Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes

The super-kdr insecticide resistance trait of the house fly confers resista nce to pyrethroids and DDT by reducing the sensitivity of the Ay nervous sy stem. The super-kdr genetic locus is tightly linked to the Vssc1 gene, whic h encodes a voltage-sensitive sodium channel ct subunit that is the princip al site of pyrethroid action. DNA sequence analyses of Vssc1 alleles from s everal independent super-kdr fly strains identified two amino acid substitu tions associated with the super-kdr trait: replacement of leucine at positi on 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of the se mutations by expressing house fly sodium channels containing them in Xen opus laevis oocytes and by characterizing the biophysical properties and py rethroid sensitivities of the expressed channels using two-electrode voltag e clamp. House fly sodium channels that were specifically modified by site- directed mutagenesis to contain the M918T/L1014F double mutation Save reduc ed levels of sodium current expression in oocytes but otherwise exhibited f unctional properties similar to those of wildtype channels and channels con taining the L1014F substitution. However, M918T/L1014F channels were comple tely insensitive to high concentrations of the pyrethroids cismethrin and c ypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in ass ociation with the L1014F mutation, gave very small sodium currents in oocyt es. Assays of these currents in the presence of high concentrations of cism ethrin suggest that this mutation alone is sufficient to abolish the pyreth roid sensitivity of house fly sodium channels. These results define the fun ctional significance of the Vssc1 mutations associated with the super-kdr t rait of the house fly and are consistent with the hypothesis that the super -kdr trait arose by selection of a second-site mutation (M918T) that confer s to flies possessing it even greater resistance than the X-dr allele conta ining the L1014F mutation. (C) 1999 Elsevier Science Ltd. All rights reserv ed.