Activation of Drosophila Sodium Channels Promotes Modification by Deltamethrin Reductions in Affinity Caused by Knock-Down Resistance Mutations

kdr and super-kdr are mutations in houseflies and other insects that confer 30and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F 1 M918T) mutations in segment IIS6 and linker II(S4–S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by , 10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata , which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action. key words: insecticide • pyrethroid • para mutation • voltage gated I N T R O D U C T I O N Voltage-activated sodium channels provide selective and rapidly activating ion pathways required for action potential generation and propagation. The a subunit of these channels contains multiple binding sites for neurotoxins and therapeutically important drugs (Catterall, 1992). The molecular nature of many of these binding sites has been identified by systematic sitedirected mutagenesis of the a subunit of mammalian sodium channels (Terlau et al., 1991; Ragsdale et al., 1994; Rogers et al., 1996; Cestele et al., 1998; Wang and Wang, 1998). Much less is known about the structure– function relationships of insect sodium channels because these proteins have only recently been cloned and the conditions for their functional expression have only recently been identified (Feng et al., 1995; Warmke et al., 1997). This heterologous expression provides new opportunities for structure–function studies because modified ligand binding sites can be identified by selecting for insects with resistance to neurotoxic ligands, especially synthetic chemicals that target insect sodium channels (Bloomquist, 1996; Narahashi, 1998). Comparative studies between mammalian and insect sodium channels can also provide insight into structure–function relationships because insect sodium channels are particularly sensitive to a number of neurotoxins such as pyrethroids (Narahashi, 1996; Vais et al., 1997; Warmke et al., 1997). Pyrethroids are commonly used as insecticides in crop protection, animal health, and the control of insects that endanger human health. These insecticides combine high insecticidal activity with low mammalian toxicity and constitute . 25% of the world insecticide market. The intensive use of pyrethroids over the last 20 yr has led to the development of resistance in many insect species (Sawicki, 1985) and this now represents the single most serious threat to their continued, effective use in many pest control programs. An important mechanism of resistance, termed knockdown resistance (or kdr ), 1 confers cross resistance to the entire class of pyrethroids and is characterized by a reduced sensitivity of the insect nervous system to these compounds (Bloomquist, 1993). This type of resistance has been reported in many important pest species, but is best characterized in the housefly, where several variAddress correspondence to Dr. Charles Cohen, Merck Research Laboratories, P.O. Box 2000, Rm. 80N-31C, Rahway, NJ 07065. Fax: 732594-3925; E-mail: [email protected] 1 Abbreviations used in this paper: ATX-II, isoleucine isoform of toxin II from Anemonia sulcata ; kdr mutation, knockdown resistance mutation. on Jne 9, 2017 D ow nladed fom Published February 28, 2000