P05, a New Leiurotoxin I-like Scorpion Toxin: Synthesis and Structure-Activity Relationships of the α-Amidated Analog, a Ligand of Ca²⁺-Activated K⁺ Channels with Increased Affinity

The venom of the scorpion Androctonus mauretanicus mauretanicus contains a toxin, P05, which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a blocker of the apamin-sensitive Ca2+-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges. A C-terminal carboxyl-amidated analog of P05 (sP05-NH2) was chemically synthesized by the solid-phase technique and fully characterized. Toxicity assays in vivo established that sP05-NH2, like native P05, is a potent and lethal neurotoxic agent in mice (LD50 of 20 ng per mouse). Pharmacological assays in vitro however showed that, unlike P05 which has a binding affinity of 2 × 10-11 M, sP05-NH2 apparently binds irreversibly to the apamin receptor. Iodination at the C-terminal His gave diiodo-sP05- NH2, which had a binding affinity similar to that of native P05. The disulfide bridge pairings were chemically determined for sP05-NH2 and thereby deduced for P05 and leiurotoxin I: linkages were between Cys3 and Cys21, Cys8 and Cys26, and Cys12 and Cys28. Molecular dynamics refinement of P05 also using data from leiurotoxin I suggests that P05 is mainly composed of a double-stranded, antiparallel β-sheet (from Leu18 to Val29) linked to an α-helix (from Arg6 to Gly6) by two disulfides (Cys8-Cys26 and Cys12-Cys28) and to an extended fragment (from Thr1 to Leu5) by the third disulfide (Cys3-Cys21). In agreement with the model, circular dichroism analysis of sP05-NH2 showed that the toxin structure is highly rigid. A common Arg-Arg-Cys-Gln sequence was identified in the helical region of both P05 and apamin. Two Arg-substituted analogs of sP05-NH2 ([Lys6,Lys7] sP05-NH2 and [Leu6,Leu7] sP05-NH2) were synthesized and tested for bioactivity. The results indicate that, as for apamin, Arg residues of the motif are required in the binding and expression of the leiurotoxin I/apamin-like biological properties of P05. © 1993, American Chemical Society. All rights reserved.