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Definition
Leiurotoxin I (Lei-NH2), a neurotoxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca2+-activated K+ channels.

 

Discovery
Chicchi et al., in 1988 purified an inhibitor of apamin binding to homogeneity in three chromatographic steps from the venom of the scorpion, Leiurus quinquestriatus hebraeus. The inhibitor was named leiurotoxin I, represents less than 0.02% of the venom protein. It is a 3.4-kDa peptide with little structural homology to apamin although it has some homology to other scorpion toxins such as charybdotoxin, noxiustoxin, and neurotoxin P2 1.

 

Structural Characteristics
All scorpion toxins are disulphide-containing peptide neurotoxins, with three disulphide bonds in "short" and four in "long" neurotoxins. All scorpion venom toxins have a distinct structural motif, with a dense core of secondary structural elements comprising disulphide bonds stabilizing the structure 2. Leiurotoxin is 31-residue polypeptide chain reticulated by three disulfide bridges, i.e. Cys3-Cys21, Cys8-Cys26 and Cys12-Cys28. A proton NMR study at 500 MHz of leiurotoxin I in water was initially carried out to elucidate the secondary structure of toxin. Protein is formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges 4.

 

Mode of Action
A distinct class of small-conductance Ca2+-activated K+ channel is blocked by leiurotoxin-1. Leiurotoxin I completely inhibits 125I-apamin binding to rat brain synaptosomal membranes (Ki = 75 pM). Thus, it is 10-20-fold less potent than apamin. Leiurotoxin I is not a strictly competitive inhibitor of this binding reaction. Like apamin, leiurotoxin I blocks the epinephrine-induced relaxation of guinea pig teniae coli (ED50 = 6.5 nM), while having no effect on the rate or force of contraction in guinea pig atria or rabbit portal vein preparations. Leiurotoxin I of scorpion venom and apamin of honeybee venom demonstrate similar activities in a variety of tissues, but are structurally unrelated peptides. Leiurotoxin is useful in elucidating the role of the small conductance, Ca2+-activated K+ channels in different tissues 6.

 

The role and toxicity of these disulfides were analysed, analogs of Lei-NH2 lacking one disulfide bridge were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two a-aminobutyrate (Abu) residues. 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. It was found that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH 5.

 

Functions
Leiurotoxin blocks the apamin-sensitive K+ channel in guinea pig hepatocytes7- a potent inhibitor of apamin binding to rat brain synaptosomal membranes. This peptide, like apamin, blocks the epinephrine-induced relaxation of guinea pig. Leiurotoxin I is a mixed-type inhibitor of apamin binding to rat brain synaptosomal membranes since it increases the apparent KO for lZ5I-apamin binding and reduces the number of binding sites. The similarity in the activities of leiurotoxin I and apamin in the guinea pig teniae coli assay indicates that, like apamin, leiurotoxin I acts as a blocker of a Ca2+- activated K+ channel 1. Only two native disulfide bonds in leiurotoxin I are sufficient to preserve a native like and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure 6.

 

Although molecular biology approaches have been employed to identify and characterize several species of voltagegated K+ channels, toxins directed against a particular channel can still be useful in defining the physiological role of that channel in a particular tissue. In addition, for those K+ channels which are not yet successfully probed by molecular biology techniques, toxins can be used as biochemical tools with which to purify the target protein of interest 6.

 

References

1.     Chicchi GG, Gimenez-Gallego G, Ber E, Garcia ML, Winquist R, Cascieri MA (1988). Purification and characterization of a unique, potent inhibitor of apamin binding from Leiurus quinquestriatus hebraeus venom. J Biol Chem., 263(21):10192-10197.

2.     Narayanan P (1999). Common structural elements in 'scorpion-toxin' type proteins. J Postgrad Med., 45(1):23-27.

3.     Martins JC, Zhang W, Tartar A, Lazdunski M, Borremans FA. (1990). Solution conformation of leiurotoxin I (Scyllatoxin) by 1H nuclear magnetic resonance: Resonance assignment and secondary structure. FEBS Lett., 260(2):249-253.

4.     Calabro V, Sabatier JM, Blanc E, Lecomte C, Van Rietschoten J, Darbon H (1997). Differential involvement of disulfide bridges on the folding of a scorpion toxin.  Journal of Peptide Research., 50(1):39-47.

5.     Sabatier JM, Lecomte C, Mabrouk K, Darbon H, Oughideni R, Canarelli S, Rochat H, Martin-Eauclaire MF, Rietschote JV (1996). Synthesis and Characterization of Leiurotoxin I Analogs Lacking One Disulfide Bridge:  Evidence That Disulfide Pairing 3-21 Is Not Required for Full Toxin Activity. Biochemistry., 35 (33):10641-10647.

6.     Zhu Q, Liang S, Martin L, Gasparini S, Me´nez A, Vita C (2002). Role of Disulfide Bonds in Folding and Activity of Leiurotoxin I: Just Two Disulfides Suffice. Biochemistry  41(38):11488-11494.

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