Chemical synthesis and structure-activity relationships of Ts kappa, a novel scorpion toxin acting on apamin-sensitive SK channel

Tityus kappa (Ts kappa), a novel toxin from the venom of the scorpion Tityu s serrulatus, is a 35-residue polypeptide crosslinked by three disulphide b ridges and acts on small-conductance calcium-activated potassium channels ( SK channels). Ts kappa was chemically synthesized using the solid-phase met hod and characterized. The synthetic product, sTs kappa, was indistinguisha ble from the natural toxin when tested in vitro in competition assay with r adiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nM). Th e sTs kappa was further tested in vivo for lethal activity to mice followin g intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cy stine pairings were formerly established by enzyme-based cleavage of sTs ka ppa; they were between Cys(7)-Cys(28), Cys(13)-Cys(33) and Cys(17)-Cys(35), which is a disulphide bridge pattern similar to that of other short scorpi on toxins. According to previous studies on SK channel-acting toxins, the p utative influence of certain basic residues of Ts kappa (i.e. Arg(6), Arg(9 ), Lys(18), Lys(19)) 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 ana lysis of the synthetic analogues by H-1-NMR, suggest that Arg(6), and to a lesser extent Arg(9), are important residues for an high-affinity interacti on of this toxin with SK channels; interestingly these residues are located outside the alpha-helical structure, whereas the pharmacologically importa nt basic residues from other SK channel-specific toxins had been located in side the alpha-helix.