Sodium channels posses receptor sites for many neurotoxins, of which several groups were shown to inhibit sodium current inactivation. Receptor sites that bind ␣-and ␣-like scorpion toxins are of particular interest since neurotoxin binding at these extracellular regions can affect the inactivation process at intramembranal segments of the channel. We examined, 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 both mammalian and insect sodium channels. As specific probes for rat and insect sodium channels, we used the radiolabeled ␣-scorpion toxins AaH II and Lqh␣IT, the most active ␣-toxins on mammals and insect, respectively. We demonstrate that the different scorpion toxins may be classified to several groups, according to their in vivo and in vitro activity on mammalian and insect sodium channels. Analysis of competitive binding interaction reveal that each group may occupy a distinct receptor site on sodium channels. The ␣-mammal scorpion toxins and the anti-insect Lqh␣IT bind to homologous but not identical receptor sites on both rat brain and insect sodium channels. Sea anemone toxin ATX II, previously considered to share receptor site 3 with ␣-scorpion toxins, is suggested to bind to a partially overlapping receptor site with both AaH II and Lqh␣IT. Competitive binding interactions with other scorpion toxins suggest the presence of a putative additional receptor site on sodium channels, which may bind a unique group of these scorpion toxins (Bom III and IV), active on both mammals and insects. We suggest the presence of a cluster of receptor sites for scorpion toxins that inhibit sodium current inactivation, which 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 cluster.

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor-trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.

Maurotoxin is a toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus. It is a 34-amino-acid peptide cross-linked by four disulfide bridges. Maurotoxin competes with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes. Due to its very low concentration in venom (0.6% of the proteins), maurotoxin was chemically synthesized by means of an optimized solid-phase technique. The synthetic maurotoxin was characterized. It was lethal to mice following intracerebroventricular injection (LD,,, 80 ng/mouse). The synthetic maurotoxin competed with '*'I-apamin and 'Z51-kaliotoxin for binding to rat-brain synaptosomes with half-maximal effects at concentrations of 5 nM and 0.2 nM, respectively. Synthetic maurotoxin was tested on K' channels and was found to block the Kvl.1, Kv1.2, and Kv1.3 currents with half-maximal blockage (I&) at 37, 0.8 and 150 nM, respectively. Thus, maurotoxin is a scorpion toxin with four disulfide bridges that acts on K' channels. The half-cystine pairings of synthetic maurotoxin were identified by enzymatic cleavage. The pairings were Cys3 -Cys24, Cys9-Cys29, Cysl3-Cysl9 and Cys31 -Cys34. This disulfide organization is unique among known scorpion toxins. The physicochemical and pharmacological properties of synthetic maurotoxin were indistinguishable from those of natural maurotoxin, which suggests that natural maurotoxin adopts the same half-cystine pairing pattern. The conformation of synthetic maurotoxin was investigated by means of circular dichroism spectroscopy and molecular modeling. In spite of its unusual half-cystine pairings, the synthetic-maurotoxin conformation appears to be similar to that of other short scorpion toxins.Keywords: maurotoxin; scorpion toxin; half-cystine pairing ; apamin-sensitive K' channels ; voltage-gated K' channels.Because polypeptide animal toxins interact with ion channels and modulate their activities [ 1 -31, these toxins are useful pharmacological probes to investigate ion-specific channel proteins and their function. In recent years, toxins acting on various K+ channels have been isolated from diverse scorpion venoms [4]. Maurotoxin has recently been purified from the venom of the chactoid scorpion Scorpio maurus, and characterized (Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results). Maurotoxin is a basic toxin of 34 amino acid residues cross-linked by four disulfide bridges. Maurotoxin was found to compete with radiolabeled apamin and kaliotoxin for binding to rat-brain synaptosomes (Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results). Thus, it is a scorpion toxin with four disulfide bridges that acts on K' channels. Due to its sequence, maurotoxin does not belong to any of the four groups of K+-channel Maurotoxin is only 0.6% of the total proteins in a crude venom, which is not readily available. Thus, we performed chemical solid-phase synthesis of this toxin to e...

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