1.7 sodium channel α subunit is expressed together with the β1 and β2 subunits in DRG neurons [21
], but the native subunit structure of Nav
1.7-containing sodium channel complexes is not known. Expression of Nav
1.7 channels in Xenopus
oocytes in the absence of β subunits gives channels with abnormally slow inactivation kinetics [21
]. The first study of Nav
1.7 expression in oocytes [21
] reported no effect of either the β1 or β2 subunit on the properties of expressed channels, but a subsequent study [28
] found that coexpression with the β1 subunit accelerated channel inactivation and altered the gating properties of the channel. We chose to express Nav
1.7 channels in combination with the β1 and β2 subunits to facilitate direct comparison of our results with the results of previous studies of rat Nav
1.3 and Nav
1.6 channels in this system [15
]. The kinetics of inactivation and the voltage dependence of activation of Nav
1.7+β1+β2 channels in the present study agree well with the results obtained for Nav
1.7+β1 channels [28
] but the voltage dependence of steady-state fast inactivation of Nav
1.7+β1+β2 channels was shifted by approximately 10 mV in the direction of depolarization compared to Nav
1.7+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 Nav
1.7 channels with effects on other rat sodium channel isoforms under the same assay conditions [11
]. 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 [29
Tefluthrin modified Nav
1.7 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
]. Modification of Nav
1.7 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 Nav
1.3 and Nav
1.6 channels whose modification by tefluthrin was enhanced two- to four-fold by repetitive depolarization [15
]. By contrast, repetitive depolarization did not significantly enhance the modification of Nav
1.8 channels by tefluthrin [11
]. This difference may reflect an intrinsic difference in the properties of Nav
1.8 channels compared to the other four isoforms, but it also may reflect the omission of β subunits in experiments with the Nav
1.7 sodium channels were distinguished principally by their very low sensitivity to modification by tefluthrin. compares the extent of both resting and use-dependent (after 100 prepulses) modification of Nav
1.7 channels by 100 µM tefluthrin to corresponding data from previous studies of the rat Nav
1.6 and Nav
1.8 channels [11
]. Our results identify Nav
1.7 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 Nav
1.7 channels in DRG neurons, it is likely that they contribute significantly to the TTX-sensitive, pyrethroid-resistant current found in these cells [19
Fig. 6 Comparison of the resting (0 prepulses) and use-dependent (after 100 prepulses) modification of five rat sodium channel isoforms expressed in Xenopus oocytes. Data for rat Nav1.7 channels are taken from the data set shown in . Data for rat Nav1.2, (more ...)
Prior to the present study, Nav
1.2 was the only rat isoform identified as being pyrethroid-resistant. The identification of Nav
1.7 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 Nav
1.6 and Nav
1.8) and the two pyrethroid-resistant isoforms (rat Nav
1.2 and Nav
1.7) 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 () is a conserved valine in the sensitive isoform sequences but is replaced with an isoleucine in the Nav
1.2 and Nav
1.7 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
]. Moreover, Vssc1 sodium channels containing the V410M mutation were resistant to modification by cismethrin when expressed in Xenopus
]. It is of interest that such a wide variety of substitutions are tolerated at this valine residue despite its highly conserved sequence context.
Fig. 7 Identification of a sodium channel amino acid sequence polymorphism associated with differential pyrethroid sensitivity. Left: diagram of sodium channel homology domain I showing the location of a conserved block of amino acid sequence in transmembrane (more ...)
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 Nav
1.3 channels in the oocyte expression system [15
] revealed that the human channel was much less sensitive to modification by tefluthrin (similar in sensitivity to the rat Nav
1.7 channel described here) than the rat Nav
1.3 ortholog. The rat and human Nav
1.3 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.
- We expressed rat Nav1.7 voltage-gated sodium channels in Xenopus laevis oocytes.
- The effects of tefluthrin on Nav1.7 channels were enhanced by repeated depolarization.
- Nav1.7 channels were relatively insensitive to tefluthrin compared to other isoforms.
- Pyrethroid sensitivity was correlated with an amino acid polymorphism in domain IS6.
- The domain IS6 polymorphism occurs at a site of knockdown resistance mutations.