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 (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, 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 LqhIT 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 LqhIT, 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. LqhIT 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 LqhIT 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 LqhIT/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.

EXPERIMENTAL PROCEDURES

Toxins

Neuronal Membrane Preparations

Radioiodination

Binding Assay

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-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 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 LqhIT, respectively, and consist 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 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.

Electrophysiological Experiment

Rat Neuronal Cells

Insect Axon

In Vivo Animal Bioassays

RESULTS

Correlation between Toxicity and Binding of Scorpion Toxins

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 LqhIT (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.

Electrophysiological Activity of -Like Scorpion Toxins

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.

Competitive Inhibition of LqhIT Binding on Cockroach and Locust Sodium Channels

The activity of these -like toxins on both mammals and insects allowed the examination of their interaction with LqhIT binding on insect sodium channels. LqhIT 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-LqhIT 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 LqhIT (Kopeyan et al., 1993; Fig. 1), reveals similar IC to that of LqhIT 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 LqhIT 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-LqhIT binding by - and -like scorpion toxins. Insect neuronal membranes were incubated with I-LqhIT and increasing concentrations of the other toxins (as described under ``Experimental Procedures''). The amount of I-LqhIT 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-LqhIT (``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-LqhIT. 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-LqhIT 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 LqhIT binding on both cockroach and locust membranes, but at concentrations higher by about 3-4 orders of magnitude than LqhIT (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 LqhIT.

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 LqhIT in both locust and cockroach at moderate concentrations (Fig. 5D). Unlike the increase in IC detected between cockroach and locust for LqhIT 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 LqhIT.

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 LqhIT to cockroach sodium channels (Fig. 5C), suggesting similarity between their receptor sites.

Allosteric Modulation of LqhIT Binding by Veratridine and Brevetoxin

Figure 6: Effects of concurrent presence of brevetoxin PbTx-1 and veratridine on the binding of I-LqhIT to locust and cockroach neuronal membranes. A, effect of veratridine in the presence of brevetoxin on I-LqhIT binding. Locust neuronal membranes were incubated with 0.1 nMI-LqhIT, 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-LqhIT bound in the presence of brevetoxin alone. The increase in I-LqhIT 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-LqhIT 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 pMI-LqhIT 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-LqhIT binding. Locust neuronal membranes were incubated with 0.1 nMI-LqhIT 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-LqhIT 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 LqhIT 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 LqhIT binding (see Fig. 6B). Thus, veratridine enhances in a synergic manner the binding of LqhIT 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 LqhIT binding may be explained by the increase in concentration of LqhIT receptor sites previously observed in the presence of 100 µM veratridine (Gordon and Zlotkin, 1993). All the available receptor sites for LqhIT are, in turn, modified to a higher affinity state by PbTx-1, resulting in an apparent cooperative increase in LqhIT binding (see Cestele et al.(1995) and Fig. 6B). The effect of brevetoxin on the binding of LqhIT 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 LqhIT binding is additive (Fig. 6B). Brevetoxin was shown to increase the affinity of LqhIT with no effect on the receptor concentration (Cestele et al., 1995). No effect is detected on the binding of LqhIT on cockroach sodium channels under any conditions or combinations tested (Fig. 6). The differences in allosteric modulation of LqhIT binding indicate the presence of structural differences between locust and cockroach sodium channels.

DISCUSSION

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.

Several Groups of -Like Toxins Are Revealed by Activity in Vivo and in Vitro

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 LqhIT. 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 LqhIT 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, LqhIT and Lqq III are considered anti-insect -scorpion toxins.

Scorpion Toxins Receptor Sites Are Homologous But Not Identical on Mammal and Insect Sodium Channels

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 LqhIT 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 LqhIT 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 LqhIT binding inhibition in cockroach as compared to locust neuronal membranes; Fig. 5and Table 3), which conforms with the shift in affinity of LqhIT 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 LqhIT 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 LqhIT in cockroach versus locust (Fig. 5C) conforms that the receptor site for ATX II is highly similar to that of LqhIT on the two insect sodium channels, but different (at least in part) from the one of AaH II. The membrane potential-independent binding of LqhIT is comparable to the ability of ATX II to compete in a potential-independent manner with LqhIT 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 LqhIT 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.

Other Receptor Sites Are Revealed by -Like Toxin Binding

Receptor Site of Lqq IV

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 LqhIT.

Receptor Site for Bom III and IV

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 LqhIT 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 LqhIT.

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 LqhIT, 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; LqhIT and AaH II are preferably active on insect and mammalian sodium channels, respectively).

Comparison between Locust and Cockroach Sodium Channels

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 LqhIT, 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 LqhIT to locust sodium channels (Cestele et al., 1995) (Fig. 6), but reveal no effect on LqhIT 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 LqhIT 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.

FOOTNOTES

*

This work was supported in part by Research Grant 93-00294 from the U.S.A.-Israel Binational Science Foundation (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.

§

Recipient of a fellowship from the French Commissariat a l'Energie Atomique (CEA, Saclay). To whom correspondence should be addressed. Tel.: 33-91-96-20-67; Fax: 33-91-65-75-95; gordon{at}bisance.citi2.fr.

¶

Recipient of a fellowship from the Ministere de la Recherche et de la Technologie.

()

The abbreviations used are: TTX, tetrodotoxin; AaIT, excitatory insect-selective toxin from the scorpion Androctonus australis Hector, also called AaH IT; AaH I-III, -toxins I, II, and III from the venom of the scorpion A. australis Hector; ATX II, toxin II from the sea anemone Anemonia sulcata; Bom III and Bom IV, toxin III and IV from the venom of the scorpion Buthus occitanus mardochei from Mexico; BSA, bovine serum albumin; Css II and Css VI, scorpion -toxins II and VI from the venom of the Mexican scorpion Centruroides suffusus suffusus; E, membrane potential; E, holding potential; LqhIT, -toxin specific to insects, from the venom of the scorpion Leiurus quinquestriatus hebraeus; LqhIT, depressant insect-selective toxin from the scorpion L. quinquestriatus hebraeus; Lqq III-V, -toxins III, IV, and V from the venom of the scorpion L. quinquestriatus quinquestriatus (Lqq V is called also LqTx or ScTx); PbTx-1, brevetoxin from the marine dinoflagellate Ptychodiscus brevis; TxVIA, -conotoxin-TxVIA from Conus textile.

()

D. Gordon and M. Fainzilber, unpublished results.

ACKNOWLEDGEMENTS

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