Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel

Abstract

Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNa v 1-1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNa v 1-1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors.

The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides

“…2. Homology modeling and ligand docking were performed using the ZMM program (45) and Monte Carlo-minimization protocol (46) as described elsewhere (6). Molecular images were created using the PyMOL Molecular Graphics System, Version 0.99rc6 (Schrödinger, LLC, New York, NY).…”

“…Identification of these sodium channel mutations has facilitated the development of molecular markers for early detection of pyrethroid resistance in various populations around the world (5). Furthermore, these mutations help define the receptor sites for pyrethroids on sodium channels and reveal the molecular mechanisms of the differential sensitivities of insect and mammalian sodium channels to pyrethroids (6).…”

“…Phe-1764 and Tyr-1771 in rat Na v 1.2 and Phe-1579 and Tyr-1586 in Na v 1.4, are consistently critical for the binding and action of a wide range of LAs and related drugs on mammalian sodium channels. To facilitate recognition of ligand-sensing residues among different sodium channels, here we labeled these two LA-sensing residues in IVS6 as F 4i15 and Y 4i22 using a nomenclature universal for P-loop ion channels (6,15). Similar to the effect on the action of LAs, alanine substitution, F 4i15 A, resulted in a significant reduction in the ability of DCJW and an experimental SCBI, RH3421, to inhibit Na v 1.4 sodium channels expressed in Xenopus oocytes (16).…”

Rotational Symmetry of Two Pyrethroid Receptor Sites in the Mosquito Sodium Channel

“…To overcome the problem of different residue numbers in homologous positions of sodium channels in different organisms and to highlight symmetric locations of residues in different channel domains, we used a residue-labeling scheme, which is universal for P-loop channels (Zhorov and Tikhonov, 2004;Du et al, 2013). A residue label includes the domain number (1-4), segment type (k, the linker-helix L45; i, the inner helix S6; and o, the outer helix S5), and relative number of the residue in the segment (see Fig.…”

A Mutation in the Intracellular Loop III/IV of Mosquito Sodium Channel Synergizes the Effect of Mutations in Helix IIS6 on Pyrethroid Resistance

“…In recent years, using X-ray structures of a bacterial potassium channel KcsA (Doyle et al, 1998), the mammalian voltage-gated potassium channel K v 1.2 crystallized in the open state (Long et al, 2005) and a bacterial sodium channel, Na v Ab, crystallized in the closed state (Payandeh et al, 2011) as templates, homology models of eukaryotic sodium channels have been developed to predict binding sites of sodium channel neurotoxins (Lipkind and Fozzard, 2005;O'Reilly et al, 2006;Zhorov, 2007, 2012;Du et al, 2013). Mutational analyses coupled with computer modeling show that pyrethroids bind to two analogous receptor sites.…”