Characterization of Four Toxins from Buthus Martensi Scorpion Venom, which Act on Apamin‐Sensitive Ca²⁺‐Activated K⁺ Channels

Abstract

Four peptidyl inhibitors of the small-conductance Ca2+-activated K' channels (SK,,) have been isolated from the venom of the Chinese scorpion Buthus martensi. These peptides were identified by screening C,, HPLC fractions of the crude venom by means of mass analysis by matrix-assisted-laser-desorption/ ionization time-of-flight mass spectrometry, and toxicological tests in mice. Edman degradation analysis of the purified peptides showed sequences of 28-31 amino acids including 6 cysteine residues. Three of the sequences were similar to the PO1 peptides from Androctonus scorpions, showing 76% sequence similarity for the most closely related, named BmPO1, and 46% for the other two, named BmP02 and BmP03. Like the PO1 peptides, these molecules showed a low toxic activity in mice after intracerebroventricular injection, and competed (K,,, > 1 pM) with iodinated apamin for binding to its receptor site from rat brain, which has been proved to be the SK,, channels. The fourth toxin was structurally related to the POS/leiurotoxin I toxin family, with 90% similarity, and was named BmPO5. This toxin exhibited a high toxic activity with lethal effects in mice. Due to its small representation in the venom [less than 0.01% (by mass)], its biological properties have been assessed on the synthetic analogue of BmPOS, which was assembled on a solid phase by means of Fmoc methodology. The synthetic peptide was physicochemically identical to the natural peptide, as shown by comparison of their molecular masses and amino acid compositions, and by their coelution after coinjection on capillary electrophoresis. These results confirmed the primary structure of BmPO5 including an amidated C-terminus. Similarly to natural BmPO5, synthetic BmPO5 produced toxic and lethal effects after intracerebroventricular injection in mice (LD,,, = 37 ng), and was able to compete with iodinated apamin for binding to its receptor in rat brain

Sequence and electrophysiological characterization of two insect‐selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi

“…1 g of dry crude venom. Venom was extracted in 0.5 M acetic acid, centrifuged and ¢ltered on 45 Wm ¢lters as previously described [4].…”

“…Bulk venom fractionation (100 mg) was performed on a semi-preparative reversed-phase column (Shiseido C18, 10U300 mm) using an acetonitrile/water/0.1% TFA gradient (0^45% CH 3 CN in 102 min, 3 ml/min) as previously described [4]. Fractions 32 and 33 were submitted to cation-exchange HPLC on a Tosoh SP5PW column (4.6U70 mm), with a gradient of NaCl (0^1 M NaCl in 88 min) in 10 mM phosphate bu¡er (pH 6.7).…”

“…Peptides were sequenced on a Shimadzu PPSQ-10 gas-phase sequencer either without prior derivatization or after reduction and 4-vinyl-pyridine alkylation [4]. Reduced alkylated toxins were submitted to proteolysis by (a) TPCK-treated trypsin (Sigma Co., USA), 2% w/w, 37³C, 14 h in 100 mM ammonium bicarbonate, 0.1 mM CaCl 2 pH 8.1 (for Bm32-VI, a second digestion was performed with 2% w/w of enzyme added after 4 h and a total reaction time of 24 h), (b) asparagine endopeptidase (Asp-N, Takara Shuzo, Japan), 5% w/w, 37³C, 14 h in 50 mM sodium phosphate bu¡er pH 8.0, and (c) Achromobacter protease I (API, Wako, Japan), 5% w/w, 37³C, 14 h in 100 mM Tris bu¡er, pH 8.0 and 12.5%, 37³C, 14 h in 100 mM ammonium bicarbonate pH 8.0.…”

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).…”

Gene Expression, Mutation, and Structure−Function Relationship of Scorpion Toxin BmP05 Active on SKCa Channels

“…The study of K + channels is of high value for the understanding of many physiological processes. Rat adrenal chromaffin cells (RACC) 1 express SK Ca channels (2), which produce slow after-hyper-polarization and play an important role in spacing action potentials and in limiting the frequency of repetitive firing.…”