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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 100 µM 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 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 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.
Actions on voltage-gated sodium channels are intrinsic to both the insecticidal effects of pyrethroids and their neurotoxic effects in mammals . Pyrethroids disrupt nerve function by altering the kinetics of the transitions between conducting (open) and nonconducting (closed; inactivated) states of voltage-gated sodium channels that underlie the generation of nerve action potentials . Pyrethroid modification causes persistent channel opening, which in turn causes repetitive nerve firing following single stimuli or use-dependent conduction block depending on the duration of the open time of the pyrethroid-modified channel.
Native sodium channels in mammalian tissues are heteromultimeric complexes comprised of one large (~260 kDa) α subunit and either one or two smaller (33–36 kDa) auxiliary β subunits [3,4]. Sodium channel α subunits form the ion pore and confer the fundamental functional and pharmacological properties of the channel . Sodium channel β subunits modulate channel function, regulate channel expression at the level of the plasma membrane, and contribute to cell adhesion and cell-cell communication .
Mammalian genomes contain nine genes encoding sodium channel α subunit isoforms, designated Nav1.1 – Nav1.9 [5,6], and four genes encoding sodium channel β subunits, designated β1 – β4 . Overlapping patterns of sodium channel α subunit expression in the central and peripheral neurons [7,8] limit the utility of native neuronal preparations to identify isoform-dependent differences in pharmacology. This limitation can be overcome by the heterologous expression of cloned individual sodium channel isoforms in the unfertilized oocytes of the frog Xenopus laevis. This system has been employed to assess the pyrethroid sensitivity of four rat sodium channel isoforms. Nav1.2 sodium channels exhibit very low sensitivity to modification by deltamethrin and other pyrethroids [9,10]. By contrast rat Nav1.3, Nav1.6 and Nav1.8 channels are more sensitive to pyrethroid modification [11–16].
The dorsal root ganglion (DRG), a cluster of cell bodies of afferent sensory nerves, has been widely used to study the electrical properties of mammalian peripheral neurons . Electrophysiological studies with cultured DRG neurons have identified two populations of sodium channels that are distinguished by their gating properties and their sensitivity to block by tetrodotoxin (TTX) . The TTX-resistant channel population is sensitive to pyrethroid modification, whereas the TTX-sensitive population is relatively insensitive to pyrethroids [19,20]. The TTX-resistant sodium current has been correlated with expression of the Nav1.8 isoform, but the coordinate expression of multiple TTX-sensitive isoforms within individual DRG cells  complicates the identification of the molecular basis of the TTX-sensitive current that is resistant to pyrethroids.
The Nav1.7 sodium channel isoform (formerly designated PN1) was first identified by cDNA cloning from peripheral nervous tissue [21,22] and is abundantly expressed in the dorsal root ganglion . Here we describe the action of the potent pyrethroid insecticide tefluthrin on the rat the Nav1.7 sodium channel α subunit isoform coexpressed with the rat β1 and β2 subunits in Xenopus oocytes. Our results identify Nav1.7 as a pyrethroid-resistant isoform that is likely to contribute to the TTX-sensitive, pyrethroid-resistant sodium current of DRG neurons.
The rat Nav1.7 voltage-gated sodium channel α subunit cDNA was provided by G. Mandel (State University of New York, Stony Brook, NY) and the β1 and β2 subunit cDNAs were provided by W.A. Catterall (University of Washington, Seattle, WA). Plasmid cDNAs were digested with restriction enzymes to provide linear templates for cRNA synthesis in vitro using a commercial kit (mMessage mMachine, Ambion, Austin, TX). The integrity of synthesized cRNA was determined by electrophoresis in 1% (w/v) agarose – formaldehyde gels.
Stage V–VI oocytes were removed from female X. laevis frogs (Nasco, Ft. Atkinson, WI) as described elsewhere . This procedure was performed in accordance with National Institutes of Health guidelines and followed a protocol that was approved by the Cornell University Institutional Animal Care and Use Committee. Each data set was derived from oocytes isolated from at least two different frogs. Oocytes were injected with a 1:1:1 (mass ratio) mixtures of α subunit, β1 subunit and β2 subunit cRNAs (0.5 – 5 ng/oocyte); this mixture provided a ~9-fold molar excess of β1 and β2 cRNAs to ensure the preferential expression of the desired ternary α+β complex . Injected oocytes were incubated in ND-96 medium (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES; adjusted to pH 7.6 at room temperature with NaOH) supplemented with 5% horse serum (Sigma-Aldrich), 1% streptomycin/penicillin, and 1% sodium pyruvate  at 19°C for 3–5 days until electrophysiological analysis of sodium currents.
Sodium currents were recorded from oocytes perfused with ND-96 at 21–23°C in the two-electrode voltage clamp configuration using an Axon Geneclamp 500B amplifier (Molecular Devices, Foster City, CA). Microelectrodes were pulled from borosilicate glass capillary tubes (1.0 mm O.D.; 0.5 mm I.D.; World Precision Instruments Inc., Sarasota, FL) and filled with 3 M KCl. Filled electrodes had resistances of 0.7–1.5 MΩ when immersed in ND-96 medium. Currents were filtered at 2 kHz with a low-pass 4-pole Bessel filter and digitized at 50 kHz (Digidata 1320A; Molecular Devices). To determine the voltage dependence of activation, oocytes were clamped at a membrane potential of −100 mV and currents were measured during a 40-ms depolarizing test pulse to potentials from −60 mV to 20 mV in 5-mV increments. Maximal peak transient currents were obtained upon depolarization to −10 mV. To determine the voltage dependence of steady-state inactivation, oocytes were clamped at a membrane potential of −140 mV followed by a 100-ms conditioning prepulse to potentials from −100 mV to 5 mV in 5-mV increments and then a 40-ms test pulse to −10 mV. For determinations of use dependence, oocytes were given trains of 1 to 100 5-ms conditioning prepulses to 10 mV, separated by a 10-ms interpulse interval at the holding potential, followed by a 40-ms test pulse to −10 mV. All experiments employed 10-s intervals between pulses or pulse trains to permit complete recovery from tefluthrin modification. Capacitive transients and leak currents were subtracted using the P/4 method .
Tefluthrin (98.8% purity; Syngenta, Bracknell, Berks., UK) was prepared as a stock solution in dimethyl sulfoxide (DMSO) and diluted with ND-96 immediately before use to a final concentration of 100 µM. The final DMSO concentration in the bath did not exceed 0.1%, a concentration that had no effect on sodium currents. Oocytes were perfused at 0.45 ml/min with tefluthrin in ND-96 for 6 min and then with ND-96 for 3 min prior to recording. Washout of tefluthrin during perfusion with ND-96 for up to 30 min was negligible under these conditions. All experiments with pyrethroids employed a disposable capillary perfusion system  and custom-fabricated single-use recording chambers  to prevent cross-contamination between oocytes.
Data were acquired and analyzed using pClamp 10.2 (Molecular Devices, Burlingame, CA) and Origin 8.0 (OriginLab Corp., Northampton, MA). The Boltzmann equation [y = (A1 − A2) / (1+e(x−x0)/dx) + A2] was used to fit conductance-voltage and sodium current inactivation data. Time constants for fast sodium channel inactivation were obtained by using the Chebyshev method in Origin 7.0 to fit the falling phase of the peak transient current to a double exponential decay model. Similarly, time constants for pyrethroid-induced sodium tail currents were obtained from fits of currents measured following 40-ms step depolarizations from −100 mV to −10 mV to a double exponential decay model. The conductance of the pyrethroid-induced sodium tail current, extrapolated to the moment of repolarization and normalized to the conductance of the peak current measured in the same oocyte in the absence of pyrethroid, was employed to calculate the fraction of sodium channels modified by each compound in each experiment as described previously . Statistical analyses were performed using the Prism software package (GraphPad Software, La Jolla, CA).
We expressed the Nav1.7 α subunit in combination with the β1 and β2 auxiliary subunits to give channel complexes of comparable subunit composition to those employed in our previous studies of other rat sodium channel isoforms [15,16]. Figure 1A shows a typical sodium current recorded from an oocyte expressing Nav1.7 channels. These currents activated and inactivated within 10 ms of depolarization under conditions that produced maximal peak transient currents. The voltage dependent gating of Nav1.7 channels is illustrated in Fig. 1B and summarized in Table 1.
We employed 100 µM tefluthrin, the highest concentration achievable in ND-96 perfusion medium, to maximize the extent of sodium channel modification by this pyrethroid. We assessed the modification of channels by tefluthrin in the resting state by holding the oocyte membrane at a hyperpolarized potential during perfusion and measuring the pyrethroid-modified current during the first depolarizing pulse. Figure 2 show representative currents recorded from a single oocyte expressing Nav1.7 channels before and after exposure to 100 µM tefluthrin. Tefluthrin modification was evident in the prolonged and incomplete inactivation of the peak current during a depolarizing pulse, resulting in a marked "late current" at the end of a 40-ms depolarization, and in the induction of a sodium tail current following repolarization.
The decay of peak transient currents was fitted best by a two-component, single exponential decay model that yielded first-order decay constants for the fast initial phase (τfast) and slower secondary phase (τslow) of inactivation. Figure 3 shows the effect of tefluthrin on the kinetic components of fast inactivation at test potentials near those producing maximal peak transient currents. Tefluthrin slightly accelerated τfast at all potentials (Fig. 3A) but retarded τslow (Fig. 3B).
Tefluthrin at 100 µM had no significant effect on the voltage dependence of peak sodium current activation or steady-state fast inactivation (Table 1). However, the altered voltage dependence of activation of tefluthrin-modified channels was indirectly evident in the effects of tefluthrin on the insecticide-induced late current. We determined the late current/peak current (Ilate/Ipeak) ratios in the absence or presence of tefluthrin at test potentials surrounding that producing the maximal peak current for unmodified channels (Fig. 4). In the absence of tefluthrin the Ilate/Ipeak ratio was small and did not vary greatly with test potentials in the range of −20 mV to 5 mV. However, the Ilate/Ipeak ratio measured in the presence of tefluthrin not only was much larger but also exhibited a marked voltage dependence with a strong enhancement of Ilate relative to Ipeak at more negative test potentials.
The decay of tefluthrin-induced tail currents (as in Fig. 2) was fitted best by a two-component, single exponential decay model that yielded first-order time constants for the fast initial phase and slower secondary phase of decay with values of 6.6 ± 0.5 and 37.8 ± 2.2 ms, respectively (means ± SE, n = 15). We employed the normalized conductance of the tefluthrin-induced sodium tail current to calculate the fraction of sodium channels that were modified by tefluthrin . By this criterion tefluthrin at 100 µM modified only 8.9 ± 0.7% (mean ± SE, n = 10) of Nav1.7 sodium channels in the resting state.
Modification of both insect and mammalian sodium channels by some pyrethroids is enhanced by repeated depolarization . We therefore determined the effect of repeated high-frequency depolarization on the extent of modification of Nav1.7 sodium channels by tefluthrin. In the absence of tefluthrin, repeated stimulation caused an initial decline in the amplitudes of peak transient sodium currents obtained during the first five depolarizing pulses, after which current amplitudes declined more slowly and then stabilized at ~80% of the initial amplitude (Fig. 5A). Tefluthrin had no effect on the stability of peak currents following repeated depolarizing prepulses (Fig. 5A). Fig. 5B shows the effect of 0–100 depolarizing prepulses on the extent of modification of Nav1.7 sodium channels by 100 µM tefluthrin, corrected for the use-dependent changes in control peak current stability shown in Fig. 5A. The extent of modification increased as a function of prepulse number over the first 40 prepulses and then stabilized. Maximal use-dependent modification (at 60 prepulses) was 18.0 ± 1.6% (n = 10), approximately two-fold greater than resting modification.
The Nav1.7 sodium channel α subunit is expressed together with the β1 and β2 subunits in DRG neurons [21,27], but the native subunit structure of Nav1.7-containing sodium channel complexes is not known. Expression of Nav1.7 channels in Xenopus oocytes in the absence of β subunits gives channels with abnormally slow inactivation kinetics [21,28]. The first study of Nav1.7 expression in oocytes  reported no effect of either the β1 or β2 subunit on the properties of expressed channels, but a subsequent study  found that coexpression with the β1 subunit accelerated channel inactivation and altered the gating properties of the channel. We chose to express Nav1.7 channels in combination with the β1 and β2 subunits to facilitate direct comparison of our results with the results of previous studies of rat Nav1.2, Nav1.3 and Nav1.6 channels in this system [15,16]. The kinetics of inactivation and the voltage dependence of activation of Nav1.7+β1+β2 channels in the present study agree well with the results obtained for Nav1.7+β1 channels  but the voltage dependence of steady-state fast inactivation of Nav1.7+β1+β2 channels was shifted by approximately 10 mV in the direction of depolarization compared to Nav1.7+β1 channels. This difference may be due to the inclusion of the β2 subunit in our experiments.
We employed 100 µM tefluthrin, the highest nominal concentration achievable in ND-96 perfusion medium, to maximize the extent of sodium channel modification and to facilitate comparison of the effects of tefluthrin on Nav1.7 channels with effects on other rat sodium channel isoforms under the same assay conditions [11,15,16]. Studies of pyrethroid action modification of sodium currents under voltage clamp conditions, such as those in the present study, typically employ high concentrations of insecticide to produce detectable populations of pyrethroid-modified channels. However, the concentrations of pyrethroid required to disrupt normal action potential generation in neurons are orders of magnitude lower because only a small fraction of the available population of channels must be modified in order to disrupt electrical signaling .
Tefluthrin modified Nav1.7 sodium channels by causing a slowly-inactivating late current during a depolarizing pulse and a prominent tail current following repolarization. These effects, which are the hallmarks of pyrethroid action on sodium channels in native neurons and heterologous expression systems, were also qualitatively similar to the effects of tefluthrin on other rat sodium channel isoforms in the oocyte system [11,15,16]. Modification of Nav1.7 channels by tefluthrin was enhanced by approximately two-fold by the application of trains of brief depolarizing prepulses. This result is in good agreement with previous studies of Nav1.2, Nav1.3 and Nav1.6 channels whose modification by tefluthrin was enhanced two- to four-fold by repetitive depolarization [15,16]. By contrast, repetitive depolarization did not significantly enhance the modification of Nav1.8 channels by tefluthrin . This difference may reflect an intrinsic difference in the properties of Nav1.8 channels compared to the other four isoforms, but it also may reflect the omission of β subunits in experiments with the Nav1.8 isoform.
Nav1.7 sodium channels were distinguished principally by their very low sensitivity to modification by tefluthrin. Figure 6 compares the extent of both resting and use-dependent (after 100 prepulses) modification of Nav1.7 channels by 100 µM tefluthrin to corresponding data from previous studies of the rat Nav1.2, Nav1.3, Nav1.6 and Nav1.8 channels [11,15,16]. Our results identify Nav1.7 channels as having the lowest sensitivity to pyrethroid modification of any of the five rat isoforms studied to date. In view of the widespread expression of Nav1.7 channels in DRG neurons, it is likely that they contribute significantly to the TTX-sensitive, pyrethroid-resistant current found in these cells [19,20].
Prior to the present study, Nav1.2 was the only rat isoform identified as being pyrethroid-resistant. The identification of Nav1.7 as a second pyrethroid-resistant isoform provided the opportunity to search for amino acid sequence differences between channels characterized as either pyrethroid-sensitive or pyrethroid-resistant. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1 and rat Nav1.3, Nav1.6 and Nav1.8) and the two pyrethroid-resistant isoforms (rat Nav1.2 and Nav1.7) and found only a single site in the sodium channel amino acid sequence at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This residue, located in transmembrane segment 6 of homology domain I (Fig. 7) is a conserved valine in the sensitive isoform sequences but is replaced with an isoleucine in the Nav1.2 and Nav1.7 sequences. The site of this polymorphism lies within a block of highly conserved sequence in a region of the channel that is thought to form part of the inner pore. It is also noteworthy that this residue (Val410 in Vssc1) has been identified as the site of multiple point mutations resulting in replacement by leucine, alanine, glycine or methionine in sodium channel sequences from pyrethroid-resistant insects [30–32]. Moreover, Vssc1 sodium channels containing the V410M mutation were resistant to modification by cismethrin when expressed in Xenopus oocytes . It is of interest that such a wide variety of substitutions are tolerated at this valine residue despite its highly conserved sequence context.
Whereas our results implicate this amino acid polymorphism in domain IS6 as a determinant of relative pyrethroid sensitivity among rat sodium channel isoforms it is not the only such determinant in mammalian sodium channels. Comparisons of rat and human Nav1.3 channels in the oocyte expression system  revealed that the human channel was much less sensitive to modification by tefluthrin (similar in sensitivity to the rat Nav1.7 channel described here) than the rat Nav1.3 ortholog. The rat and human Nav1.3 amino acid sequences differ at only 54 of 1951 amino acid sequence positions and share the valine residue in domain IS6 that is identified in the present study as being associated with pyrethroid sensitivity. We conclude that other determinants of differential sensitivity among mammalian sodium channel isoforms remain to be discovered.
This work was supported in part by a grant (R01-ES013686) from the National Institute of Environmental Health Sciences, National Institutes of Health. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environmental Health Sciences. We thank P. Adams and S. Kopatz for technical assistance, and we thank R. Araujo, S. McCavera and R. von Stein for critical reviews of the manuscript.
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