Functional reconstitution of rat Nav1.6 sodium channels in vitro for studies of pyrethroid action.

The ability to reconstitute sodium channel function and pharmacology in vitro using cloned subunits of known structure has greatly enhanced our understanding of the action of pyrethroid insecticides at this target and the structural determinants of resistance and interspecies selectivity. However, the use of reconstituted channels raises three critical questions: (1) Which subunits and subunit combinations should be used? (2) Which heterologous expression system is preferred? (3) Which combination of subunits and expression system best represents the function of native neuronal channels in the organism of interest? This review considers these questions from the perspective of recent research in this laboratory on the action of pyrethroid insecticides on rat Nav1.6 sodium channels by comparing the effects of heteroligomeric complex composition on channel function and insecticide response when channels are expressed in either Xenopus oocytes or stably-transformed HEK293 cells. These comparisons provide new insight into the influence of cellular context on the functional and pharmacological properties of expressed channels, the modulatory effects of sodium channel auxiliary subunits on the action of pyrethroids, and the relative fidelity of the Xenopus oocyte and HEK293 cell expression systems as model systems for studying of channel function and pyrethroid action.

Effects of the ß1 auxiliary subunit on modification of Rat Na(v)1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin.

We expressed rat Nav1.6 sodium channels with or without the rat ß1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Nav1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~18 mV for tefluthrin and ~24 mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~10-14 mV in the voltage dependence of steady-state inactivation and increased in the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Nav1.6 with the ß1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the ß1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons.

Resmethrin, the first modern pyrethroid insecticide.

The discovery of resmethrin almost five decades ago was the seminal event in the development of pyrethroid insecticides as important pest management tools, the value of which endures to this day. This brief review considers the development of pyrethroids from the perspective of the discovery of resmethrin. I describe the pathway to the discovery of resmethrin and the unique properties that differentiated it from the pyrethrins and earlier synthetic pyrethroids is described. I also summarize information on metabolic fate and mechanisms of selective toxicity, first elucidated with resmethrin, that have shaped our understanding of pyrethroid toxicology since that time. Finally, I review the discovery pathway that led from resmethrin to the development of the first photostable, agriculturally useful pyrethroids that established the importance of this insecticide class.

Functional expression of Rat Nav1.6 voltage-gated sodium channels in HEK293 cells: modulation by the auxiliary ß1 subunit.

The Nav1.6 voltage-gated sodium channel α subunit isoform is abundantly expressed in the adult rat brain. To assess the functional modulation of Nav1.6 channels by the auxiliary ß1 subunit we expressed the rat Nav1.6 sodium channel α subunit by stable transformation in HEK293 cells either alone or in combination with the rat ß1 subunit and assessed the properties of the reconstituted channels by recording sodium currents using the whole-cell patch clamp technique. Coexpression with the ß1 subunit accelerated the inactivation of sodium currents and shifted the voltage dependence of channel activation and steady-state fast inactivation by approximately 5-7 mV in the direction of depolarization. By contrast the ß1 subunit had no effect on the stability of sodium currents following repeated depolarizations at high frequencies. Our results define modulatory effects of the ß1 subunit on the properties of rat Nav1.6-mediated sodium currents reconstituted in HEK293 cells that differ from effects measured previously in the Xenopus oocyte expression system. We also identify differences in the kinetic and gating properties of the rat Nav1.6 channel expressed in the absence of the ß1 subunit compared to the properties of the orthologous mouse and human channels expressed in this system.

Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels.

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.

Compound-specific effects of mutations at Val787 in DII-S6 of Nav 1.4 sodium channels on the action of sodium channel inhibitor insecticides.

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Na(v)1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A and V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Na(v)1.4/V787A, Na(v)1.4/V787C, and Na(v)1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary ß1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 µM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 µM metaflumizone was significantly reduced in Na(v)1.4/V787A channels and completely abolished in Na(v)1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depend on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding.

Differential state-dependent modification of inactivation-deficient Nav1.6 sodium channels by the pyrethroid insecticides S-bioallethrin, tefluthrin and deltamethrin.

Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Na(v)1.6+ß1+ß2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Na(v)1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Na(v)1.6Q3+ß1+ß2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Na(v)1.6Q3 following short (10ms) depolarizations, but prolonged depolarizations (up to 150ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Na(v)1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure-activity relationships.

Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances.

Synthetic pyrethroid insecticides were introduced into widespread use for the control of insect pests and disease vectors more than three decades ago. In addition to their value in controlling agricultural pests, pyrethroids are at the forefront of efforts to combat malaria and other mosquito-borne diseases and are also common ingredients of household insecticide and companion animal ectoparasite control products. The abundance and variety of pyrethroid uses contribute to the risk of exposure and adverse effects in the general population. The insecticidal actions of pyrethroids depend on their ability to bind to and disrupt voltage-gated sodium channels of insect nerves. Sodium channels are also important targets for the neurotoxic effects of pyrethroids in mammals but other targets, particularly voltage-gated calcium and chloride channels, have been implicated as alternative or secondary sites of action for a subset of pyrethroids. This review summarizes information published during the past decade on the action of pyrethroids on voltage-gated sodium channels as well as on voltage-gated calcium and chloride channels and provides a critical re-evaluation of the role of these three targets in pyrethroid neurotoxicity based on this information.

Role of the local anesthetic receptor in the state-dependent inhibition of voltage-gated sodium channels by the insecticide metaflumizone.

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Na(v)) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Na(v)1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Na(v)1.4/F1579A and Na(v)1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Na(v)1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Na(v)1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class.

Differential state-dependent modification of rat Na(v)1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin.

We expressed rat Na(v)1.6 sodium channels in combination with the rat ß1 and ß2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na(v)1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent "late" currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were ~25mV for tefluthrin and ~20mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of ~5-10mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na(v)1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by ~2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na(v)1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na(v)1.6 channels in HEK293 cells differ from the effects of these compounds on Na(v)1.6 channels in Xenopus oocytes and more closely reflect the actions of pyrethroids on channels in their native neuronal environment.

Actions of Tefluthrin on Rat Na(v)1.7 Voltage-Gated Sodium Channels Expressed in Xenopus Oocytes.

In rats expression of the Na(v)1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Na(v)1.7 sodium channel α subunit together with the rat auxiliary ß1 and ß2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 µM modified of Na(v)1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked "late current" at the end of a 40-ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to two-fold by the application of a train of up to 100 5-ms depolarizing prepulses. These effects of tefluthrin on Na(v)1.7 channels were qualitatively similar to its effects on rat Na(v)1.2, Na(v)1.3 and Na(v)1.6 channels assayed previously under identical conditions. However, Na(v)1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Na(v)1.3 and Na(v)1.6 channels. It is likely that Na(v)1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Na(v)1.3, Na(v)1.6 and Na(v)1.8) and two pyrethroid-resistant isoforms (rat Na(v)1.2 and Na(v)1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary ß1 and ß2 subunits.

The Na(v)1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na(v)1.6 exist predominantly as Na(v)1.6+ß1+ß2 heterotrimeric complexes. We assessed the independent and joint effects of the rat ß1 and ß2 subunits on the gating and kinetic properties of rat Na(v)1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The ß1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The ß2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The ß1 and ß2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na(v)1.6 sodium channels. These results identify unique effects of the ß1 and ß2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.

Coexpression with Auxiliary ß Subunits Modulates the Action of Tefluthrin on Rat Na(v)1.6 and Na(v)1.3 Sodium Channels.

We expressed the rat Na(v)1.3 and Na(v)1.6 sodium channel α subunit isoforms in Xenopus oocytes either alone or with the rat ß1 and ß2 auxiliary subunits in various combinations and assessed the sensitivity of the expressed channels to resting and use-dependent modification by the pyrethroid insecticide tefluthrin using the two-electrode voltage clamp technique. Coexpression with the ß1 and ß2 subunits, either individually or in combination, did not affecting the resting sensitivity of Na(v)1.6 channels to tefluthrin. Modification by tefluthrin of Na(v)1.6 channels in the absence of ß subunits was not altered by the application of trains of high-frequency depolarizing prepulses. By contrast, coexpression of the Na(v)1.6 channel with the ß1 subunit enhanced the extent of channel modification twofold following repeated depolarization. Coexpression of Na(v)1.6 with the ß2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Na(v)1.6 with both ß subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Na(v)1.6+ß1 channels. In contrast to Na(v)1.6, the resting modification by tefluthrin of Na(v)1.3 channels expressed in the absence of ß subunits was reduced by repeated depolarization. However, tefluthrin modification of the Na(v)1.3 α subunit expressed with both ß subunits was enhanced 1.7-fold by repeated depolarization, thereby confirming that ß subunit modulation of use-dependent effects was not confined to the Na(v)1.6 isoform. These results show that the actions of pyrethroids on mammalian sodium channels in the Xenopus oocyte expression system are determined in part by the interactions of the sodium channel α subunit with the auxiliary ß subunits that are part of the heteromultimeric sodium channel complexes found in neurons and other excitable cells.

State-Dependent Modification of Voltage-Gated Sodium Channels by Pyrethroids.

Pyrethroids disrupt nerve function by altering the rapid kinetic transitions between conducting and nonconducting states of voltage-gated sodium channels that underlie the generation of nerve action potentials. Recent studies of pyrethroid action on cloned insect and mammalian sodium channel isoforms expressed in Xenopus laevis oocytes show that in some cases pyrethroid modification is either absolutely dependent on or significantly enhanced by repeated channel activation. These use-dependent effects have been interpreted as evidence of preferential binding of at least some pyrethroids to the open, rather than resting, state of the sodium channel. This paper reviews the evidence for state-dependent modification of insect and mammalian sodium channels expressed in oocytes by pyrethroids and considers the implications of state-dependent effects for understanding the molecular mechanism of pyrethroid action and the development and testing of models of the pyrethroid receptor.

Divergent actions of the pyrethroid insecticides S-bioallethrin, tefluthrin, and deltamethrin on rat Na(v)1.6 sodium channels.

We expressed rat Na(v)1.6 sodium channels in combination with the rat beta(1) and beta(2) auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin, and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a type I structure, produced transient modification evident in the induction of rapidly decaying sodium tail currents, weak resting modification (5.7% modification at 100 microM), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a type II structure, produced sodium tail currents that were ~9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 microM), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 microM), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration-effect data showed that repetitive depolarization increased the potency of tefluthrin approximately 15-fold and that tefluthrin was approximately 10-fold more potent than deltamethrin as a use-dependent modifier of Na(v)1.6 sodium channels. Concentration-effect data from parallel experiments with the rat Na(v)1.2 sodium channel coexpressed with the rat beta(1) and beta(2) subunits in oocytes showed that the Na(v)1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Na(v)1.2 isoform. These results implicate sodium channels containing the Na(v)1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids.

Human embryonic kidney (HEK293) cells express endogenous voltage-gated sodium currents and Na v 1.7 sodium channels.

Human embryonic kidney (HEK293) cells are widely used for the heterologous expression of voltage- and ligand-gated ion channels. Patch clamp analysis of HEK293 cells in the whole-cell configuration identified voltage-gated, rapidly inactivating inward currents. Peak current amplitudes ranged from less than 100 pA to more than 800 pA, with the majority (84 of 130 cells) in the 100-400 pA range. Transient inward currents were separated into three components on the basis of sensitivity to cadmium and tetrodotoxin (TTX). Application of cadmium (300 microM) reduced current amplitude to 65% of control, consistent with the existence of current carried by a cadmium-sensitive nonspecific cation channel previously identified in HEK293 cells. Application of TTX (500 nM) reduced current amplitude by 47%, consistent with the existence of current carried by a TTX-sensitive voltage-gated sodium channel. Joint application of cadmium and TTX was additive, reducing current amplitude to 28% of control. The residual cadmium- and TTX-resistant currents represent a third pharmacologically distinct component of the rapidly inactivating inward current that was not characterized further. The pyrethroid insecticide tefluthrin (10 microM) prolonged the inactivation of transient currents and induced slowly decaying tail currents, effects that are characteristic of sodium channel modification by pyrethroids. The use of sodium channel isoform-specific primers in polymerase chain reaction amplifications on HEK293 cell first-strand cDNA detected the consistent expression of the human Na(v)1.7 sodium channel isoform in cells that expressed the TTX-sensitive component of current. These results provide evidence for an endogenous TTX-sensitive sodium current in HEK293 cells that is associated primarily with the expression of the Na(v)1.7 sodium channel isoform.

Evidence for a separate mechanism of toxicity for the Type I and the Type II pyrethroid insecticides.

Neurotoxicity and mechanistic data were collected for six alpha-cyano pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and lambda-cyhalothrin) and up to six non-cyano containing pyrethroids (bifenthrin, S-bioallethrin [or allethrin], permethrin, pyrethrins, resmethrin [or its cis-isomer, cismethrin] and tefluthrin under standard conditions. Factor analysis and multivariate dissimilarity analysis were employed to evaluate four independent data sets comprised of (1) fifty-six behavioral and physiological parameters from an acute neurotoxicity functional observatory battery (FOB), (2) eight electrophysiological parameters from voltage clamp experiments conducted on the Na(v)1.8 sodium channel expressed in Xenopus oocytes, (3) indices of efficacy, potency and binding calculated for calcium ion influx across neuronal membranes, membrane depolarization and glutamate released from rat brain synaptosomes and (4) changes in chloride channel open state probability using a patch voltage clamp technique for membranes isolated from mouse neuroblastoma cells. The pyrethroids segregated into Type I (T--syndrome-tremors) and Type II (CS syndrome--choreoathetosis with salivation) groups based on FOB data. Of the alpha-cyano pyrethroids, deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin arrayed themselves strongly in a dose-dependent manner along two factors that characterize the CS syndrome. Esfenvalerate and fenpropathrin displayed weaker response profiles compared to the non-cyano pyrethroids. Visual clustering on multidimensional scaling (MDS) maps based upon sodium ion channel and calcium influx and glutamate release dissimilarities gave similar groupings. The non-cyano containing pyrethroids were arrayed in a dose-dependent manner along two different factors that characterize the T-syndrome. Bifenthrin was an outlier when MDS maps of the non-cyano pyrethroids were based on sodium ion channel characteristics and permethrin was an outlier when the MDS maps were based on calcium influx/glutamate release potency. Four of six alpha-cyano pyrethroids (lambda-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of lambda-l-cyhalothrin reduced open channel probability whereas the S-isomers, antagonized the action of the R-isomers. None of the non-cyano pyrethroids reduced open channel probability, except bioallethrin, which gave a weak response. Overall, based upon neurotoxicity data and the effect of pyrethroids on sodium, calcium and chloride ion channels, it is proposed that bioallethrin, cismethrin, tefluthrin, bifenthrin and permethrin belong to one common mechanism group and deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.

Human and rat Nav1.3 voltage-gated sodium channels differ in inactivation properties and sensitivity to the pyrethroid insecticide tefluthrin.

Voltage-gated sodium channels are important sites for the neurotoxic actions of pyrethroid insecticides in mammals. The pore-forming alpha subunits of mammalian sodium channels are encoded by a family of 9 genes, designated Nav1.1-Nav1.9. Native sodium channels in the adult central nervous system (CNS) are heterotrimeric complexes of one of these 9 alpha subunits and two auxiliary (beta) subunits. Here we compare the functional properties and pyrethroid sensitivity of the rat and human Nav1.3 isoforms, which are abundantly expressed in the developing CNS. Coexpression of the rat Nav1.3 and human Nav1.3 alpha subunits in combination with their conspecific beta1 and beta2 subunits in Xenopus laevis oocytes gave channels with markedly different inactivation properties and sensitivities to the pyrethroid insecticide tefluthrin. Rat Nav1.3 channels inactivated more slowly than human Nav1.3 channels during a depolarizing pulse. The rat and human channels also differed in their voltage dependence of steady-state inactivation. Exposure of rat and human Nav1.3 channels to 100 microM tefluthrin in the resting state produced populations of channels that activated, inactivated and deactivated more slowly than unmodified channels. For both rat and human channels, application of trains of depolarizing prepulses enhanced the extent of tefluthrin modification approximately twofold; this result implies that tefluthrin may bind to both the resting and open states of the channel. Modification of rat Nav1.3 channels by 100 microM tefluthrin was fourfold greater than that measured in parallel assays with human Nav1.3 channels. Human Nav1.3 channels were also less sensitive to tefluthrin than rat Nav1.2 channels, which are considered to be relatively insensitive to pyrethroids. These data provide the first direct comparison of the functional and pharmacological properties of orthologous rat and human sodium channels and demonstrate that orthologous channels with a high degree of amino acid sequence conservation differ in both their functional properties and their sensitivities to pyrethroid insecticides.

Pyrethroids, knockdown resistance and sodium channels.

Knockdown resistance to DDT and the pyrethrins was first described in 1951 in the housefly (Musca domestica L.). This trait, which confers reduced neuronal sensitivity to these insecticides, was subsequently shown to confer cross-resistance to all synthetic pyrethroid insecticides that have been examined to date. As a consequence, the worldwide commercial development of pyrethroids as a major insecticide class over the past three decades has required constant awareness that pyrethroid overuse has the potential to reselect this powerful resistance mechanism in populations that previously were resistant to DDT. Demonstration of tight genetic linkage between knockdown resistance and the housefly gene encoding voltage-sensitive sodium channels spurred efforts to identify gene mutations associated with knockdown resistance and understand how these mutations confer a reduction in the sensitivity of the pyrethroid target site. This paper summarizes progress in understanding pyrethroid resistance at the molecular level, with particular emphasis on studies in the housefly.