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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+β1+β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 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 β1 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 β2 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 β1 and β2 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 β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (2010 Elsevier Inc. All rights reserved.)

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.

Fluvalinate is a pyrethroid insecticide that is widely used in the control of the varroa mite (Varroa destructor), an ecto-parasite of the honeybee. Previously we identified four fluvalinate-resistance-associated mutations in the sodium channel gene of the varroa mite. One of the mutations caused a leucine (L) to proline (P) change at 1770 in the linker connecting domains III and IV of the sodium channel. Interestingly, at the position corresponding to the L to P mutation, all known insect (including honeybee) sodium channel proteins already naturally contain a P residue (e.g., P1577 in the cockroach sodium channel BgNa(v)). To determine whether insect sodium channels are less sensitive to fluvalinate than arachnid sodium channels, we replaced P1577 with an L in a BgNa(v) variant (BgNa(v)1-1) and examined the sensitivity of the recombinant channel to fluvalinate. The P1577L substitution did not alter the gating properties of the BgNa(v)1-1 channel expressed in Xenopus oocytes. However, the BgNa(v)1-1(P1577L) channel was five-fold more sensitive to fluvalinate compared with the BgNa(v)1-1 channel. These results not only implicate the L to P mutation in fluvalinate resistance in varroa mites, but also suggest a possible contribution of L1770 to the higher sensitivity of varroa mites to fluvalinate than their insect hosts.

The voltage-gated sodium channel is the primary target site of pyrethroids, which constitute a major class of insecticides used worldwide. Pyrethroids prolong the opening of sodium channels by inhibiting deactivation and inactivation. Despite numerous attempts to characterize pyrethroid binding to sodium channels in the past several decades, the molecular determinants of the pyrethroid binding site on the sodium channel remain elusive. Here, we show that an F-to-I substitution at 1519 (F1519I) in segment 6 of domain III (IIIS6) abolished the sensitivity of the cockroach sodium channel expressed in Xenopus laevis oocytes to all eight structurally diverse pyrethroids examined, including permethrin and deltamethrin. In contrast, substitution by tyrosine or tryptophan reduced the channel sensitivity to deltamethrin only by 3- to 10-fold, indicating that an aromatic residue at this position is critical for the interaction of pyrethroids with sodium channels. The F1519I mutation, however, did not alter the action of two other classes of sodium channel toxins, batrachotoxin (a site 2 toxin) and Lqhalpha-IT (a site 3 toxin). Schild analysis using competitive interaction of pyrethroid-stereospecific isomers demonstrated that the F1519W mutation and a previously known pyrethroid-resistance mutation, L993F in IIS6, reduced the binding affinity of 1S-cis-permethrin, an inactive isomer that shares the same binding site with the active isomer 1R-cis-permethrin. Our results provide the first direct proof that Leu993 and Phe1519 are part of the pyrethroid receptor site on an insect sodium channel.

Sodium channels play an essential role in generating the action potential in eukaryotic cells, and their transcripts, especially those in insects, undergo extensive A-to-I RNA editing. The functional consequences of RNA editing of sodium channel transcripts, however, have yet to be determined. We characterized 20 splice variants of the German cockroach sodium channel gene BgNa(v). Functional analysis revealed that these variants exhibited a broad range of voltage-dependent activation and inactivation. Further analysis of two variants, BgNa(v)1-1 and BgNa(v)1-2, which activate at more depolarizing membrane potentials than other variants, showed that RNA editing events were responsible for variant-specific gating properties. Two U-to-C editing sites identified in BgNa(v)1-1 resulted in a Leu to Pro change in segment 1 of domain III (IIIS1) and a Val to Ala change in IVS4. The Leu to Pro change shifted both the voltage dependence of activation and steady-state inactivation in the depolarizing direction. Two A-to-I editing events in BgNa(v)1-2 resulted in a Lys to Arg change in IS2 and an Ile to Met change in IVS3. The Lys to Arg change shifted the voltage dependence of activation in the depolarizing direction. Moreover, these RNA editing events occurred in a tissue-specific and development-specific manner. Our findings provide direct evidence that RNA editing is an important mechanism generating tissue-/cell type-specific functional variants of sodium channels.

Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels.

Pyrethroid insecticide resistance due to reduced nerve sensitivity, known as knockdown resistance (kdr or kdr-type), is linked to multiple point mutations in the para-homologous sodium channel genes. Previously we demonstrated that two mutations (E434K and C764R) in the German cockroach sodium channel greatly enhanced the ability of the L993F mutation (a known kdr -type mutation) to reduce sodium channel sensitivity to deltamethrin, a pyrethroid insecticide. Neither E434K nor C764R alone, however, altered sodium channel sensitivity. To examine whether E434K and C764R also enhance the effect of pyrethroid resistance-associated sodium channel mutations identified in other insects, we introduced a V to M mutation (V409M) into the cockroach sodium channel protein at the position that corresponds to the V421M mutation in the Heliothis virescens sodium channel protein. We found that the V409M mutation alone modified the gating properties of the sodium channel and reduced channel sensitivity to deltamethrin by 10-fold. Combining the V409M mutation with either the E434K or C764K alone did not reduce the V409M channel sensitivity to deltamethrin further. However, the triple mutation combination (V409M, E434K and C764R) dramatically reduced channel sensitivity by 100-fold compared with the wild-type channel. These results suggest that the E434K and C764R mutations are important modifiers of sodium channel sensitivity to pyrethroid insecticides.

Abstract: We expressed the rat Nav1.3 and Nav1.6 sodium channel α subunit isoforms in Xenopus oocytes either alone or with the rat β1 and β2 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 β1 and β2 subunits, either individually or in combination, did not affecting the resting sensitivity of Nav1.6 channels to tefluthrin. Modification by tefluthrin of Nav1.6 channels in the absence of β subunits was not altered by the application of trains of high-frequency depolarizing prepulses. By contrast, coexpression of the Nav1.6 channel with the β1 subunit enhanced the extent of channel modification 2-fold following repeated depolarization. Coexpression of Nav1.6 with the β2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Nav1.6 with both β subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Nav1.6+β1 channels. In contrast to Nav1.6, the resting modification by tefluthrin of Nav1.3 channels expressed in the absence of β subunits was reduced by repeated depolarization. However, tefluthrin modification of the Nav1.3 α subunit expressed with both β subunits was enhanced 1.7-fold by repeated depolarization, thereby confirming that β subunit modulation of use-dependent effects was not confined to the Nav1.6 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 β subunits that are part of the heteromultimeric sodium channel complexes found in neurons and other excitable cells. [Copyright &y& Elsevier]

Abstract: In rats expression of the Nav1.7 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 Nav1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 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 100μM modified of Nav1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked “late current” at the end of a 40ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to twofold by the application of a train of up to 100 5ms depolarizing prepulses. These effects of tefluthrin on Nav1.7 channels were qualitatively similar to its effects on rat Nav1.2, Nav1.3 and Nav1.6 channels assayed previously under identical conditions. However, Nav1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Nav1.3 and Nav1.6 channels. It is likely that Nav1.7 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 Nav1.3, Nav1.6 and Nav1.8) and two pyrethroid-resistant isoforms (rat Nav1.2 and Nav1.7) 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. [Copyright &y& Elsevier]

Abstract: We expressed rat Nav1.6 sodium channels in combination with the rat β1 and β2 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 100μM), 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 100μM), 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 100μM), 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 ~15-fold and that tefluthrin was ~10-fold more potent than deltamethrin as a use-dependent modifier of Nav1.6 sodium channels. Concentration–effect data from parallel experiments with the rat Nav1.2 sodium channel coexpressed with the rat β1 and β2 subunits in oocytes showed that the Nav1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Nav1.2 isoform. These results implicate sodium channels containing the Nav1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids. [Copyright &y& Elsevier]

Abstract: Voltage-gated sodium channels are important sites for the neurotoxic actions of pyrethroid insecticides in mammals. The pore-forming α 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 α subunits and two auxiliary (β) 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 α subunits in combination with their conspecific β1 and β2 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 100μM 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 100μM 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. [Copyright &y& Elsevier]