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

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

SPECIFIC AIMS

The most toxic polypeptides in Buthoid scorpion venom are long-chain neurotoxins that affect electrical excitability by modulating the gating of voltage-gated sodium channels (NaChs) in animal excitable cells. These toxins are divided into and ßbeta; classes according to their mode of action and binding sites. -Toxins prolong the action potential by inhibition of the fast inactivation of NaChs upon binding to receptor site-3, which involves extracellular loops in domain 4 of the channel. ßbeta;-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 NaChs. Coinjection of and ßbeta;-toxins has been shown to produce synergic effects in blowfly and lepidopetra larvae. Our aim was to clarify at the molecular level the basis of this synergism.

PRINCIPAL FINDINGS

1. Effects of in vivo co-administration of scorpion and ßbeta;-toxins
Mixtures of and ßbeta;-toxins active on insects were injected to blowfly larvae and their effective dose 50% (ED₅₀) was determined (sustained paralysis after 10–15 min was considered a positive score). A synergic effect was considered maximal when the doses of toxins in the mixture were minimal. Injection of mixtures of - and ßbeta;-toxins reduced 4- to 10-fold the dose required for each toxin separately to generate an effect. These results indicated a positive cooperativity between and ßbeta;-toxins in vivo. Notably, the increase in toxicity of the anti-insect selective depressant ßbeta;-toxin LqhIT2 was larger (10-fold) than that of the anti-insect selective excitatory ßbeta;-toxin, Bj-xtrIT, when co-administrated with either of the -toxins active on insects, LqhIT or Lqh3.

2. Effects of LqhIT2 and Bj-xtrIT on LqhIT binding to locust neuronal membranes
Binding of ¹²⁵I-LqhIT to receptor site-3 is enhanced by 150–180% in the presence of low concentrations (nM range) of the ßbeta;-toxin LqhIT2 and vice versa, indicating a mutual allosteric interaction between their receptor sites on locust NaChs. Although binding of ¹²⁵I-LqhIT is also enhanced (150%) by low concentrations of Bj-xtrIT (Fig. 1 ), LqhIT in concentrations of up to 5 µM did not enhance the binding of ¹²⁵I-Bj-xtrIT. This may explain the higher synergic effect between LqhIT2 and the -toxins compared to Bj-xtrIT.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effects of Bj-xtrIT and Bj-xtrIT-E15R on 125I-LqhIT binding to locust neuronal membranes. Membranes (160 µg) were incubated with 0.1 nM 125I-LqhIT for 45 min at room temperature. Nonspecific binding, measured in the presence of 1 µM of unlabeled LqhIT (comprised 10–15% of total binding), was subtracted. The dashed line indicates maximal specific binding of 125I-LqhIT under control conditions. The Ki value of LqhIT was 1.1 ± 0.2 nM (n=3). A maximal increase in 125I-LqhIT 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-LqhIT binding by 30%. In the presence of PbTx2, the maximal increase of 125I-LqhIT 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.

3. Enhancement of LqhIT binding and toxicity by the nontoxic mutant Bj-xtrIT-E15R
In light of the allosteric enhancement of -toxin binding to receptor site-3 by the binding of a ßbeta;-toxin to receptor site-4, we examined the effect of a nontoxic mutant, Bj-xtrIT-E15R, which retained high affinity for receptor site-4, on LqhIT binding. Remarkably, this mutant increased ¹²⁵I-LqhIT binding to receptor site-3 equally well as the native excitatory toxin (Fig. 1) . Moreover, low concentrations of Bj-xtrIT-E15R also increased by 3-fold the toxicity of LqhIT and Lqh3 when coinjected to blowfly larvae (Fig. 2 ), though this increase was smaller from that induced by Bj-xtrIT (5 fold).

View larger version (15K): [in this window] [in a new window]   Figure 2. The toxicity of LqhIT and Lqh3 is enhanced by Bj-xtrIT-E15R. Dose response curves for toxicity of LqhIT and Lqh3 upon injection to blowfly larvae in the presence of increasing concentrations of Bj-xtrIT-E15R (in ng/100 mg body wt). Forty larvae were used to generate each data point, which represents the ED50 equivalent of the -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 LqhIT 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 that it acts as a competitive antagonistic of Bj-xtrIT. ED50 values of LqhIT, Lqh3, and Bj-xtrIT are 12, 26, and 14, respectively (in ng/100 mg body wt).

Since lipid-soluble NaCh activators such as brevetoxins (PbTxs, which bind to receptor site-5), have been shown to allosterically increase the binding of LqhIT to locust NaChs, we examined the effect of Bj-xtrIT-E15R on ¹²⁵I-LqhIT binding in the presence of saturating concentrations of PbTx-2. The increase in ¹²⁵I-LqhIT binding to receptor site-3 by simultaneous binding of PbTx-2 and Bj-xtrIT–E15R was additive (Fig. 1) , indicating that the conformational changes they produce are different and independent.

CONCLUSIONS AND SIGNIFICANCE

Simultaneous binding of scorpion - and ßbeta;-toxins has shown that receptor sites 3 and 4 of insect sodium channels are able to interact allosterically. These interactions contribute to the synergic effects in toxicity obtained upon their co-administration (Fig. 3 ). Since LqhIT enhances allosterically the binding of LqhIT2 but not of Bj-xtrIT, it may be concluded that the two ßbeta;-toxins interact differently with receptor site-4 of the insect sodium channel.

View larger version (69K): [in this window] [in a new window]   Figure 3. A flowchart that describes a scorpion and its long chain toxins, which recognize two distinct receptor sites on the sodium channel (in linear schematic presentation). Coinjection of - and ßbeta;-toxins, which bind to receptor sites 3 and 4, respectively, induce a synergic toxic effect in the insect (e.g., locust). The allosteric interaction between both receptor sites accounts for the enhancement in binding leading to enhanced toxicity.

Synergic effects between - and ßbeta;-toxins result from a combination of mutual physiological effects on nerve firing in vivo and allosteric interactions between receptor sites on the same channel that enhance toxin binding. Such allosteric interactions may explain the presence of a wide array of both and ßbeta; toxins, which vary greatly in toxicity in scorpion venom.

The allosteric interactions between receptor sites of - and ßbeta;-toxins observed here suggest cooperative interactions between domains 2 and 4 of insect sodium channels (Fig. 3) . This phenomenon may be used to clarify novel allosteric interactions between distant channel regions and lead to a better understanding of channel gating modulation. It may also be applied in developing drug enhancers, which by themselves are inactive, but their co-administration may reduce the required doses of potent drugs.

Our results demonstrate that binding per se of the nontoxic ligand Bj-xtrIT-E15R to receptor site-4 on the insect sodium channel is sufficient to render an allosteric effect that enhances both the toxicity and binding of scorpion -toxins to receptor site-3. This result shows that mere binding of a ligand may enhance other toxin activity and implies a novel role for weakly active or nontoxic polypeptides in arthropod venom as enhancers of the effect of other active neurotoxins.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5545fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5545fjev1 20/11/1933    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cohen, L. Articles by Gordon, D. Search for Related Content PubMed PubMed Citation Articles by Cohen, L. Articles by Gordon, D.