Introduction Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue11.Nov2000:

Insect Biochemistry and Molecular Biology 30 2000 1051–1059 www.elsevier.comlocateibmb Point mutations in domain III of a Drosophila neuronal Na channel confer resistance to allethrin R.L. Martin a , B. Pittendrigh b , J. Liu a , R. Reenan c , R. ffrench-Constant d , D.A. Hanck a, a Department of Medicine MC6094, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA b Department of Entomology, Purdue University, West Lafayette, IN 47907, USA c University of Connecticut Health Center-MC3301, Department of Genetics and Developmental Biology, 263 Farmington Avenue, Farmington, CT 06030, USA d Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK Received 3 December 1999; received in revised form 30 March 2000; accepted 30 March 2000 Abstract Voltage-gated sodium channels are the presumed site of action of pyrethroid insecticides and DDT. We screened several mutant sodium channel Drosophila lines for resistance to type I pyrethroids. In insecticidal bioassays the para 74 and para DN7 fly lines showed greater than 4-fold resistance to allethrin relative to the allethrin sensitive Canton-S control line. The amino acid substitutions of both mutants are in domain III. The point mutation associated with para 74 lies within the S6 transmembrane region and the amino acid substitution associated with para DN7 lies within the S4–S5 linker region. These sites are analogous to the mutations in domain II underlying knockdown resistance kdr and super-kdr, naturally occurring forms of pyrethroid resistance found in house- flies and other insects. Electrophysiological studies were performed on isolated Drosophila neurons from wild type and para 74 embryos placed in primary culture for three days to two weeks. The mutant para 74 sodium currents were kinetically similar to wild type currents, in activation, inactivation and time to peak. The only observed difference between para 74 and wild-type neurons was in the affinity of the type I pyrethroid, allethrin. Application of 500 nM allethrin caused removal of inactivation and prolonged tail currents in wild type sodium channels but had little or no effect on para 74 mutant sodium channels.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Sodium channel; Allethrin; Drosophila; Para mutants; Electrophysiology

1. Introduction

The voltage-gated sodium channel is responsible for the rapid upstroke of the action potential in many excit- able tissues, including most neurons. In Drosophila, the α -subunit is encoded by the para gene Loughney et al., 1989 and it is highly homologous to sodium channels in other insects. The α -subunit of the sodium channel is comprised of four domains, each with six transmem- brane spanning regions Fig. 1. Several years ago pyr- ethroid and DDT resistant mutants in house fly were described Williamson et al., 1996; Miyazaki et al., 1996, which were identified to result from point mutations in domain II of that voltage-gated sodium Corresponding author. E-mail address: dottiehearts.bsd.uchicago.edu D.A. Hanck. 0965-174800 - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 8 0 - 1 channel Smith et al., 1997; Lee et al., 1999b. The kdr knockdown resistant mutation is located in S6 Fig. 1. In a second pesticide resistant allele, the kdr mutation was found paired with another amino acid change in the intracellular linker between S4 and S5. The double mutant conferred much greater resistance to pyrethroids and was named super-kdr. Pittendrigh et al. 1997 identified two mutations simi- lar to kdr and super-kdr in domain III of the voltage- gated Drosophila sodium channel, which confer resist- ance to pyrethroids and DDT. First, the para 74 mutant fly line has a mutation analogous to that of kdr, but in the third domain, with a Met to Ile M1536I change within ten amino acid positions of the analogous position that kdr occupies in the second domain. Second, the par- a DN7 fly line has a Ala to Val A1422V change in the intracellular loop between S4 and S5 of the III domain, only a single amino acid position away from the anal- 1052 R.L. Martin et al. Insect Biochemistry and Molecular Biology 30 2000 1051–1059 S2 S3 S4 S5 S1 S6 para74 paraDN7 S2 S3 S4 S5 S1 S6 S2 S3 S4 S5 S1 S6 S2 S3 S4 S5 S1 S6 super kdr kdr A1422V M1536I Fig. 1. A schematic diagram of the para voltage gated sodium channel indicating the location of the two amino acid replacements in DDT resistant para ts mutants. A1422V in para DN7 and M1536I in para 74 numbering is according to GenBank accession number AAB59195. The relative location of the kdr and super-kdr replacements documented in its homologs in the house fly and the German cockroach Dong and Scott, 1994; Williamson et al., 1996; Miyazaki et al., 1996; Dong, 1997 are also shown. ogous position in domain II of super-kdr. In this study we performed lethality assays on the domain III mutants and compared the electrophysiological properties of the wild type Canton-S voltage-gated sodium channel and the affinity of allethrin with the para 74 mutant in Droso- phila neurons. Some of these data have been presented in abstract form Martin et al., 1998; Pittendrigh et al., 1998.

2. Methods