Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons.

In this study, we isolated and pharmacologically characterized the first a-like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDA-YIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGR-YGNACWCIKLPDEVPIRIPGKCR. Using the two-electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage-gated Na + channels expressed in Xenopus laevis oocytes (Na v 1.2/b 1 , Na v 1.5/b 1 , para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC 50 = 80 ± 14 nM) while Na v 1.2/ b 1 still was not affected at concentrations up to 5 lM. Na v 1.5/b 1 was influenced at micromolar concentrations.

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7 -37 sequences, were obtained by stepwise solid-phase synthesis using W-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(I1) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.096. Each protein was characterised chemically. structurally and functionally. 'H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall threedimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca'+-activated K' channels and on rat brain synaptosomal voltage-dependent K' channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alp fold can be engineered with only 31 amino acid residues.Protein toxins are used by venomous animals to subdue prey. These molecules, which generally have a molecular mass ranging between 2-12 kDa, are active towards different molecular targets [1-41 and are characterised by the presence of several disulfide bridges. Scorpion venoms contain a variety of proteins known to inhibit Na' and K' channels. Charybdotoxin, present as a minor component (= 0.2%) in the venom of the Israeli Lpiurus quinquestriatus scorpion, was the first discovered high-affinity inhibitor of high-conductance Ca'+-activated K' channels present in skelctal muscles [5, 61. proteins 11, 3, 4, 20, 211 with subtly different specificities and which therefore represent valuable tools in order to study diffcrcnt K' channels.We have recently reported the three-dimensional solution structure of charybdotoxin solved by two-dimensional NMR [20, 211 and the refined and detailed description of the sidechain organisation [22]. This analysis revealed the presence of a well ordered structure containing three antiparallel strands fo...

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We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltagedependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and QDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.