Insect Sodium Channels Research Articles

Identification, Characterization, and Modeling of a Bioinsecticide Protein Isolated from Scorpion Venom gland: A Three-Finger Protein.

The majority of insecticides target sodium channels. The increasing emergence of resistance to the current insecticides has persuaded researchers to search for alternative compounds. Scorpion venom gland as a reservoir of peptides or proteins, which selectively target insect sodium channels. These proteins would be an appropriate source for finding new suitable anti-insect components. Transcriptome of venom gland of scorpion Mesobuthus eupeus was obtained by RNA extraction and complementary DNA library synthesis. The obtained transcriptome was blasted against protein databases to find insect toxins against sodium channel based on the statistically significant similarity in sequence. Physicochemical properties of the identified protein were calculated using bioinformatics software. The three-dimensional structure of this protein was determined using homology modeling, and the final structure was assessed by molecular dynamics simulation. The sodium channel blocker found in the transcriptome of M. eupeus venom gland was submitted to the GenBank under the name of meuNa10, a stable hydrophilic protein consisting of 69 amino acids, with the molecular weight of 7721.77 g/mol and pI of 8.7. The tertiary structure of meuNa10 revealed a conserved LCN-type cysteine-stabilized alpha/beta domain stabilized by eight cysteine residues. The meuNa10 is a member of the 3FP superfamily consisting of three finger-like beta strands. This study identified meuNa10 as a small insect sodium channel-interacting protein with some physicochemical properties, including stability and water-solubility, which make it a good candidate for further in vivo and in vitro experiments in order to develop a new bioinsecticide.

Pyrethroids in an AlphaFold2 Model of the Insect Sodium Channel.

Simple SummaryPyrethroids are a major class of insecticides for controlling arthropod pests and human disease transmitting vectors. Resistance to pyrethroid insecticides presents a serious obstacle to effective pest control. One major mechanism of pyrethroid resistance is caused by mutations in the voltage-gated sodium channel, the target of pyrethroid toxic action. Previous mutational analyses, coupled with homology modeling, predicted two pyrethroid receptor sites on insect sodium channels. Mutations that are located in or close to these receptor sites likely confer resistance by directly reducing pyrethroid binding affinity. However, many mutations appear to be far from the receptor sites, and how these mutations confer pyrethroid resistance remains unknown. In this study, we employed the AlphaFold2 neural network to generate new models of mosquito and cockroach sodium channels. We then used computational methods to dock pyrethroids in the open and closed states of sodium channels to understand the atomic mechanisms of interactions between pyrethroids and sodium channels. Our analysis suggests that mutations residing beyond the receptor sites alter the action of pyrethroids by allosterically affecting the geometry of the receptor sites. The information gained from this study could be valuable for a structure-based design of novel pyrethroids.Pyrethroid insecticides stabilize the open state of insect sodium channels. Previous mutational, electrophysiological, and computational analyses led to the development of homology models predicting two pyrethroid receptor sites, PyR1 and PyR2. Many of the naturally occurring sodium channel mutations, which confer knockdown resistance (kdr) to pyrethroids, are located within or close to these receptor sites, indicating that these mutations impair pyrethroid binding. However, the mechanism of the state-dependent action of pyrethroids and the mechanisms by which kdr mutations beyond the receptor sites confer resistance remain unclear. Recent advances in protein structure prediction using the AlphaFold2 (AF2) neural network allowed us to generate a new model of the mosquito sodium channel AaNav1-1, with the activated voltage-sensing domains (VSMs) and the presumably inactivated pore domain (PM). We further employed Monte Carlo energy minimizations to open PM and deactivate VSM-I and VSM-II to generate additional models. The docking of a Type II pyrethroid deltamethrin in the models predicted its interactions with many known pyrethroid-sensing residues in the PyR1 and PyR2 sites and revealed ligand-channel interactions that stabilized the open PM and activated VSMs. Our study confirms the predicted two pyrethroid receptor sites, explains the state-dependent action of pyrethroids, and proposes the mechanisms of the allosteric effects of various kdr mutations on pyrethroid action. The AF2-based models may assist in the structure-based design of new insecticides.

Characterization of two kdr mutations at predicted pyrethroid receptor site 2 in the sodium channels of Aedes aegypti and Nilaparvata lugens

Pyrethroid insecticides prolong the opening of insect sodium channels by binding to two predicted pyrethroid receptor sites (PyR), PyR1 and PyR2. Many naturally-occurring sodium channel mutations that confer pyrethroid resistance (known as knockdown resistance, kdr) are located at PyR1. Recent studies identified two new mutations, V253F and T267A, at PyR2, which co-exist with two well-known mutations F1534C or M918T, at PyR1, in pyrethroid-resistant populations of Aedes aegypti and Nilaparvata lugens, respectively. However, the role of the V253F and T267A mutations in pyrethroid resistance has not been functionally examined. Here we report functional characterization of the V253F and T267A mutations in the Ae. aegypti sodium channel AaNav2-1 and the N. lugens sodium channel NlNav1 expressed in Xenopus oocytes. Both mutations alone reduced channel sensitivity to pyrethroids, including etofenprox. We docked etofenprox in a homology model of the pore module of the NlNav1 channel based on the crystal structure of an open prokaryotic sodium channel NavMs. In the low-energy binding pose etofenprox formed contacts with V253, T267 and a previously identified L1014 within PyR2. Combining of V253F or T267A with F1534C or M918T results in a higher level of pyrethroid insensitivity. Furthermore, both V253F and T267A mutations altered channel gating properties. However, V253F- and T267A-induced gating modifications was not observed in the double mutant channels. Our findings highlight the first example in which naturally-found combinational mutations in PyR1 and PyR2 not only confer higher level pyrethroid insensitivity, but also reduce potential fitness tradeoff in pyrethroid-resistant mosquitoes caused by kdr mutation-induced sodium channel gating modifications.

De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator.

The brown widow spider, Latrodectus geometricus, is a predator of a variety of agricultural insects and is also hazardous for humans. Its venom is a true pharmacopeia representing neurotoxic peptides targeting the ion channels and/or receptors of both vertebrates and invertebrates. The lack of transcriptomic information, however, limits our knowledge of the diversity of components present in its venom. The purpose of this study was two-fold: (1) carry out a transcriptomic analysis of the venom, and (2) investigate the bioactivity of the venom using an electrophysiological bioassay. From 32,505 assembled transcripts, 8 toxin families were classified, and the ankyrin repeats (ANK), agatoxin, centipede toxin, ctenitoxin, lycotoxin, scorpion toxin-like, and SCP families were reported in the L. geometricus venom gland. The diversity of L. geometricus venom was also uncovered by the transcriptomics approach with the presence of defensins, chitinases, translationally controlled tumor proteins (TCTPs), leucine-rich proteins, serine proteases, and other important venom components. The venom was also chromatographically purified, and the activity contained in the fractions was investigated using an electrophysiological bioassay with the use of a voltage clamp on ion channels in order to find if the neurotoxic effects of the spider venom could be linked to a particular molecular target. The findings show that U24-ctenitoxin-Pn1a involves the inhibition of the insect sodium (Nav) channels, BgNav and DmNav. This study provides an overview of the molecular diversity of L. geometricus venom, which can be used as a reference for the venom of other spider species. The venom composition profile also increases our knowledge for the development of novel insecticides targeting voltage-gated sodium channels.

Charge substitutions at the voltage-sensing module of domain III enhance actions of site-3 and site-4 toxins on an insect sodium channel

Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion β-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1–S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT3-c, on BgNav1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.

Mapping the interaction surface of scorpion β-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant β-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion β-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

New Sea Anemone Toxin RTX-VI Selectively Modulates Voltage-Gated Sodium Channels.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.

Two classic mutations in the linker‐helix IIL45 and segment IIS6 of Apolygus lucorum sodium channel confer pyrethroid resistance

Pyrethroids are classified as type I and type II for distinct symptomology. Voltage-gated sodium channel is a primary target of pyrethroids. Mutations of the insect sodium channel have been identified to result in resistance to pyrethroids. Double mutation (L1002 F/M906 I) was detected in field-strain of Apolygus lucorum (Meyer-Dür). Although, it was illuminated the function of the same position mutation in other pests, it is necessary to demonstrate the role in A. lucorum . In this study, we examined the effects of mutations on channel gating and pyrethroid sensitivity in Xenopus oocytes. L1002 F, M906 I and L1002 F/M906 I all shifted the voltage dependence of activation in the depolarizing direction. L1002 F, M906 I and L1002 F/M906 I all reduced the amplitude of tail currents induced by type I (bifenthrin and permethrin) and type II (λ-cyhalothrin and deltamethrin). The double mutation, L1002 F/M906 I, reduced integral channel modification by 10-fold compared with the L1002 F and M906 I mutations alone, respectively. Computational analysis based on the model of dual pyrethroid receptors, the two resistance mutations, L1002 F and M906 I are facing two opposite sides of this newly identified pocket. Both mutations affect the optimal binding of the ligands by changing the shape of the pocket but in different ways. Our results illustrate the distinct effect of mutations on pyrethroids. It is predicted with computer modeling that these mutations allosterically affect pyrethroid binding. © 2020 Society of Chemical Industry.

Cloning and expression of OdTx12, a β excitatory toxin from Odontobuthus doriae, in Escherichia coli and evaluation of its bioactivity in Locusta migratoria

The venom of Odontobuthus doriae contains several peptide toxins that interfere with the sodium channel function of cell membranes, some of which specifically act on the insect's sodium channel without affecting mammalian cells. In this study sodium channel toxins of Odontobuthus doriae were aligned to other closely related toxins by BLAST and ClustalW servers. Among these toxins, NaTx12 (OdTx12) showed more than 90% similarity to the most known beta excitatory toxin, AaHIT1; furthermore, our modeling studies confirmed high tertiary structure similarity of these proteins. OdTx12 was cloned and expressed in E.coli, using pET26-b and pET28-a expression vectors. Tris-tricine SDS-PAGE and Western blot analysis showed OdTx12 expression by pET28-a, only. After purification, bioactivity of the purified protein was analyzed by injection and oral administration to Locusta migratoria larvae, and toxicity to mammals was tested on mice. Injection of OdTx12 resulted in the killing of larvae with LD50 of 0.4 and 0.2 after 48 and 72 h respectively, but oral administration of OdTx12 had no significant effect on Locusta migratoria, nor did the injection to mice show any signs of toxicity. These results showed that OdTx12, as a novel β excitatory toxin can be considered as a candidate for insect control purposes.

Mutational analysis of state-dependent contacts in the pore module of eukaryotic sodium channels

Voltage-gated sodium channels have residues that change or may change contacts upon gating. Contributions of individual contacts in stability of different states are incompletely understood. Pore-lining inner helices contain exceptionally conserved asparagines in positions i20. Here we explored how mutations in positions i20 and i29 affect electrophysiological properties of insect sodium channels. In repeat interfaces I/IV, III/II and IV/III, alanine substitutions caused positive activation shifts in positions i20 and i29, negative shifts of slow inactivation in positions i20 and positive shifts of slow inactivation in positions i29. The results support the hypothesis on open state inter-repeat H-bonding of residues i20 and i29. The shift magnitudes vary between interfaces, reflecting structural asymmetry of the channels. Mutations in positions i20 of repeats III and IV caused much longer recovery delay from the slow and fast inactivation than other mutations. In repeat IV, alanine substitution of tyrosine i30 rescued positive activation shift of mutation in position i29. Our data suggest that polar residues in positions i29 are involved in stabilization of both the open and slow-inactivated states. Transition between the states may involve switching of H-bonding partners of residues i29 from the conserved asparagines to currently unknown residues.

Positive Modulation of Human Nav Channels by Pyrethroids

Voltage-gated sodium channels (Nav) are crucial to the initiation and propagation of action potentials in electrically excitable cells. Inhibitors of such ion channels have been developed and approved as drugs to treat a variety of indications. In contrast, sodium channel activators have not been considered relevant and safe in a therapeutic setting because of their high risk of toxicity and side effects. Nevertheless, there is a recent interest in activators of e.g. Nav1.1 (SCN1A) for the treatment of neurological indications, i.e. that selective positive modulation of the Nav1.1 sodium channel may hold therapeutic potential for epilepsy, schizophrenia, and Alzheimer's disease (Jensen et al., 2014). Research aimed at the identification of novel, selective Nav positive modulators, however, appears to be hampered by a deficit in suitable tool compounds with certain modulating features to initiate and validate screening assays. Although sodium channel modifying pyrethroids have been in use as important pest management tools in agriculture, public health, and a variety of household applications, their action on human Nav has not been studied in detail. Therefore we tested a set of Type 1 pyrethroids that have been described to have positive modulatory effects on insect and rodent sodium channels. Utilizing the Syncropatch 96 automated patch clamp system we were able to identify several pyrethroids activating human Nav 1.1 and/or Nav1.5. Confirmation and characterization by manual patch-clamp revealed pyrethroids with different types of positive modulation. Both, compounds acting via a delay of the inactivation time constant (similar to those published recently by Crestey et al., 2015) as well as such promoting a sustained channel opening (Veratridine-like) were identified for Nav1.1 and Nav1.5, respectively.

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

Mutations in sodium channels are known to confer knockdown resistance to pyrethroid insecticides, such as permethrin and cypermethrin, in various agricultural pests and disease vectors. Double mutations, D3i28V and E3i32G, were detected in cypermethrin-resistant Helicoverpa armigera and Heliothis virescens populations. However, the role of the two mutations in pyrethroid resistance remains unclear. In this study, we introduced the mutations into the cockroach sodium channel, BgNav1-1a, and examined their effects on channel gating and pyrethroid sensitivity in Xenopus oocytes. D3i28V alone and the double mutation, D3i28V/E3i32G, shifted the voltage dependence of activation in the depolarizing direction by 15 mV and 20 mV, respectively, whereas E3i32G had no significant effect. D3i28V reduced the amplitude of tail currents induced by permethrin and NRDC 157 (Type I pyrethroids) and deltamethrin and cypermethrin (Type II pyrethroids), whereas E3i32G alone had no effect. Intriguingly, the amplitude of Type II pyrethroid-induced tail current from D3i28V/E3i32G channels was similar to that of BgNav1-1a channels, but the decay of the tail currents was accelerated. Such effects were not observed for Type I pyrethroid-induced tail currents. Computational analysis based on the model of dual pyrethroid receptors on insect sodium channels predicted D3i28V and E3i32G exert their effects on channel gating and pyrethroid action via allosteric mechanisms. Our results not only illustrate the distinct effect of the D3i28V/E3i32G double mutations on Type I vs. Type II pyrethroids, but also reinforce the concept that accelerated decay of tail currents can be an effective mechanism of pyrethroid resistance to Type II pyrethroids.

Elucidation of pyrethroid and DDT receptor sites in the voltage-gated sodium channel

DDT and pyrethroid insecticides were among the earliest neurotoxins identified to act on voltage-gated sodium channels. In the 1960s, equipped with, at the time, new voltage-clamp techniques, Professor Narahashi and associates provided the initial evidence that DDT and allethrin (the first commercial pyrethroid insecticide) caused prolonged flow of sodium currents in lobster and squid giant axons. Over the next several decades, continued efforts by Prof. Narahashi’s group as well as other laboratories led to a comprehensive understanding of the mechanism of action of DDT and pyrethroids on sodium channels. Fast forward to the 1990s, genetic, pharmacological and toxicological data all further confirmed voltage-gated sodium channels as the primary targets of DDT and pyrethroid insecticides. Modifications of the gating kinetics of sodium channels by these insecticides result in repetitive firing and/or membrane depolarization in the nervous system. This mini-review focuses on studies from Prof. Narahashi’s pioneer work and more recent mutational and computational modeling analyses which collectively elucidated the elusive pyrethroid receptor sites as well as the molecular basis of differential sensitivities of insect and mammalian sodium channels to pyrethroids.

Insight into the Mode of Action of Haedoxan A from Phryma leptostachya.

Haedoxan A (HA) is a major active ingredient in the herbaceous perennial plant lopseed (Phryma leptostachya L.), which is used as a natural insecticide against insect pests in East Asia. Here, we report that HA delayed the decay rate of evoked excitatory junctional potentials (EJPs) and increased the frequency of miniature EJPs (mEJPs) on the Drosophila neuromuscular junction. HA also caused a significant hyperpolarizing shift of the voltage dependence of fast inactivation of insect sodium channels expressed in Xenopus oocytes. Our results suggest that HA acts on both axonal conduction and synaptic transmission, which can serve as a basis for elucidating the mode of action of HA for further designing and developing new effective insecticides.

Mapping Receptor Sites for Sodium Channel Blocking Insecticides DCJW and Metaflumizone in an Insect Sodium Channel

Acquired pest resistance to long-used insecticides, such as sodium channel agonists DDT and pyrethroids, motivates development of new insecticides with different mechanisms of action. Indoxacarb, its insect-specific active metabolite DCJW, and metaflumizone (MFZ) are sodium channel blocker insecticides (SCBIs). They preferably bind to and trap sodium channels in the slow-inactivated state, a mode of action similar to that of local anesthetics (LAs). Previous studies identified several SCBI-sensing residues that face the inner pore, but the atomic mechanism of action of SCBIs is unknown. Unlike classical LAs, DCJW and MFZ are uncharged molecules. They may chelate a permeant ion to form a cationic blocking particle. Here we have built a homology model of the open cockroach sodium channel BgNav1-1 using as templates the X-ray structures of an open potassium channel Kv1.2 and a closed sodium channel NavAb. We then generated libraries of conformers of sodium ion-bound SCBIs and docked them into the channel model imposing ligand-sidechain distance constraints between SCBIs and known SCBI-sensing residues. Our computations predicted that SCBIs bind in the inner pore with the SCBI-chelated sodium ion located at the cation-attractive focus of P1 helices and an aromatic moiety extended into the III/IV repeat interface that has insect-specific residues. Model-driven mutagenesis and electrophysiology identified five new SCBI-sensing residues, including residues that are specific to either DCJW or MFZ. Our study supports the following conclusions. (1) Electroneutral SCBIs chelate a sodium ion by the common C=N-NH-C=O fragment to form a cationic blocking particle. (2) The blocking particle preferably binds in the inner pore of sodium-deficient slow-inactivated channels. (3) Selective toxicity of MFZ is due to binding to insect-specific residues in the III/IV repeat interface. Supported by NIH and NSERC.

Differential effects of the recombinant toxin PnTx4(5-5) from the spider Phoneutria nigriventer on mammalian and insect sodium channels.

The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl-d-aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P.nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50=212.5nM), and at 1μM caused an increase on current peak amplitude of 105.2±3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43±8.04%, IC50=1.5μM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.

Mutations in the transmembrane helix S6 of domain IV confer cockroach sodium channel resistance to sodium channel blocker insecticides and local anesthetics

Indoxacarb and metaflumizone are two sodium channel blocker insecticides (SCBIs). They preferably bind to and trap sodium channels in the slow-inactivated non-conducting state, a mode of action similar to that of local anesthetics (LAs). Recently, two sodium channel mutations, F1845Y (F4i15Y) and V1848I (V4i18I), in the transmembrane segment 6 of domain IV (IVS6), were identified to be associated with indoxacarb resistance in Plutella xylostella. F4i15 is known to be critical for the action of LAs on mammalian sodium channels. Previously, mutation F4i15A in a cockroach sodium channel, BgNav1-1a, has been shown to reduce the action of lidocaine, a LA, but not the action of SCBIs. In this study, we introduced mutations F4i15Y and V4i18A/I individually into the cockroach sodium channel, BgNav1-1a, and conducted functional analysis of the three mutants in Xenopus oocytes. We found that both the F4i15Y and V4i18I mutations reduced the inhibition of sodium current by indoxacarb, DCJW (an active metabolite of indoxacarb) and metaflumizone. F4i15Y and V4i18I mutations also reduced the use-dependent block of sodium current by lidocaine. In contrast, substitution V4i18A enhanced the action metaflumizone and lidocaine. These results show that both F4i15Y and V4i18I mutations may contribute to target-site resistance to SCBIs, and provide the first molecular evidence for common amino acid determinants on insect sodium channels involved in action of SCBIs and LA.

β/δ-PrIT1, a highly insecticidal toxin from the venom of the Brazilian spider Phoneutria reidyi (F.O. Pickard-Cambridge, 1897).

A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. β/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-β/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes β/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, β/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, β/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for β/δ-PrIT1, leading to two different pharmacological responses. β/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.

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

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 Kv1.2-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.

Distinct modulating effects of TipE-homologs 2–4 on Drosophila sodium channel splice variants

The Drosophila melanogaster TipE protein is thought to be an insect sodium channel auxiliary subunit functionally analogous to the β subunits of mammalian sodium channels. Besides TipE, four TipE-homologous proteins (TEH1–4) have been identified. It has been reported that TipE and TEH1 have both common and distinct effects on the gating properties of splice variants of the Drosophila sodium channel, DmNav. However, limited information is available on the effects of TEH2, TEH3 and TEH4 on the function of DmNav channel variants. In this study, we found that TEH2 increased the amplitude of peak current, but did not alter the gating properties of three examined DmNav splice variants expressed in Xenopus oocytes. In contrast, TEH4 had no effect on peak current, yet altered the gating properties of all three channel variants. Furthermore, TEH4 enhanced persistent current and slowed sodium current decay. The effects of TEH3 on DmNav variants are similar to those of TEH4, but the data were collected from a small portion of oocytes because co-expression of TEH3 with DmNav variants generated a large leak current in the majority of oocytes examined. In addition, TEH3 and TEH4 enhanced the expression of endogenous currents in oocytes. Taken together, our results reveal distinct roles of TEH proteins in modulating the function of sodium channels and suggest that TEH proteins might provide an important layer of regulation of membrane excitability in vivo. Our results also raise an intriguing possibility of TEH3/TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels.