Chemical synthesis and structure–activity relationships of Ts κ, a novel scorpion toxin acting on apamin‐sensitive SK channel

Abstract

Tityus kappa (Ts kappa), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts K was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs kappa, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nM). The sTs kappa was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs kappa; they were between Cys7-Cys28, Cys13-CyS33 and Cys17-Cys35, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts kappa (i.e. Arg6, Arg9, Lys18, Lys19) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by 1H-NMR, suggest that Arg6, and to a lesser extent Arg9, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the alpha-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the alpha-helix.

Potassium channel blockers from the venom of the Brazilian scorpion Tityus serrulatus ( )

“…According to what was demonstrated for previous studies on SK Ca channel-acting toxins, the positionsArg 6 , Arg 9 , Lys 18 and Lys 19 play a crucial role in its pharmacological activity (Lecomte et al, 1999). It was confirmed by binding assay and 1 H-NMR conformational analysis of the synthetic analogues that the Arg 6 residue and, to a lesser extent the Arg 9 residue, are responsible for a high-affinity interaction of this toxin with SK Ca channels.…”

“…A glance at the primary structure can help ( Figure 4A). According to point mutations and 1 H-NMR data above cited (Lecomte et al, 1999) the Arg 6 is mainly responsible for the high-affinity interaction of Ts9 with SK Ca channels, by creating a strong positive electrostatic interaction. The interaction of Ts9 with the SK Ca channel was modelled according to the charge anisotropy of the ligand and it was found that a basic surface, encompassing residues Lys 18 and Lys 19 on one side and Arg 9 and Tyr 8 on the other, would be in direct interaction with the channel (Blanc et al, 1997).…”

“…The residues in red in Ts7 are found in direct interaction with the receptor after analysis of the complex KTX/K v 1.3 chimera, using solid-phase NMR (Lange et al, 2006). The two Arg residues in red in Ts9 (R6 and R9) have been shown crucial for the SK2 recognition (Lecomte et al, 1999). The two Lys residues in blue (K18 and K19) are Amino acids sequence of Ts11 (P0C174/ gi|90101747), Ts12 (P0C175/ gi|90101748) and…”

Tamapin, a Venom Peptide from the Indian Red Scorpion (Mesobuthus tamulus) That Targets Small Conductance Ca2+-activated K+ Channels and Afterhyperpolarization Currents in Central Neurons

“…Both scorpions are found in desert regions of North Africa and the Eastern Mediterranean. Other more recently identified scorpion toxins that compete for apamin-binding sites with high affinity include toxin from the venom of the Central American scorpion Tityus serrulatus (12)(13)(14) and BmPO5 from the Asian scorpion Buthus martensii Karsch, widely distributed throughout China (15). Finally, toxins with greatly reduced binding affinities include PO1 and BmPO1 (15,16).…”

Therapy and prophylaxis of inhaled biological toxins

“…Also has K + channel blocking action (Simpson et al, 1993) α-Tityustoxins (K + channel specific)* Polypeptides of 30-70 amino acids with three or four disulphide bridges. TsTx K-alpha contains 37 amino acids (Rogowski et al, 1992;Becerril et al, 1997) and TsTx kappa has 35 amino acids (Lecomte et al, 1999) 3.9 kD TsTx K-alpha reacts at an extracellular site on voltagegated non-inactivating K + channels. Toxin does not occlude pore (Werkman et al, 1993) Tricothecene mycotoxins (T-2)* T-2 has a tetracyclic sesquiterpene structure, including a six-membered oxygen-containing ring, an epoxide group in the 12,13 position and an olefinic bond in the 9,10 position.…”