Abstract

Batrachotoxin (BTX), a steroidal alkaloid, and pyrethroid insecticides bind to distinct but allosterically coupled receptor sites on voltage-gated sodium channels and cause persistent channel activation. BTX presumably binds in the inner pore, whereas pyrethroids are predicted to bind at the lipid-exposed cavity formed by the short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6. The alkylamide insecticide (2E,4E)-N-(1,2-dimethylpropyl)-6-(5-bromo-2-naphthalenyl)-2,4-hexadienamide (BTG 502) reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. However, a pyrethroid-sensing residue, Phe³ⁱ¹⁷ in IIIS6, which does not face the pore, is essential for the activity of BTG 502 but not for BTX. In this study, we found that three additional deltamethrin-sensing residues in IIIS6, Ile³ⁱ¹², Gly³ⁱ¹⁴, and Phe³ⁱ¹⁶ (the latter two are also BTX-sensing), and three BTX-sensing residues, Ser³ⁱ¹⁵ and Leu³ⁱ¹⁹ in IIIS6 and Phe⁴ⁱ¹⁵ in IVS6, are all critical for BTG 502 action on cockroach sodium channels. Using these data as constraints, we constructed a BTG 502 binding model in which BTG 502 wraps around IIIS6, probably making direct contacts with all of the above residues on the opposite faces of the IIIS6 helix, except for the putative gating hinge Gly³ⁱ¹⁴. BTG 502 and its inactive analog DAP 1855 antagonize the action of deltamethrin. The antagonism was eliminated by mutations of Ser³ⁱ¹⁵, Phe³ⁱ¹⁷, Leu³ⁱ¹⁹, and Phe⁴ⁱ¹⁵ but not by mutations of Ile³ⁱ¹², Gly³ⁱ¹⁴, and Phe³ⁱ¹⁶. Our analysis revealed a unique mode of action of BTG 502, its receptor site overlapping with those of both BTX and deltamethrin.