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(Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Martin-Eauclaire, M. F., Rochat, H. and El Ayeb, M., unpublished results).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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Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfidebridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1^C5, C2Ĉ6, C3^C4 and C7^C8, versus C1^C5, C2^C6, C3^C7 and C4Ĉ 8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys 13 ) and C4 (Cys 19 ), results in conventional Pi1-and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A 12 ,A 20 ]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog. ß