Differential state-dependent modification of inactivation-deficient Nav1.6 sodium channels by the pyrethroid insecticides S-bioallethrin, tefluthrin and deltamethrin

Abstract

Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Na(v)1.6+β1+β2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Na(v)1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Na(v)1.6Q3+β1+β2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Na(v)1.6Q3 following short (10ms) depolarizations, but prolonged depolarizations (up to 150ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Na(v)1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure-activity relationships.

Fig. 1

(A) Diagram of the extended transmembrane structure of a voltage-gated sodium channel α subunit protein showing the location of the IFM/QQQ mutation introduced into the Na_v1.6Q3 channel. (B) Representative sodium current trace recorded from an oocytes expressing Na_v1.6 channels during a 40-ms step depolarization from −100 mV to −10 mV. (C) Representative sodium current trace recorded from an oocytes expressing Na_v1.6Q3 channels during a 40-ms step depolarization from −100 mV to −10 mV.

Fig. 2

Voltage dependence of activation of Na_v1.6 and Na_v1.6Q3 sodium channels expressed in Xenopus oocytes. 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. Values for G/G_max were plotted as a function of test potential and curves were drawn by fitting the mean values to the Boltzmann equation. Each data point is the mean of 10 (Na_v1.6) or 14 (Na_v1.6Q3) determinations with different cells; bars show SE values larger than the data point symbols.

Fig. 3

Pyrethroid-modified currents carried by Na_v1.6Q3 channels expressed in Xenopus oocytes. (A) Representative sodium currents recorded from a single oocyte before (control) and after equilibration with 100 μM tefluthrin using the indicated pulse protocol. (B) Comparison of representative sodium tail currents recorded from different oocytes either before pyrethroid exposure (control) or after equilibration with S-bioallethrin (100 μM), tefluthrin (100 μM) or deltamethrin (10 μM). Currents were recorded for 20 ms immediately following a 40-ms step depolarization from −100 mV to −10 mV. The dashed line indicates zero current.

Fig. 4

Effects of S-bioallethrin (100 μM), tefluthrin (100 μM), and deltamethrin (10 μM) on the voltage dependence of activation of Na_v1.6Q3 sodium channels expressed in Xenopus oocytes. Normalized conductance (G/G_max) values were plotted as a function of test potential and curves were drawn by fitting the mean values to the Boltzmann equation. Each data point is the mean of 5 (S-bioallethrin), 9 (tefluthrin) or 6 (deltamethrin) determinations with different cells; bars show SE values larger than the data point symbols. The dashed line shows the curve (from Fig. 2) obtained by fitting mean control values to the Boltzmann equation.

Fig. 5

Effect of increasing depolarization time on the amplitude of control (left) and tefluthrin-induced (right) sodium tail currents. Representative traces were recorded from a single oocyte expressing Na_v1.6Q3 channels either before or after equilibration with tefluthrin (100 μM) during and after depolarizations of 5–25 ms in 5-ms increments from a holding potential of −100 mV to a test potential of −10 mV.

Fig. 6

Effect of increasing depolarization time on sodium tail currents recorded from oocytes expressing Na_v1.6Q3 sodium channels before (control) or after equilibration with tefluthrin (100 μM). Each data point is the mean of 6 determinations with different cells; bars show SE values larger than the data point symbols.

Fig. 7

Effect of increasing depolarization time on the pyrethroid-specific component of sodium tail currents recorded from oocytes expressing Na_v1.6Q3 sodium channels. (A) Normalized net tail currents measured following equilibration with S-bioallethrin (100 μM), tefluthrin (100 μM) or deltamethrin (10 μM) and step depolarizations of 10–150 ms in 10-ms increments. (B) Normalized net tail currents measured following equilibration with S-bioallethrin (100 μM) and step depolarizations of 3–18 ms in 3-ms increments. Values are means of 6 determinations with different cells and are corrected for control tail currents measured prior to pyrethroid exposure in the same oocyte; bars show SE values larger than the data point symbols.