Purification, Characterization, and Synthesis of Three Novel Toxins from the Chinese ScorpionButhus martensi, Which Act on K⁺Channels

Abstract

Three novel toxins belonging to the scorpion K+ channel-inhibitor family were purified to homogeneity from the venom of the Chinese scorpion Buthus martensi. They have been identified according to their molecular mass (3800-4300 Da) and their neurotoxicity in mice and characterized as 37-amino acid peptides. One of them shows 81-87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an amidated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD50s of 40-95 ng per mouse). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [125I]kaliotoxin for its binding to rat brain synaptosomes (IC50s of 0.05-1 nM) and to block the cloned voltage-gated K+ channel Kv1.3 from rat brain, expressed in Xenopus oocytes (IC50s of 0.6-1.6 nM). BmTX1 and BmTX2 were also shown to compete with [125I]charybdotoxin for its binding to the high-conductance Ca2+-activated K+ channels present on bovine aorta sarcolemmal membranes (IC50s of 0.3-0.6 nM). These new sequences show multipoint mutations when compared to the other related scorpion K+ channel toxins and should prove to be useful probes for studying the diverse family of K+ channels.

Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels

“…An additional Gly residue was found at the C-terminus of the mature peptide before the stop codon, which was removed during post-translational processing and was absolutely required for the C-terminal amidation of the mature toxin. The deduced amino acid sequence of BmKTX was consistent with that determined [12]. The polyadenylation signal, aataaa, was found in the 3h untranslated region at position 18 upstream of the poly(A).…”

“…All short-chain toxins exhibit a similar overall three-dimensional structure with an α-helix and a three-stranded β-sheet constrained by three or four disulphide bridges [7,9,11]. The K + channel toxins can further be classified into at least five subgroups sharing a low sequence similarity and having their own comparative selectivities towards each corresponding subtype of the target channels [4,8,9,12,13]. They therefore prove to be useful probes for investigating the diverse subtype of K + channels.…”

“…From the Chinese scorpion Buthus martensi Karsch, toxins named BmP01, BmP02, BmP03 and BmP05 acting on apaminsensitive small-conductance Ca# + -activated K + channels were first separated and purified [4], and a further three novel toxins, named BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2 acting on the voltage-gated K + channels were subsequently characterized [12]. As BmTX1 and BmTX2 were also shown to compete with "#&I-charybdotoxin (ChTX) and shared 65-70 % identity with its related toxins, they should be considered to belong to the ChTX subgroup, which is characterized by the activity towards high-conductance Ca# + -activated K + channels, despite their broad specificity [12]. To answer the question of whether all these toxins come from a common ancestor, it seems Abbreviations used : ChTX, charybdotoxin ; KTX, kaliotoxin ; RACE, rapid amplification of cDNA ends.…”

Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom

“…PBTx3 shows similarities with ChTx (41%) [29], Lqh 15-1 (44%) [30] and ChTx-Lq-2 (38%) [11] from Leiurus quinquestriatus var. Hebraeus, BmTx 1 (55%) and 2 (41%) [31] from Buthus martensi Karsch, HgTx 2 (55%) [32] and LbTx (50%) [33] from Centruroides limbatus, IbTx (47%) [34] and TmTx (52%) [35] from Buthus tamulus. Alignment of the cysteine residues (C 6 -C 27 , C 12 -C 32 , C 16 -C 34 ) showed that it was a novel toxin and that the cysteine motif was highly conserved.…”

BmBKTx1, a Novel Ca2+-activated K+ Channel Blocker Purified from the Asian Scorpion Buthus martensi Karsch

“…␣-KTx1.x peptides are listed to the left of BmBKTx1 and SK blockers. BmBKTx1, BmTx1, BmTx2, and BmP05 were from scorpion B. martensi Karsh (39,40), ChTx (charybdotoxin) and LeTxI (scyllatoxin) from scorpion Leiurus quiquestriatus var. Hebraeus (23,41), IbTx (iberiotoxin) from scorpion Buthus tamales (42), LbTx (limbatotoxin) from scorpion Centruroides limbatus (43), and P05 from scorpion Androctonus mauretanicus mauretanicus (22).…”