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(The FASEB Journal. 2006;20:1933-1935.)
© 2006 FASEB

Allosteric interactions between scorpion toxin receptor sites on voltage-gated Na channels imply a novel role for weakly active components in arthropod venom

Lior Cohen, Noa Lipstein and Dalia Gordon1

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel.

1Correspondence: Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel. E-mail: dgordon{at}post.tau.ac.il

ABSTRACT

Scorpion beta and alpha-toxins modify the activation and inactivation of voltage-gated sodium channels. Although the two types of toxin bind at two distinct receptor sites on the same sodium channel, they exhibit synergic effects when coinjected into insects. To clarify the basis of this synergism we examined the mutual effects of alpha and beta toxin representatives in radio-ligand binding assays. We found positive allosteric interactions between receptor site-4 of the excitatory Bj-xtrIT and the depressant LqhIT2 beta toxins and receptor site-3 of the alpha toxin Lqh{alpha}IT, on locust neuronal membranes. Unexpectedly, a nontoxic mutant Bj-xtrIT-E15R, which binds with high affinity to receptor site-4, was able to enhance Lqh{alpha}IT binding and toxicity similarly to the unmodified Bj-xtrIT. This result indicates that mere binding of a nontoxic ligand to receptor site-4 ("silent binding") induces a conformational change that does not alter channel gating, but influences toxin binding at receptor site-3 leading to enhanced toxicity. This finding suggests a new functional role for weakly toxic polypeptides in that they enhance the effect of other active neurotoxins in the arthropod venom. Such silent binding may have also valuable implications in attempts to improve drug efficacy by combining potent drugs with nonactive allosteric enhancers.—Cohen, L., Lipstein, N., Gordon, D. Allosteric interactions between scorpion toxin receptor sites on voltage-gated Na channels imply a novel role for weakly active components in arthropod venom.


Key Words: ligand binding • toxicity to insects • gating modifiers • scorpion alpha-toxin • beta-toxin

VOLTAGE-GATED SODIUM CHANNELS (NaCh) are critical for generation and propagation of action potentials in excitable cells, and are targeted by a large variety of chemically distinct compounds that bind to several receptor sites on the pore-forming {alpha}-subunit (1 , 2) . Most lipid-soluble NaCh activators, including pyrethroid insecticides, toxic alkaloids (e.g., veratridine and batrachotoxin), and marine cyclic polyether toxins (e.g., brevetoxins), affect NaChs of both insects and mammals. However, several scorpion toxins show a preference for distinct NaCh subtypes of mammals or insects (3 4 5) and may serve as a guide in the design of selective drugs and insecticides.

Scorpion toxins that modulate NaCh gating are divided into {alpha} and ß classes according to their mode of action and binding features to distinct receptor sites (3 , 6) . {alpha}-Toxins prolong the action potential by inhibition of the fast inactivation of NaChs upon binding to receptor site-3 (1 , 6 7 8) , which involves extracellular loops in domains 1 and 4 of the channel (1 , 9) . These toxins are divided into three groups according to their preference for neuronal NaChs of insects and mammals: 1) anti-mammalian {alpha}-toxins (e.g., Aah2 from Androctonus australis hector, and Lqh2 from Leiurus quinquestriatus hebraeus), which are highly toxic in mammalian brain and bind with high affinity to rat brain NaChs (6 , 10 , 11) ; 2) anti-insect {alpha}-toxins (e.g., Lqh{alpha}IT), which are highly toxic to insects and show weak activity in mammalian brain (7 , 8) ; and 3) {alpha}-like toxins (e.g., Bom3 and Bom4 from Buthus occitanus mardochei, and Lqh3), which show considerable toxicity by injection into rat brain and to insects, compete well with Lqh{alpha}IT on the insect receptor site-3 but bind weakly to rat brain synaptosomes (4 , 8 , 10 11 12) .

ß-Toxins shift the voltage dependence of channel activation to more negative membrane potentials upon binding to receptor site-4, assigned mainly to external loops in domain 2 of mammalian and insect NaChs (5 , 13 14 15) . Like {alpha}-toxins, these toxins are divided into 1) anti-mammalian ß-toxins (e.g., Css2 and Css4 from Centruroides suffusus suffusus; refs. 3 , 6 ); 2) ß-toxins that bind to and affect both insect and mammalian NaChs (e.g., Ts1 from Tityus serrulatus and Lqhß1; refs. 6 , 16 ); and 3) anti-insect selective depressant and excitatory ß-toxins, which have been characterized and typified by the symptoms they produce in blowfly larvae and their structures (3 , 17 18 19) . Depressant toxins induce a slow progressive onset of flaccid paralysis preceded by a short transient phase of contraction (e.g., LqhIT2 and Lqh-dprIT3; refs. 20 , 21 ). Excitatory toxins induce an immediate contraction paralysis upon injection (e.g., AahIT; (17) and Bj-xtrIT from Buthotus judaicus; 2 ), and are unique in structure (18 ,23) .

Alterations in the binding of {alpha}-toxins to receptor site-3 on rat brain and insect NaChs have been shown to be induced allosterically by a variety of NaCh lipophilic activators (e.g., site-2 toxins like veratridine, site-5 toxins like brevetoxin, and site-7 toxins like pyrethroids; refs. 1 , 4 , 8 , 24 25 26 27 ). Also, the binding of scorpion ß-toxins was shown to be enhanced by brevetoxins (27 , 28) , and a decrease in binding of the delta-conotoxin {delta}-TxVIA (from the marine snail Conus textile) to receptor site-6 on NaChs was detected in the presence of veratridine (29) . These reports demonstrated the sensitivity and flexibility of the NaCh protein for alterations induced by ligand binding at various sites. Moreover, synergic effects between pharmacologically different scorpion toxins, which do not compete in binding (30) , were observed when coinjected into blowfly and lepidopetra larvae (31) . However, no explanation for this synergism was provided.

Using quantitative toxicity and binding assays, we show for the first time allosteric interactions between the scorpion toxin receptor sites 3 and 4, which bind {alpha}- and ß-toxins, on insect NaChs. We also analyzed whether a nontoxic mutant, Bj-xtrIT-E15R, which binds receptor site-4 on insect NaChs with affinity almost as high as that of the unmodified toxin and acts as a competitive antagonist of Bj-xtrIT in vivo (32) , has an effect on the activity of {alpha}-toxins. Our results shed new light on the synergic effects of {alpha}- and ß-toxins on neuronal excitability in vivo and demonstrate that mere binding of a ligand, independent of its toxicity, to receptor site-4 enhances {alpha}-toxin activity. These results imply a novel role for weakly active or nontoxic polypeptides in animal venoms as enhancers of the effect of other active neurotoxins.

MATERIALS AND METHODS

Toxins
Bj-xtrIT, LqhIT2, Lqh-dprIT3, and Lqh{alpha}IT were produced in a recombinant form as previously described (21 , 22) . Brevetoxin PbTx-2 (from the marine dinoflagellate Ptychodiscus brevis), Lqhß1, and Lqh3 were purchased from Latoxan (Valence, France, E-mail: latoxan{at}latoxan.com).

Toxicity assays
Four-day-old blowfly larvae (Sarcophaga falculata; 150±20 mg body wt) were injected intersegmentally with five to seven concentrations of each toxin or toxin combinations (nine larvae in each group) in three independent experiments. ED50 (effective dose 50%) values were calculated as previously described (32 , 33) . A positive result for Lqh{alpha}IT, Lqh3, and Bj-xtrIT was scored when a characteristic contraction paralysis was observed 1–5 min postinjection (longer durations did not change the ED50 scores). Toxins were mixed in various ratios and the ED50 of each mixture was scored. Toxin mixtures in ratios providing the maximal enhancement of toxicity are presented in Table 1 (mixture ED50). When a toxin mixture was injected at doses close to the mixture ED50 values (Lqh{alpha}IT+Bj-xtrIT and Lqh3+Bj-xtrIT), the effect developed more slowly, and so the contraction paralysis was scored after 10–15 min. For toxins LqhIT2, Lqh-dprIT3, and Lqhß1, the positive effect was a characteristic transient immobilization and contraction replaced by gradually increasing flaccidity observed 1–5 min after injection. For toxin mixtures, the positive effect was scored as sustained paralysis (contraction and/or flaccidity) and observed for up to 20 min after injection.


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  		Table 1. Toxicity of scorpion toxins to blowfly larvaea

Binding experiments
Locust synaptosomes were prepared from dissected brains and ventral nerve cords of adult locusts (Locusta migratoria) using an established method (8) . Membrane protein concentration was determined by a Bio-Rad protein assay, using BSA as standard. All toxins were radio-iodinated by lactoperoxidase (Sigma, St. Louis, MO, USA; Cat. No., L8257; 7 U per 60 µl reaction mix) using 10 µg toxin and 0.5 mCi carrier-free Na125I (Amersham, UK) following a published protocol (34) . The monoiodotoxin was purified using an analytical Resource RP-HPLC column (6.4x100 mm, 15 µm particle size; Amersham, Sweden). The concentration of the radiolabeled toxin was determined according to the specific activity of the 125I corresponding to 2500 to 3000 dpm/fmol of monoiodotoxin, depending on the age of the radiotoxin and by estimation of its biological activity (usually 70–80%; ref. 35 ). The composition of the media used in the binding assays and termination of the reactions have been described (35) . Nonspecific toxin binding was determined in the presence of 1–30 µM of the unlabeled toxin, as specified in the figure legends. Equilibrium competition binding assays were performed using increasing concentrations of unlabeled toxin in the presence of a constant low concentration of 125I-toxin and analyzed by the computer program KaleidaGraph (Synergy Software, Essex Junction, VT, USA) using a nonlinear Hill equation (for IC50 determination, with Hill coefficient set to 1). The Ki values were calculated by the equation Ki = IC50/(1+(L*/Kd)), where L* is the concentration of radioiodinated toxin and Kd is its dissociation constant. Each experiment was performed in duplicate and repeated at least three times as indicated (n). Data are presented as mean ± SE of the number of independent experiments (35) .

RESULTS

Effects of in vivo co-administration of scorpion {alpha}- and ß-toxins
We first examined the mutual effects of scorpion {alpha}- and ß-toxins on insects by using potent insecticidal toxins: Lqh{alpha}IT and Lqh3 alpha anti-insect and {alpha}-like toxins (7 , 11) , Bj-xtrIT, an anti-insect selective excitatory ß-toxin (22) , LqhIT2 and Lqh-dprIT3 anti-insect depressant ß-toxins (20 , 21) , and Lqhß1, which induces typical effects of depressant toxins upon injection into blowfly larvae (16) . We quantified the effects of mixtures containing toxins of the same pharmacological class (Lqh{alpha}IT and Lqh3 or Bj-xtrIT and LqhIT2) by injection into blowfly larvae (Table 1) . The mixture ED50 values obtained indicated that the effects produced by toxins of the same class were additive (Table 1) . In contrast, injection of mixtures of {alpha}- and ß-toxins (Lqh{alpha}IT+Bj-xtrIT; Lqh3+Bj-xtrIT; Lqh{alpha}IT+LqhIT2; Lqh3+LqhIT2; Lqh{alpha}IT+Lqh-dprIT3; Lqh{alpha}IT+Lqhß1) reduced by 4–10 fold the dose required for each participating toxin to generate an effect, as manifested in the mixture ED50 values (Table 1) . This indicated a positive cooperativity between the {alpha}- and ß-toxins in vivo.

Effects of Bj-xtrIT on Lqh{alpha}IT binding to locust neuronal membranes
To clarify the basis of the synergic effect observed in toxicity, we analyzed the effect of Bj-xtrIT on 125I-Lqh{alpha}IT binding. Bj-xtrIT increased the specific binding of 125I-Lqh{alpha}IT to locust synaptosomes to 147 ± 12%, with an EC50 (EC50) of 43 ± 8.5 nM (n=3, Fig. 1 ). This result indicated an allosteric interaction between receptor sites 3 and 4 on locust sodium channels. To further analyze the enhancement in Lqh{alpha}IT binding, we used the nontoxic mutant Bj-xtrIT-E15R (no effect on blowfly larvae in doses 10,000-fold higher than the ED50 of the unmodified toxin) (32) . Unexpectedly, Bj-xtrIT-E15R increased the binding of 125I-Lqh{alpha}IT to locust synaptosomes to 152 ± 18% with an EC50 of 21 ± 3.5 nM (n=3), which was similar to the effect obtained with the unmodified toxin (Fig. 1) . Since brevetoxins (PbTxs that bind to receptor site-5) have been shown to allosterically increase the binding of Lqh{alpha}IT to locust synaptosomes (8) , we examined the effect of Bj-xtrIT-E15R on 125I-Lqh{alpha}IT binding in the presence of saturating concentrations of PbTx-2. Brevetoxin PbTx-2 (1 µM) alone increased 125I-Lqh{alpha}IT binding by 30%, and the enhancement by Bj-xtrIT–E15R was additive throughout the entire concentration range (Fig. 1) . These results indicate that the effects of Bj-xtrIT-E15R and brevetoxin are independent, which is in concert with an earlier report showing that the receptor sites of Bj-xtrIT and brevetoxin do not interact allosterically in this neuronal preparation (27) .


Figure 1
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  		Figure 1. Effects of Bj-xtrIT and Bj-xtrIT-E15R on 125I-Lqh{alpha}IT binding to locust neuronal membranes. Membranes (160 µg) were incubated with 0.1 nM 125I-Lqh{alpha}IT for 45 min at room temperature. Nonspecific binding, measured in the presence of 1 µM of unlabeled Lqh{alpha}IT (comprised 10–15% of total binding), was subtracted. The dashed line indicates maximal specific binding of 125I-Lqh{alpha}IT under control conditions. The Ki value of Lqh{alpha}IT was 1.1 ± 0.2 nM (n=3). Maximal increase in 125I-Lqh{alpha}IT binding in the presence of Bj-xtrIT or Bj-xtrIT-E15R was 147 ± 12% and 152 ± 18% of the control, respectively, with effective concentrations 50% (EC50 values) of 43 ± 8.5 and 21 ± 3.5 nM (n=3), respectively. Brevetoxin PbTx2 at 1 µM increased 125I-Lqh{alpha}IT binding by 30%. In the presence of PbTx2, the maximal increase of 125I-Lqh{alpha}IT binding by Bj-xtrIT-E15R was 180 ± 14%, with no significant change in its EC50 value (23±4.5 nM, n=3). Data points represent mean ±SE of 3 independent experiments.

Enhancement of Lqh{alpha}IT toxicity by Bj-xtrIT-E15R
The unexpected enhancement of 125I-Lqh{alpha}IT binding by the nontoxic Bj-xtrIT-E15R mutant prompted us to examine its effect on the toxicity of {alpha}-toxins. Whereas injection of increasing concentrations of Bj-xtrIT-E15R together with Bj-xtrIT raised its ED50 >10-fold, coinjection of this mutant with Lqh{alpha}IT or Lqh3 decreased progressively the {alpha}-toxin dose needed for paralysis (Fig. 2 ). This positive cooperativity was obtained at a low EC50 of Bj-xtrIT-E15R (12–15 ng per 100 mg larvae, equivalent to 15–18 nM), which correlated well with the high binding affinity of this mutant to insect sodium channels (32) . Yet the maximal enhancement in Lqh{alpha}IT and Lqh3 toxicity (3-fold, Fig. 2 ) was significantly smaller than that obtained with the wild-type Bj-xtrIT (~5-fold, Table 1 ). Together with the binding assays, these results imply that binding per se of Bj-xtrIT-E15R to receptor site-4 on the insect sodium channel renders an allosteric effect that enhances both the binding and the toxicity of scorpion {alpha}-toxins (Table 1 and Figs. 1 and 2 ).


Figure 2
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  		Figure 2. The toxicity of Lqh{alpha}IT and Lqh3 is enhanced by Bj-xtrIT-E15R. Dose response curves for toxicity of Lqh{alpha}IT and Lqh3 upon injection into blowfly larvae in the presence of increasing concentrations of Bj-xtrIT-E15R (ng/100 mg body wt). Forty larvae were used to generate each data point, which represents the ED50 equivalent of the {alpha}-toxin in the presence of the indicated dose of Bj-xtrIT-E15R. The half-maximal dose of Bj-xtrIT-E15R (EC50) that increases toxicity (decrease in ED50) of Lqh{alpha}IT and Lqh3 is 12.6 and 14.7 ng per 100 mg, respectively. Similar doses of Bj-xtrIT-E15R increase the ED50 of Bj-xtrIT (shown on the right Y axis), indicating it acts as a competitive antagonistic of Bj-xtrIT. The ED50 values for each toxin are shown in Table 1 .

The effects produced by simultaneous binding of toxins at receptors sites 3 and 4
The synergic effects obtained on co-administration of Lqh{alpha}IT and LqhIT2 (Table 1) were analyzed in binding studies. A low LqhIT2 concentration increased the binding of 125I-Lqh{alpha}IT to locust synaptosomes to 184 ± 16%, with an EC50 of 1.8 ± 0.5 nM (n=3, Fig. 3 A). In a reciprocal assay we examined the effects of Lqh{alpha}IT on the binding of 125I-LqhIT2 and 125I-Bj-xtrIT to receptor site-4 on the locust neuronal preparation. LqhIT2 was shown to bind to two noninteracting receptor sites on insect neuronal membranes, a high-affinity site corresponding to the insect sodium channel receptor site-4, and a low-affinity/high-capacity site of yet unidentified nature (36) . Indeed, the apparent Ki of the recombinant LqhIT2 to its high-affinity site is 1.3 ± 0.5 nM (n=3), whereas its affinity for its second site is low (340±66 nM) (Fig. 3B ). Excitatory toxins have been shown to displace LqhIT2 only from its high-affinity site, which comprises ~40% of 125I-LqhIT2 specific binding to locust neuronal membranes (Fig. 3B ; 22,36). The Ki value of Bj-xtrIT to the high-affinity site of LqhIT2 is 1.8 ± 0.6 nM (Fig. 3B ), which corresponds to its apparent affinity to locust sodium channels (Fig. 3C ). Yet low concentrations of Lqh{alpha}IT increased 125I-LqhIT2 binding to 123 ± 12% with an EC50 of 11 ± 2.5 nM (n=3; Fig. 3B ). To clarify whether this increase resulted from binding to the high- and/or low-affinity sites of LqhIT2, the assay was performed with very high concentrations of both Bj-xtrIT, which occupied all the high-affinity sites, and Lqh{alpha}IT (Fig. 3B ). Since Lqh{alpha}IT had no additional effects on 125I-LqhIT2 binding, we concluded it did not affect the binding to the low-affinity site (Fig. 3B ). Thus, the Lqh{alpha}IT-induced increase in LqhIT2 binding to its high-affinity site (receptor site-4) is 158 ± 15% (Fig. 3B right Y axis). In contrast, Lqh{alpha}IT did not affect the specific binding of 125I-Bj-xtrIT to locust neuronal membranes (Fig. 3C ). These results indicate that in contrast to Bj-xtrIT, the positive allosteric effects on binding between Lqh{alpha}IT and LqhIT2 are reciprocal.


Figure 3
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  		Figure 3. Mutual allosteric modulations of Lqh{alpha}IT and ß-toxins binding to locust neuronal membranes. A) Scorpion ß-toxins Bj-xtrIT and LqhIT2 enhance 125I-Lqh{alpha}IT binding. Binding of 125I-Lqh{alpha}IT was measured as specified in the legend of Fig. 1 . The maximal increase in 125I-Lqh{alpha}IT binding obtained in the presence of Bj-xtrIT and LqhIT2 was 147 ± 12% and 184 ± 16%, respectively. The effects were obtained with EC50 values of 43 ± 8.5 and 1.8 ± 0.5 nM (n=3) for Bj-xtrIT and LqhIT2, respectively. B) Enhancement of 125I-LqhIT2 binding by increasing concentrations of Lqh{alpha}IT. Membranes (160 µg) were incubated with 0.1 nM 125I-LqhIT2 for 45 min at room temperature. Nonspecific binding, measured in the presence of 30 µM of unlabeled LqhIT2 (comprising 50% of total binding), was subtracted. The dashed line indicates the maximal specific binding of 125I-LqhIT2 under control conditions. The Ki values of LqhIT2 were 1.3 ± 0.5, and 340 ± 66 nM (n=3) for its high- and low-affinity sites, respectively. 30 µM Bj-xtrIT inhibited 40% of 125I-LqhIT2 specific binding with Ki of 1.8 ± 0.6 nM (n=3) (Bj-xtrIT competes with LqhIT2 only for its high-affinity site). Lqh{alpha}IT increased 125I-LqhIT2 binding to 123 ± 12% of the control, with EC50 of 11 ± 2.5 nM (n=3). Incubation of 125I-LqhIT2 with high concentrations of both Bj-xtrIT and Lqh{alpha}IT (open circles) inhibited 40% of 125I-LqhIT2 binding. Therefore, we normalized the right Y-axis such that 100% refers to 125I-LqhIT2 binding to its high-affinity site. Accordingly, Lqh{alpha}IT increased 125I-LqhIT2 binding to 158 ± 15%. C) 125I-Bj-xtrIT binding is not affected by Lqh{alpha}IT. Membranes (160 µg) were incubated with 0.1 nM 125I- Bj-xtrIT as in panel A. Nonspecific binding, measured in the presence of 1 µM of unlabeled Bj-xtrIT (comprising 30% of total binding), was subtracted. The dashed line indicates the maximal specific binding of 125I-Bj-xtrIT. The Ki value of Bj-xtrIT was 1.6 ± 0.3 nM (n=3). D) Enhancement in 125I-Lqh{alpha}IT binding is reversed at high concentrations of Bj-xtrIT and LqhIT2. Data are from the same experiments shown in A, except that the concentration range was expanded to 10 µM. All data points represent mean ±SE of 3 independent experiments.

It is noteworthy that the enhancement in Lqh{alpha}IT binding, observed at relatively low concentrations of LqhIT2 and Bj-xtrIT, declines progressively to the initial level (100%) of 125I-Lqh{alpha}IT specific binding, when examined at higher concentrations (≥1 µM) of ß-toxins (Fig. 3D ). This result could be explained by an additional low-affinity site for ß-toxins, whose occupancy at high toxin concentrations averts the allosteric enhancement in Lqh{alpha}IT binding conferred by ß-toxin binding to the high-affinity site-4. This may explain why allosteric interactions between receptor sites 3 and 4 have never been detected in earlier studies carried out with high toxin concentrations (3 , 6 , 7 , 36 37 38) .

DISCUSSION

The receptor sites of scorpion {alpha}- and ß-toxins on insect sodium channels interact allosterically
Using binding studies, we demonstrate for the first time positive allosteric interactions between receptor sites 3 and 4 on insect sodium channels (Fig. 3) , which explain in part the synergic effects observed between {alpha}- and ß-toxins in vivo (Table 1 ; 31). The positive cooperativity observed on simultaneous binding of {alpha}- and ß-toxins increases the potency of the scorpion venom and may explain the conservation of the two toxin classes in Butoid scorpions (3 , 6 , 39) . The larger increase in Lqh{alpha}IT binding and toxicity when combined with LqhIT2 vs. Bj-xtrIT is likely to result from the reciprocal allosteric interactions between Lqh{alpha}IT and LqhIT2 receptor sites (Fig. 3 , Table 1 ). The difference in allosteric interaction (Fig. 3B , C) suggests that the excitatory and depressant ß-toxins interact differently with receptor site-4. This view is supported by differences in the mode of action of excitatory and depressant toxins on insect neurons (20 21 22 , 40 , 41) , differences in inhibition of their binding by antipeptide antibodies directed against various extracellular sodium channel regions (36) , and the fact that they do not compete reciprocally in binding to fly and lepidoptera neuronal preparations (30) .

The surprising effect of a nontoxic ligand that binds to the insect sodium channel
One of the most intriguing findings in this study is that the binding of the nontoxic ligand Bj-xtrIT-E15R to receptor site-4 (silent binding) enhances {alpha}-toxin binding to receptor site-3 and its effect in vivo in a similar manner to that induced by Bj-xtrIT (Figs. 1 , 2) . This result suggests that mere binding of Bj-xtrIT-E15R induces a conformational change on the channel that can be probed by {alpha}-toxin binding. Moreover, this result raises the question whether that conformational change is related to the ß-toxin physiological effect.

Since ligand binding at receptor site-4 and brevetoxin binding at receptor site-5 induce an additive increase in Lqh{alpha}IT binding to receptor site-3 (Fig. 1) , the conformational changes they produce are different. Receptor site-3 is mainly associated with the external loop S3-S4 in domain 4 of sodium channels (9 , 42) , whereas receptor site-4 for ß-toxins is mainly assigned to domain 2 (13 14 15) . The allosteric interactions observed here suggest cooperative interactions between domains 2 and 4 of insect sodium channels, which could also be inferred from the enhanced effect of the anti-mammalian {alpha}-toxin, Lqh2, on the rat brain channel bearing domain-2 of Drosophila para sodium channel, rBIIA-ParaD2 (15) . In addition, a cooperative movement of voltage sensors of different channel domains was reported (43) . Understanding such interactions at the molecular level requires identification of toxin receptor sites on the channel, and is an important area for future studies.

The phenomenon of silent binding of a toxin that acts as a competitive antagonist in vivo and induces a conformational change elsewhere on the channel is reminiscent of the {delta}-conotoxin TxVIA effect upon injection into rat brain. {delta}-TxVIA binds to receptor site-6 on these channels and antagonizes the toxicity of potent {delta}-conotoxins (29 , 44) . It also inhibits allosterically the binding of batrachotoxin to receptor site-2 on rat brain synaptosomes (45) . Thus, it is possible that concealed conformational changes of the channel induced by specific binding of unnecessarily active ligands (e.g., Bj-xtrIT-E15R or {delta}-TxVIA), is a common phenomenon. This phenomenon may be used to clarify novel allosteric interactions between distant channel regions and to reach a better understanding of channel gating modulation. It may also be applied in the development of drug enhancers, which by themselves are inactive, but their administration may reduce the doses of potent drugs required.

Physiological effects and allosteric interactions account for synergic activity in vivo
The enhancement of Lqh{alpha}IT toxicity by Bj-xtrIT-E15R is considerably lower than that induced by coinjection with the unmodified Bj-xtrIT (Fig. 2 and Table 1 ). This difference suggests that the synergic effect between {alpha}- and ß-toxins results from modulations of sodium channel gating that mutually enhance nerve firing in vivo, in addition to the allosteric interactions, which enhance toxin binding. {alpha}-Toxins induce long plateau potentials in axons attributed to an increase in the probability of sodium channels to remain in their open states due to inhibition of their fast inactivation. This effect increases neuronal excitability and neuromuscular activity (7 , 46 47 48 49) . Under such conditions the activation of sodium channels at more negative membrane potentials by ß-toxins could increase, as their effect is dependent and/or enhanced by membrane depolarization (5 , 14 , 15 , 50) . ß-Toxins induce repetitive activity and depolarization of the axonal membrane potential in insect nerves (20 21 22 , 40 , 51 , 52) , and hence may facilitate {alpha}-toxin action on the channels in their open states (1 , 46) . Thus, the enhancement in {alpha}-toxin effect by ß-toxin interaction with receptor site-4 results from an indirect modification of receptor site-3 and from alteration in the voltage dependence of channel activation. It is noteworthy that cooperative effects in insect neurons were observed between µ-agatoxin 4 from the spider Agelenopsis aperta (with ß-toxin effect but unidentified receptor site) and the site-3 sea anemone toxin As II from Anemonia sulcata (53) . The enhanced effect of {alpha}-toxins induced by ß-toxin binding per se, as demonstrated here with the nontoxic Bj-xtrIT-E15R, is further corroborated by the similar enhancement in Lqh{alpha}IT action when coinjected with a number of ß-toxins (Bj-xtrIT, LqhIT2, Lqh-dprIT3, and Lqhß1) that bind at subnanomolar concentrations to insect sodium channels. The similar enhancement is obtained despite the vast difference in mode of action and toxicity to blowfly larvae of the four toxins (Table 1 ; refs. 16 , 21 , 22 , 30 ). Therefore, it seems it is the binding to receptor site-4 that induces the conformational change, which leads to a similar enhancement in {alpha}-toxin action.

A novel function for weakly and nonactive polypeptides in arthropod venom
The observed allosteric interactions between {alpha}- and ß-toxin receptor sites shed new light on the complex composition of arthropod venom, which contain compounds with a wide array of activities. Of 100–300 different polypeptides that may be found in a single scorpion or spider venom, <10% have been characterized mostly by their biological activity (6 , 54 , 55) . For example, in the venom of Leiurus quinquestriatus hebraeus, Lqh, a number of {alpha}- and ß-toxins were characterized that vary greatly in toxicity for insects (e.g., Lqh{alpha}IT, Lqh2, Lqh3, Lqh4, Lqh6, Lqh7 {alpha}-toxins; 7,11,56,57; LqhIT2, LqhIT5, Lqh-dprIT3, Lqhß1 ß-toxins; 16 , 20 , 21 , 58 ). Despite these differences, the ß-toxins from Lqh venom induce similar increase in toxicity of Lqh{alpha}IT (Table 1) , and by extension probably of other, less toxic {alpha}-toxins. In this respect, a number of weakly or apparently inactive venom components with toxin-like scaffolds, that were identified in various arthropod venoms, such as toxin variants 1–3 from Centruroides sculpturatus Ewing (59 , 60) , Aah STR1 (61) , Aah IV (62) , and ACTX-Hvf17 from the Australian funnel-web spider Hadronyche versuta (63) , might contribute to envenomation in a similar way. We suggest that such polypeptides may act as allosteric enhancers of other potent venom components and facilitate adaptation of the venomous animal to the ever-changing environment and prey, which may explain their conservation.

ACKNOWLEDGMENTS

We are grateful to Prof. Michael Gurevitz, Plant Sciences, Tel Aviv University, for providing his lab facilities to conduct this work. This research was supported by the United States-Israel Binational Agricultural Research and Development grant IS-3480–03 (D.G.); by the Israeli Science Foundation, grant 1008/05 (D.G.); and by a grant from the G.I.F., the German-Israeli Foundation for Scientific Research and Development No. G-770–242.1/2002 (D.G.).

Received for publication January 15, 2006. Accepted for publication April 26, 2006.

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