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

Abstract

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Although it is well known that specific mutations in insect sodium channels confer knockdown resistance (kdr) to pyrethroids, the atomic mechanisms of pyrethroid-sodium channel interactions are not clearly understood. Previously, computer modeling and mutational analysis predicted two pyrethroid receptors, pyrethroid receptor site 1 (PyR1) (initial) and pyrethroid receptor site 2 (PyR2), located in the domain interfaces II/III and I/II, respectively. The models differ in ligand orientation and the number of transmembrane helices involved. In this study, we elaborated a revised PyR1 model of the mosquito sodium channel. Computational docking in the K v 1.2-based open channel model yielded a complex in which a pyrethroid (deltamethrin) binds between the linker helix IIL45 and transmembrane helices IIS5, IIS6, and IIIS6 with its dibromoethenyl and diphenylether moieties oriented in the intra-and extracellular directions, respectively. The PyR2 and revised PyR1 models explained recently discovered kdr mutations and predicted new deltamethrin-channel contacts. Further model-driven mutagenesis identified seven new pyrethroid-sensing residues, three in the revised PyR1 and four in PyR2. Our data support the following conclusions: 1) each pyrethroid receptor is formed by a linker-helix L45 and three transmembrane helices (S5 and two S6s); 2) IIS6 contains four residues that contribute to PyR1 and another four to PyR2; 3) seven pairs of pyrethroid-sensing residues are located in symmetric positions within PyR1 and PyR2; and 4) pyrethroids bind to PyR1 and PyR2 in similar orientations, penetrating deeply into the respective domain interfaces. Our study elaborates the dual pyrethroid-receptor sites concept and provides a structural background for rational development of new insecticides.

Mutations of two acidic residues at the cytoplasmic end of segment IIIS6 of an insect sodium channel have distinct effects on pyrethroid resistance

“…3B and C in Du et al (2013). In our model of PyR1-bound deltamethrin (Du et al, 2015) the ligand reaches residue I 3i23 , which is just five positions above D 3i28 . We suggest that in the double mutant the conformational shift of the IIIS6 C-terminal part would strengthen its contact with Type II, but not Type I pyrethroids.…”

“…In our latest model of pyrethroid receptors PyR1 and PyR2 (Du et al, 2015) residue D 3i28 is too far from the bound pyrethroids and residue E 3i32 (whose analog is absent in the X-ray structure which was used to build the model) would be even farther. Therefore, according to our models, mutations D 3i28 V and E 3i32 G would affect binding of pyrethroids to PyR1 and/or PyR2 by allosteric mechanisms, e.g.…”

“…In general, sodium channels modified by Type I pyrethroids return to the closed state very rapidly, within seconds, upon repolarization, whereas Type II pyrethroids-induce tail currents that decay very slowly, often minutes after repolarization (Narahashi, 1988). Most of the examined kdr sodium channel mutations greatly reduce the amplitude of tail currents induced by both Type I and Type II pyrethroids (Du et al, 2013, 2015; Lee et al, 1999; Tan et al, 2002a, 2005; Vais et al, 2001; Warmke et al, 1997). However, recently we found that two pyrethroid resistance-conferring alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 of Drosophila melanogaster do not reduce the amplitude of tail currents induced by Type II pyrethroids, but accelerate the decay of the tail currents (Chen et al, 2016).…”

“…1B). Previous modeling of the PyR1 and PyR2 receptors did not include D 3i28 or E 3i32 (Du et al, 2013, 2015), therefore we upgraded our open and closed models of the BgNa v 1-1a channel by using, as templates, the X-ray structures of the closed chimeric sodium channel Na v Ab/Na v 1.7 (Ahuja et al, 2015) and open chimeric potassium channel K v 1.2/K v 2.1 (Long et al, 2007). In these structures, the S6 helices include residues i32 and linker-helices L45 are resolved.…”

“…1B. We further optimized state-dependent contacts of residues D 3i28 and E 3i32 in the BgNa v 1-1a channel and its D 3i28 V and D 3i28 V/E 3i32 G mutants by Monte Carlo-minimizing energy of the cytoplasmic part of repeat III as described elsewhere (Du et al, 2015). …”