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

Abstract

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 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 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.

Fig. 1

Properties of Na_v1.7 sodium channels expressed in Xenopus oocytes. (A) Representative current trace recorded during a 40-ms step depolarization from −100 mV to −10 mV. (B) Voltage dependence of activation and steady-state inactivation. For activation, normalized conductance (G/G_max) was derived from the current-voltage relationship obtained using the indicated pulse protocol by dividing peak transient current (I_Na) by the driving force (V − V_rev) and normalizing to the maximum conductance observed in each cell. For inactivation, peak transient currents were measured using the indicated pulse protocol and normalized to the maximal peak transient current in each experiment (I/I_max). Values for G/G_max and I/I_max were plotted as a function of test (activation) or prepulse (inactivation) potential and curves were drawn by fitting mean values to the Boltzmann equation. Each data point is the mean of 17 (activation) or 15 (steady-state inactivation) determinations with different cells; bars show SE values larger than the data point symbols.

Fig. 2

Representative control and tefluthrin (100 µM)-modified sodium currents recorded from an oocyte expressing rat Na_v1.7 sodium channels using the indicated depolarization protocol.

Fig. 3

Effects of tefluthrin on the fast inactivation kinetics of Na_v1.7 sodium channels. (A) Effects of tefluthrin (100 µM) on the first-order time constants for the fast component of inactivation (τ_fast) of Na_v1.7 sodium channels obtained from currents recorded during 40-ms depolarizations from −100 mV to the indicated test potential (V_t); values are means ± SE of the indicated number of separate experiments with different oocytes. Each data point is the mean of 17 (control) or 7 (+tefluthrin) determinations with different cells; bars show SE values larger than the data point symbols. (B) Effects of tefluthrin (100 µM) on the first-order time constants for the slow component of inactivation (τ_slow) of Na_v1.7 sodium channels obtained from currents recorded during 40-ms depolarizations from −100 mV to the indicated test potential (V_t); values are means ± SE of the indicated number of separate experiments with different oocytes. Each data point is the mean of 17 (control) or 7 (+tefluthrin) determinations with different cells; bars show SE values larger than the data point symbols.

Fig. 4

Voltage dependence of late currents measured in the absence or presence of tefluthrin (100 µM). Peak transient currents (I_peak) and late currents (I_late ; measured at the end of a 40-ms step depolarization) were recorded during step depolarizations from −100 mV to the indicated test potential. Each data point is the mean of 17 (control) or 7 (+tefluthrin) determinations with different cells; bars show SE values larger than the data point symbols.

Fig. 5

Effect of repeated depolarization on sodium current stability and modification by tefluthrin of Na_v1.7 sodium channels. (A) Effect of repeated depolarizing pulses on the normalized amplitude of peak transient sodium currents measured in the absence or presence of tefluthrin (100 µM); values were normalized to the amplitude of the peak current from the same oocyte prior to repetitive depolarization and are expressed as means of 14 paired experiments with different oocytes; in all cases SE values were smaller than the data point symbols. (B) Effect of repeated depolarizing pulses on the extent of tefluthrin modification of Na_v1.7 channels; values for fractional modification were calculated from the normalized conductances of sodium tail currents and are expressed as means of 6 separate experiments with different oocytes; bars show SE values larger than the data point symbols.

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 Na_v1.7 channels are taken from the data set shown in Fig 5. Data for rat Na_v1.2, rat Na_v1.3, Na_v1.6 and Na_v1.8 channels are from previous studies in this laboratory [11,15,16]. Asterisks indicate significant use-dependent enhancement of channel modification (paired t-tests, P < 0.05).

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 segment 6. Right: Aligned amino acid sequences in transmembrane segment 6 from four pyrethroid-sensitive channels (house fly Vssc1, rat Na_v1.3, rat Na_v1.6 and rat Na_v1.8) and two pyrethroid-resistant channels (rat Na_v1.2 and rat Na_v1.7). Residues enclosed in the solid line are identical in all six sequences. Polymorphic residues aligning with V410 of Vssc1 are shown in boldface type; substitutions at the valine residue aligning with V410 of Vssc1 that are associated with pyrethroid resistance in insects are shown above the aligned sequences.