Andrias O. O'Reilly

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

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.

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT.

Mutations in the DIIS4–S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild‐type and mutant channels were expressed in Xenopus oocytes and subjected to voltage‐clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.

Knockdown resistance to DDT and pyrethroids: from target-site mutations to molecular modelling

Naturally derived insecticides such as pyrethrum and man-made insecticides such as DDT and the synthetic pyrethroids act on the voltage-gated sodium channel proteins found in insect nerve-cell membranes. The correct functioning of these channels is essential for the normal transmission of nerve impulses, and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein that inhibit the binding of the insecticide and result in the insect developing resistance. This perspective outlines the current understanding of the molecular processes underlying target-site resistance to these insecticides (termed kdr and super-kdr), and how this knowledge may in future contribute to the design of novel insecticidal compounds.

Inherited Pain SODIUM CHANNEL NAV1.7 A1632T MUTATION CAUSES ERYTHROMELALGIA DUE TO A SHIFT OF FAST INACTIVATION

Background: Mutations in the sodium channel Nav1.7 cause the inherited pain syndromes IEM and PEPD. Results: The new IEM mutation A1632T impairs channel inactivation, whereas an IEM/PEPD crossover mutation (A1632E) at the same position additionally increases resurgent sodium currents. Conclusion: Reduced inactivation without increased resurgent currents induces symptoms of IEM. Significance: Resurgent currents are likely to determine whether a mutation leads to IEM or PEPD. Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited “paroxysmal extreme pain disorder” (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079–11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.

A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation

Sodium channel Nav1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Nav1.7 produce painful disorders, whereas loss-of-function Nav1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Nav1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Nav1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pores cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Nav1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Nav1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Nav1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Nav1.7.

Predictive 3D modelling of the interactions of pyrethroids with the voltage-gated sodium channels of ticks and mites

BACKGROUND The pyrethroid insecticides are a very successful group of compounds that target invertebrate voltage-gated sodium channels and are widely used in the control of insects, ticks and mites. It is well established that some pyrethroids are good insecticides whereas others are more effective as acaricides. This species specificity is advantageous for controlling particular pest(s) in the presence of another non-target invertebrate, for example controlling the Varroa mite in honeybee colonies. RESULTS We applied in silico techniques to compare the voltage-gated sodium channels of insects versus ticks and mites and their interactions with a range of pyrethroids and DDT analogues. We identified a single amino acid difference within the pyrethroid binding pocket of ticks/mites that may have significant impact on the effectiveness of pyrethroids as acaricides. Other individual amino acid differences within the binding pocket in distinct tick and mite species may provide a basis for future acaricidal selectivity. CONCLUSIONS Three-dimensional modelling of the pyrethroid/DDT receptor site has led to a new hypothesis to explain the preferential binding of acaricidal pyrethroids to the sodium channels of ticks/mites. This is important for understanding pyrethroid selectivity and the potential effects of mutations that can give rise to resistance to pyrethroids in commercially-important pest species.

Tetrameric Bacterial Sodium Channels: Characterization of Structure, Stability, and Drug Binding †

NaChBac from Bacillus halodurans is a bacterial homologue of mammalian voltage-gated sodium channels. It has been proposed that a NaChBac monomer corresponds to a single domain of the mammalian sodium channel and that, like potassium channels, four monomers form a tetrameric channel. However, to date, although NaChBac has been well-characterized for functional properties by electrophysiological measurements on protein expressed in tissue culture, little information about its structural properties exists because of the difficulties in expressing the protein in large quantities. In this study, we present studies on the overexpression of NaChBac in Escherichia coli, purification of the functional detergent-solubilized channel, its identification as a tetramer, and characterization of its secondary structure, drug binding, and thermal stability. These studies are correlated with a model produced for the protein and provide new insights into the structure-function relationships of this sodium channel.

Thermal and chemical unfolding and refolding of a eukaryotic sodium channel.

Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with “denatured” channels, retaining significant helical secondary structure even at 77 °C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes.

A point mutation in the glutamate-gated chloride channel of Plutella xylostella is associated with resistance to abamectin

The diamondback moth, Plutella xylostella, is a global pest of cruciferous vegetables. Abamectin resistance in a field population of P. xylostella was introgressed into the susceptible Roth strain. The resulting introgression strain Roth‐Abm showed 11 000‐fold resistance to abamectin compared with Roth. An A309V substitution at the N‐terminus of the third transmembrane helix (M3) of the glutamate‐gated chloride channel of P. xylostella (PxGluCl) was identified in Roth‐Abm. The frequency of the V309 allele of PxGluCl was 94.7% in Roth‐Abm, whereas no such allele was detected in Roth. A subpopulation of Roth‐Abm was kept without abamectin selection for 20 generations to produce a revertant strain, Roth‐Abm‐D. Abamectin resistance in Roth‐Abm‐D declined to 1150‐fold compared with Roth, with the V309 allele frequency decreased to 9.6%. After treatment of the Roth‐Abm‐D strain with 80 mg/l abamectin the V309 allele frequency in the survivors increased to 55%. This demonstrates that the A309V mutation in PxGluCl is strongly associated with a 10‐fold increase in abamectin resistance in Roth‐Abm relative to Roth‐Abm‐D. Homology modelling and automated ligand docking results suggest that the A309V substitution allosterically modifies the abamectin‐binding site, as opposed to directly eliminating a key binding contact. Other resistance mechanisms to abamectin in Roth‐Abm are discussed besides the A309V mutation of PxGluCl.

G219S mutagenesis as a means of stabilizing conformational flexibility in the bacterial sodium channel NaChBac.

The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm ∼10°C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.