Abstract: 【Aim】 Investigation of the action mechanisms of selective toxicity of type III sea anemone toxin (Av3) on insects would be highly valuable for future design of highly selective anti-insect compounds. 【Methods】 Reversed-phase high performance liquid chromatogram (RP-HPLC) and electrosprayionization mass spectrum (ESI-MS) were used to identify the purity and molecular weight of chemically synthesized Av3 wild type (Av3-WT) and its mutants. The toxicities of Av3-WT and its mutants to  Blattella germanica adults and their inhibition effect on the inactivation of sodium channel BgNav1-1a were tested by bioassay and double voltage clamp, respectively. Systematically replacing the extracellular loops of BgNav1-1a with those of rat sodium channel rNav1-2a was executed to determine the key region for Av3-WT selective toxicity by double voltage clamp. 【Results】 The toxicity of three Av3-WT mutants Y7A, W8A and Y18A generated via mutation of aromatic amino acids Y7, W8 and Y18 to B. germanica adults was significantly reduced, and the median knockdown dose (KD₅₀) values of these three mutants increased by more than 10-fold compared with that of Av3 wild-type toxin (Av3-WT). As compared to the Av3-WT, the inhibition of the mutants Y7A, W8A and Y18A at the dose of 250 mmol/L on the inactivation of BgNav1-1a significantly decreased. The inhibition rates of Y7A, W8A and Y18A on channel inactivation were 12%, 23% and 8%, respectively, while that of Av3-WT was 62%. The chimera bearing DI/SS2-S6 of the rat sodium channel in BgNav1-1a almost lost sensitivity to Av3-WT, the inactivation of only 3.6% channels was inhibited. Substitution His404Y in DI/SS2-S6 completely abolished the inhibition effect of toxin on channel inactivation, and the inactivation of only 6% channels was inhibited by 1 μmol/L Av3-WT. 【Conclusion】 Three aromatic residues Tyr7, Trp8 and Tyr18 are involved in the constitution of bioactive surface of Av3-WT, DI/SS2-S6 is the key region affecting the specificity action of Av3-WT, and His404 in DI/SS2-S6 is critical for Av3-WT selective toxicity.

Key words: Blattella germanica, insecticide, sea anemone toxin, bioactive surface, sodium channel, double voltage clamp