The specificity of Av3 sea anemone toxin for arthropods is determined at linker DI/SS2-S6 in the pore module of target sodium channels

Av3 is a peptide neurotoxin from the sea anemone Anemonia viridis that shows specificity for arthropod voltage-gated sodium channels (Navs). Interestingly, Av3 competes with a scorpion α-toxin on binding to insect Navs and similarly inhibits the inactivation process, and thus has been classified as 'receptor site- 3 toxin', although the two peptides are structurally unrelated. This raises questions as to commonalities and differences in the way both toxins interact with Navs. Recently, site-3was partly resolved for scorpion α-toxins highlighting S1-S2 and S3-S4 external linkers at the DIV voltage-sensor module and the juxtaposed external linkers at the DI pore module. To uncover channel determinants involved in Av3 specificity for arthropods, the toxin was examined on channel chimaeras constructed with the external linkers of the mammalian brain Nav1.2a, which is insensitive to Av3, in the background of the Drosophila DmNav1. This approach highlighted the role of linker DI/SS2-S6, adjacent to the channel pore, in determiningAv3 specificity. Point mutagenesis at DI/SS2-S6 accompanied by functional assays highlighted Trp⁴⁰⁴and His⁴⁰⁵as a putative point of Av3 interaction with DmNav1. His⁴⁰⁵conservation in arthropod Navs compared with tyrosine in vertebrate Navsmay represent an ancient substitution that explains the contemporary selectivity of Av3. Trp⁴⁰⁴and His⁴⁰⁵localization near the membrane surface and the hydrophobic bioactive surface of Av3 suggest that the toxin possibly binds at a cleft by DI/S6. A partial overlap in receptor site-3 of both toxins nearby DI/S6 may explain their binding competition capabilities.