Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides

We report the isolation of cDNA clones containing the full 6.3-kb coding sequence of the para-type sodium channel gene of the housefly, Musca domestica. This gene has been implicated as the site of knockdown resistance (kdr), an important resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. The cDNAs predict a polypeptide of 2108 amino acids with close sequence homology (92% identity) to the Drosophila para sodium channel, and around 50% homology to vertebrate sodium channels. Only one major splice form of the housefly sodium channel was detected, in contrast to the Drosophila para transcript which has been reported to undergo extensive alternative splicing. Comparative sequence analysis of housefly strains carrying kdr or the more potent super kdr factor revealed two amino acid mutations that correlate with these resistance phenotypes. Both mutations are located in domain II of the sodium channel. A leucine to phenylalanine replacement in the hydro-phobic IIS6 transmembrane segment was found in two independent kdr strains and six super-kdr strains of diverse geographic origin, while an additional methionine to threonine replacement within the intracellular IIS4-S5 loop was found only in the super-kdr strains. Neither mutation was present in five pyrethroid-sensitive strains. The mutations suggest a binding site for pyrethroids at the intracellular mouth of the channel pore in a region known to be important for channel inactivation.