Modelling insecticide-binding sites in the voltage-gated sodium channel

Abstract

A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides, their mechanism of action and the role of mutations in the channel that are known to confer insecticide resistance. The sodium channel was modelled in an open conformation with the insecticide-binding site located in a hydrophobic cavity delimited by the domain II S4-S5 linker and the IIS5 and IIIS6 helices. The binding cavity is predicted to be accessible to the lipid bilayer and therefore to lipid-soluble insecticides. The binding of insecticides and the consequent formation of binding contacts across different channel elements could stabilize the channel when in an open state, which is consistent with the prolonged sodium tail currents induced by pyrethroids and DDT. In the closed state, the predicted alternative positioning of the domain II S4-S5 linker would result in disruption of pyrethroid-binding contacts, consistent with the observation that pyrethroids have their highest affinity for the open channel. The model also predicts a key role for the IIS5 and IIIS6 helices in insecticide binding. Some of the residues on the helices that form the putative binding contacts are not conserved between arthropod and non-arthropod species, which is consistent with their contribution to insecticide species selectivity. Additional binding contacts on the II S4-S5 linker can explain the higher potency of pyrethroid insecticides compared with DDT.

Figure 1. Transmembrane topology of the voltage-gated sodium channel

The pore-forming α-subunit consists of a single polypeptide chain with four internally homologous domains (I–IV), each having six transmembrane helices (S1–S6). The domains assemble to form a central aqueous pore, lined by the S5, S6 and S5-S6 linkers (P-loops). The identity and location of mutations associated with kdr are shown, with residues numbered according to the sequence of the housefly voltage-gated sodium channel.

Figure 2. Sequence alignments of the KvAP channel and housefly voltage-gated sodium channel (Na_v) S6 helices from domains I–IV

Glycine residues conserved in the gating-hinge position are shown in bold. Resistance-associated residues Val⁴¹⁰, Leu¹⁰¹⁴ and Phe¹⁵³⁸ are underlined. Residue numbers are shown on the right.

Figure 3. Model of the activated-state of the housefly voltage-gated sodium channel

(A) The voltage sensor domains are shown in surface-filling representation (red). The S4-S5 linkers, S5 helices, pore helices and S6 helices are shown as ribbons (yellow, cyan, brown and blue respectively). Residues Met⁹¹⁸, Leu⁹²⁵, Thr⁹²⁹ and Leu⁹³² are shown in space-filling representation (green). (B) The II S4-S5 linker showing its amphipathicity. The S4-S5 linker, S5 and S6 helices are coloured as described above. Residues on the II S4-S5 linker that are hydrophobic (Leu⁹¹¹, Leu⁹¹⁴, Ile⁹¹⁵, Ile⁹¹⁷, Met⁹¹⁸, Gly⁹¹⁹) are shown in green, and polar or charged residues (Thr⁹¹⁰, Asn⁹¹², Ser⁹¹⁶, Arg⁹²⁰) are in shown red.

Figure 4. Sequence alignments of the Kv1.2 channel and housefly voltage-gated sodium channel (Na_v) S4-S5 linker and S5 helix, with the bottom line showing identical (*), conserved (:) and semi-conserved (.) substitutions

Figure 5. Chemical structures of DDT and some pyrethroid insecticides

Figure 6. Docking predictions for the acid, ester and alcohol moieties of fenvalerate with the housefly voltage-gated sodium channel model

(A) Residues ≤4 Å from the acid moiety of fenvalerate are shown in stick representation. Residues found to be non-conserved between insect and non-insect species (see Figure 7) are shown in red. The estimated free-energy of binding (ΔG_b) from Autodock [29] for this docking prediction is −2.83 kcal/mol (4.184 cal≡1 J). (B) The program HBPLUS [47] was used to identify the presence of a hydrogen bond between the carbonyl group of fenvalerate and the hydroxyl group of Thr⁹²⁹ (ΔG_b=−0.95 kcal/mol). (C) Residues ≤4 Å from the alcohol moiety of fenvalerate are shown in stick representation (ΔG_b=−1.71 kcal/mol).

Figure 7. Sequence alignment showing the location of hydrophobic residues in the postulated pyrethroid binding site that differ (in bold) between arthropod and non-arthropod species

Figure 8. Optimized docking predictions for pyrethroids at the voltage-gated sodium channel

(A) Fenvalerate docked in the postulated pyrethroid-binding site. Binding interactions between the acid moiety, ester group and alcohol moiety as shown in Figure 6(A), 6(B) and 6(C) respectively, were retained during the manual docking of fenvalerate with the housefly sodium channel model. (B) Acrinathrin docked in the postulated pyrethroid-binding site. Residues that are ≤4 Å from the acid moiety are shown in stick representation. (C) Bifenthrin docked in the postulated pyrethroid-binding site.

Figure 9. Docking predictions for DDT

(A) The acid and alcohol moieties of fenvalerate. (B) The structure of DDT superimposed on to the acid moiety of fenvalerate (based on an original overlay of deltamethrin and DDT produced by M. S. Williamson and H. Rassmussen, Rothamsted Research). The volumes of the two structures are shown in mesh format. The two dimethyl groups of fenvalerate overlap with the two chlorine atoms on the trichloro group of DDT. (C) Docking of DDT with the housefly voltage-gated sodium channel. The structure of DDT was superimposed on to the acid moiety of the fenvalerate docking simulation shown in Figure 8(A).