New toxins acting on sodium channels from the scorpion Leiurus quinquestriatus hebraeus suggest a clue to mammalian vs insect selectivity

Abstract

Two new toxins were purified from Leiurus quinquestriatus hebraeus (Lqh) scorpion venom, Lqh II and Lqh III. Lqh II sequence reveals only two substitutions, as compared to AaH II, the most active scorpion α-toxin on mammals from Androctounus australis Hector. Lqh III shares 80% sequence identity with the α-like toxin Bom III, from Buthus occitanus mardochei. Using bioassays on mice and cockroach coupled with competitive binding studies with ¹²⁵I-labeled scorpion α-toxins on rat brain and cockroach synaptosomes, the animal selectivity was examined. Lqh II has comparable activity to mammals as AaH II, but reveals significantly higher activity to insects attributed to its C-terminal substitution, and competes at low concentration for binding on both mammalian and cockroach sodium channels. Lqh II thus binds to receptor site 3 on sodium channels. Lqh III is active on both insects and mammals but competes for binding only on cockroach. The latter indicates that Lqh III binds to a distinct receptor site. Thus, Lqh II and Lqh III represent two different scorpion toxin groups, the α- and α-like toxins, respectively, according to the structural and pharmacological criteria. These new toxins may serve as a lead for clarification of the structural basis for insect vs mammal selectivity of scorpion toxins.

Introduction

The principle toxic compounds in scorpion venoms are toxic polypeptides that interfere with the sodium conductance in mammalian excitable tissues. The sodium channel neurotoxins from scorpion venom defined a family of homologous polypeptides, composed of a single chain of 63–70 amino acid residues cross-linked by four disulfide bridges (Miranda et al., 1970; Martin-Eauclaire and Couraud, 1995; Gordon et al., in press) and were classified into several structural groups on the basis of primary structure (Rochat et al., 1979; Dufton and Rochat, 1984; Possani, 1984; Watt and Simard, 1984) and immunological (Delori et al., 1981) criteria. The four first groups (I–IV) contain the scorpion α-toxins and, the newly defined group of α-like toxins (Gordon et al., 1996), active on vertebrates (Martin-Eauclaire and Couraud, 1995; Gordon et al., in press). The phylogenetic 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, 1986). All these different toxins affect sodium conductance in various excitable tissues, and serve as important pharmacological tools for the study of excitability and sodium channel structure and function.

At least seven neurotoxin receptor sites have been identified by direct radiolabeled toxin binding and competition binding studies on the mammalian sodium channels, and additional as yet unidentified receptor sites have been noticed (Gordon, 1997a; Trainer et al., 1997). Although the identification and characterization of the distinct receptor sites have been predominantly performed using vertebrate excitable preparations (Catterall, 1980, Catterall, 1986; Strichartz et al., 1987), insect neuronal membranes have been shown to possess similar receptor sites. 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, Gordon et al., 1990, Gordon et al., 1992, Gordon et al., 1993; Moskowitz et al., 1991, Moskowitz et al., 1994) and pharmacological (Pelhate and Sattelle, 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, Zlotkin et al., 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, Gordon et al., 1992, Gordon et al., 1993, Gordon et al., 1996). 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 α-scorpion and sea anemone toxins) 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; Gordon, 1997a, Gordon, 1997b). The most studied neurotoxins that induce inhibition of sodium current inactivation are the scorpion α-toxins and sea anemone toxins, that were shown to bind to overlapping region comprising receptor site 3 on rat brain (Catterall and Beress, 1978; Couraud et al., 1978; Rogers et al., 1996) and insect (Gordon and Zlotkin, 1993; Gordon et al., 1996) sodium channels. Several scorpion α-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). Two scorpion α-toxins highly active on insects have been recently pharmacologically characterized, LqhαIT and Lqq III (Eitan et al., 1990; Gordon and Zlotkin, 1993; Gordon et al., 1996; Cestele et al., 1997). These two highly homologous α-toxins (see Fig. 3) were shown by competitive binding studies to be selective probes for receptor site 3 on insect sodium channels (Gordon and Zlotkin, 1993; Gordon et al., 1996; Cestele et al., 1997). Recently, we have described a new group of scorpion toxins that affect sodium current inactivation and is active on both mammals and insects, the so-called scorpion α-like toxins (Gordon et al., 1996). Our study suggested that scorpion toxins affecting inactivation of sodium channels may be divided to several different groups according to their mammal vs insect activities and their binding properties, each possessing a putative distinct receptor site on sodium channels (Gordon et al., in press).

In the present report we describe the purification and pharmacological characterization of two new scorpion toxins, designated Lqh II and Lqh III, from the venom of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus, that represent by their primary structure the scorpion α-toxins and the α-like scorpion toxin groups, respectively. We have used AaH II, the scorpion α-toxin most active on vertebrates, that reveals the highest affinity to rat brain synaptosomes (Jover et al., 1978), and LqhαIT, the scorpion α-toxin that reveals high activity on insects (Eitan et al., 1990; Gordon et al., 1996), as specific probes for receptor site 3 in rat brain and insect sodium channels, respectively. Interestingly, Lqh II reveals an unusual homology to AaH II, having only two amino acid substitutions at its N- and C-termini. Moreover, this toxin has comparable activity to mammals as AaH II, but reveals significantly higher activity to insects, attributed to the C-terminal substitution. The second toxin, Lqh III, reveals sequence similarity to the previously described α-like scorpion toxin group (Bom III and Bom IV, Gordon et al., 1996, and unpublished) and is active on both insects and mammals.