Effects of the β1 auxiliary subunit on modification of Rat Na(v)1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin

Abstract

We expressed rat Nav1.6 sodium channels with or without the rat β1 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 β1 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 β1 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.

Keywords: Functional reconstitution; HEK293 cells; Na(v)1.6 isoform; Pyrethroid insecticide; Voltage-gated sodium channel; β Subunits.

Fig. 1

Structures and isomeric compositions of deltamethrin and tefluthrin.

Fig. 2

Time course of modification of Na_a1.6 channels during perfusion with 1 μM tefluthrin. (A) Currents recorded from a representative cell at 3-min intervals (0–21 min) during perfusion with 1 μM tefluthrin showing the time-dependent increase in the tefluthrin-induced late and tail currents. (B) Time course of modification of Na_v1.6 channels by 10 μM tefluthrin from multiple experiments such as that shown in panel A. Peak, late and tail current amplitudes for each cell were normalized to the amplitude of the corresponding current recorded after 23 minutes. Each data point is the mean of 9 experiments with different cells; bars show SE values larger than the data point symbols.

Fig. 3

Representative control and pyrethroid-modified current traces recorded during a 40-ms step depolarization from −120 mV to −15 mV from HEK-Na_v1.6 (A) and HEK-Na_v1.6β1 (B) cells.

Fig. 4

Concentration-dependent modification of the voltage dependence of activation of sodium channels in HEK-Na_v1.6 (A, B) and HEK-Na_v1.6β1 (C, D) cells by tefluthrin (A, C) and deltamethrin (B, D). Values for the conductance of peak sodium current were plotted as a function of test potential and curves were drawn by fitting mean values to the Boltzmann equation. Values are the means of the indicated number of determinations with different cells; bars show SE values larger than the data point symbols. Dashed lines show the curve obtained by fitting mean control values to the Boltzmann equation (He and Soderlund, 2014).

Fig. 5

Effect of coexpression with β subunits on the magnitude of the shift in V_0.5 values for Nav1.6 sodium channel activation caused by tefluthrin (100 μM) or deltamethrin (10 μM). Values for HEK-Na_v1.6 and HEK-Na_v1.6β1 cells were calculated by subtracting the mean control V_0.5 values from the mean V_0.5 values measured in the presence of insecticide; bars show SE values as in Tables 1 and 2. Comparable values for HEK-Na_v1.6β1β2 cells were calculated from previously-published data (He and Soderlund, 2011) and are provided here for comparison.

Fig. 6

Voltage dependence of the control peak current and the peak and late currents induced by 100 μM tefluthrin in assays with HEK-Na_v1.6 cells. (A) Normalized current – voltage plots for control peak sodium currents (He and Soderlund, 2014) and for the peak currents (as in Fig. 3A), and late currents (measured at the end of a 40-ms depolarizing pulse) following exposure to tefluthrin. Values are the means of 64 (control) or 11 (+tefluthrin) determinations with different cells; bars show SE values larger than the data point symbols. (B) Plots of the conductance transformations of data in Fig. 6A; curves were drawn by fitting mean values to the Boltzmann equation.

Fig. 7

Concentration-dependent modification of the voltage dependence of steady-state inactivation of sodium channels in HEK-Na_v1.6 (A, B) and HEK-Na_v1.6β1 (C, D) cells by tefluthrin (A, C) and deltamethrin (B, D). Normalized amplitudes of peak sodium currents were plotted as a function of test potential and curves were drawn by fitting mean values to the Boltzmann equation. Data points in the shaded regions were omitted from the fits of data obtained in the presence of pyrethroids (see text for details). Values are the means of the indicated number of determinations with different cells; bars show SE values larger than the data point symbols. Dashed lines show the curve obtained by fitting mean control values to the Boltzmann equation (He and Soderlund, 2014).

Fig. 8

Effect of coexpression with β subunits on the magnitude of the shift in V_0.5 values for Nav1.6 sodium channel inactivation caused by tefluthrin (100 μM) or deltamethrin (10 μM). Values for HEK-Na_v1.6 and HEK-Na_v1.6β1 cells were calculated by subtracting the mean control V_0.5 values from the mean V_0.5 values measured in the presence of insecticide; bars show SE values as in Tables 1 and 2. Comparable values for HEK-Na_v1.6β1β2 cells were calculated from previously-published data (He and Soderlund, 2011) and are provided here for comparison.

Fig. 9

Effect of coexpression with the β1 subunit on tefluthrin-induced, inactivation-resistant currents carried by Na_v1.6 sodium channels. Values are means ±SE of normalized fractional current (I/I_max) measured following conditioning depolarizations to 0 mV in either HEK-Na_v1.6 or HEK-Na_v1.6β1 cells following exposure to four concentrations of tefluthrin (see also Figs. 7 and 8).

Fig. 10

Effects of tefluthrin (A, B) or deltamethrin (C, D) on sodium window currents in HEK-Na_v1.6 (A, C) and HEK-Na_v1.6β1 (B, D) cells. Each panel shows voltage dependence plots for activation and steady-state inactivation in the presence of 10 μM tefluthrin or 10 μM deltamethrin in taken from data in Figs. 4 and 7. Dashed lines show the curve obtained by fitting mean control values for Na_v1.6 and HEK-Na_v1.6β1 cells to the Boltzmann equation (He and Soderlund, 2014).

Fig. 11

Effect of coexpression with the β1 subunit on the resting (0 prepulses) and use-dependent (100 prepulses) modification of Na_v1.6 sodium channels by 10 μM deltamethrin. Values are means ± SE of 7 determinations. Comparable values for HEK-Na_v1.6β1β2 cells from previously-published data (He and Soderlund, 2011) are provided here for comparison.