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 codi ng sequence of the para-type sodium channel gene of the housefly, Musc a domestica. This gene has been implicated as the site of knockdown re sistance (kdr), an important resistance mechanism that confers nerve i nsensitivity to DDT and pyrethroid insecticides. The cDNAs predict a p olypeptide of 2108 amino acids with close sequence homology (92% ident ity) to the Drosophila para sodium channel, and around 50% homology to vertebrate sodium channels. Only one major splice form of the housefl y sodium channel was detected, in contrast to the Drosophila para tran script which has been reported to undergo extensive alternative splici ng. Comparative sequence analysis of housefly strains carrying kdr or the more potent super kdr factor revealed two amino acid mutations tha t correlate with these resistance phenotypes. Both mutations are locat ed in domain II of the sodium channel. A leucine to phenylalanine repl acement in the hydro-phobic IIS6 transmembrane segment was found in tw o independent kdr strains and six super-kdr strains of diverse geograp hic origin, while an additional methionine to threonine replacement wi thin the intracellular IIS4-S5 loop was found only in the super-kdr st rains. Neither mutation was present in five pyrethroid-sensitive strai ns. The mutations suggest a binding site for pyrethroids at the intrac ellular mouth of the channel pore in a region known to be important fo r channel inactivation.