cockroach()sodium channels. The binding of LqhIT to locust neuronal membrane has been demonstrated to be cooperatively increased by veratridine, whereby 100 µM veratridine increases both the affinity and capacity of LqhIT 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 LqhIT to Locust Sodium Channels

Brevetoxin PbTx-1 increases 1.8-fold the binding of LqhIT 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 LqhIT (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 LqhIT binding in cockroach neuronal membranes have been studied in parallel under similar conditions. Since most recently we have shown that LqhIT binds with a 10-fold higher affinity to cockroach sodium channels (K about 20-30 pM) than to locust, I-LqhIT 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 LqhIT 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-LqhIT (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 LqhIT 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 5.10M 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).

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 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.

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 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.

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).


DISCUSSION

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 -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.

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 -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.

Other brevetoxin analogs, which have been reported to be inactive on -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.

Alkaloid toxins, such as veratridine, are able to increase the affinity of the -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).

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 LqhIT 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 LqhIT 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 LqhIT 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 LqhIT 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 LqhIT receptor site observed on the cockroach sodium channels (Fig. 5).

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 LqhIT 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 LqhIT 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 LqhIT binding in locust and cockroach.

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) 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).

Hence, the lack of allosteric interaction between brevetoxin and LqhIT receptor sites on cockroach sodium channels may indicate some structural/functional differences between cockroach and locust sodium channels.

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 obtained for LqhIT 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 -Scorpion Toxin Receptor Sites

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 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.

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 -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.

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 -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

The IC 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.



FOOTNOTES

*
This work was supported by a research grant from the Franco-Israeli Association for Scientific and Technologic Research (AFIRST) (to H. R.) and from U.S.A.-Israel Binational Science Foundation Grant 93-00294 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A recipient of a fellowship from the ``Ministere de la recherche et de la technologie.''

To whom correspondence should be addressed. Tel.: 33-91-96-20-67; Fax: 33-91-65-75-95; E-mail: gordon@citi2.fr.

The abbreviations used are: BTX, batrachotoxin; AaH II, -toxin II from the venom of the scorpion A. australis Hector; [H]BTX-B, [H]batrachotoxin A 20--benzoate; CNS, central nervous system; LqhIT, -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.

D. Gordon and S. Cestèle, unpublished results.

Y. Pichon, personal communication.


ACKNOWLEDGEMENTS

Our sincere thanks to Prof. Eliahu Zlotkin from the Hebrew University of Jerusalem for the kind gift of the LqhIT toxin. We are grateful to Procida Co. (Marseille, France) for the generous gift of the P. americana.


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