Role of the local anesthetic receptor in the state-dependent inhibition of voltage-gated sodium channels by the insecticide metaflumizone

Abstract

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Na(v)) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Na(v)1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Na(v)1.4/F1579A and Na(v)1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Na(v)1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Na(v)1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class.

Fig. 1.

Structures of SCI insecticides. A, metaflumizone. B, RH-4841. C, indoxacarb and DCJW.

Fig. 2.

Effects of metaflumizone (10 μM) on the voltage dependence of activation, steady-state fast inactivation and slow inactivation. A, conductance-voltage plots of activation measured upon depolarization from −120 mV to test potentials ranging from −80 to +40 mV. Peak sodium currents were transformed to conductances (G) using the equation G = I_Na/(V_test − V_rev), where I_Na is the peak sodium current during test depolarization (V), and V_rev is the sodium current reversal potential. Data were normalized to maximum peak conductance (G_Max) and fitted using the Boltzmann equation; values are means ± S.E. from five oocytes. The asterisks indicate test potentials of submaximal activation at which the measured sodium current amplitude in the presence of metaflumizone was significantly (*, P < 0.05; **, P < 0.001; paired t test) different from the control. B, representative normalized sodium current traces recorded from an oocyte before or after metaflumizone exposure upon depolarization from −120 to −20 mV. C, voltage dependence of steady-state fast inactivation; conditioning pulses (200 ms) from −120 mV to potentials ranging from −100 to 0 mV were followed immediately by 20-ms test pulses to −10 mV. Peak sodium currents were normalized to the maximum current obtained during the inactivation protocol for that oocyte and plotted versus the conditioning potential; curves were fitted using the Boltzmann equation; values are means ± S.E. from five oocytes. D, voltage dependence of slow inactivation; amplitudes of peak transient currents measured during a 20-ms depolarization to −10 mV after a 100-s conditioning prepulse from a holding potential (V_hold) of −120 mV to potentials ranging from −90 to 0 mV and a 50-ms hyperpolarization to −120 mV were normalized to the maximum current obtained during the inactivation protocol for that oocyte and plotted as a function of the conditioning potential. Values are means ± S.E. from six (control) or five (metaflumizone) separate experiments in different oocytes; curves are fitted to the Boltzmann equation.

Fig. 3.

Inhibition of Na_v1.4 sodium currents by SCI insecticides. A to C, representative traces of sodium currents through Na_v1.4 channels recorded before and after 15 min of perfusion with 10 μM metaflumizone (A), RH-4841 (B), or DCJW (C). D, time course of sodium current inhibition caused by metaflumizone, RH-4841, and DCJW. Oocytes were held at a holding potential of −30 mV and stimulated once every minute with a test pulse (20 ms) to −10 mV that was preceded by a 2-s hyperpolarization to −120 mV. After 4 min of stable control recordings at −30 mV, insecticides were perfused into the bath for 15 min; peak sodium currents were normalized to the mean sodium current amplitude obtained during the 4 min of stable control currents before insecticide perfusion. Data were fitted using a first-order exponential decay function that yielded a single time constant (τ₁); values are means ± S.E. of four or more individual experiments in separate oocytes.

Fig. 4.

Voltage-dependent inhibition of Na_v1.4 sodium currents by SCI insecticides. Oocytes were clamped at a membrane potential of −120, −60, or −30 mV and stimulated every minute with a test pulse (20 ms) to −10 mV during 15 min of insecticide perfusion; a 2-s hyperpolarization to −120 mV preceded each test pulse in experiments at holding potentials −60 and −30 mV. Fractional inhibition was derived from the amplitude of the peak transient sodium current at the end of the perfusion period normalized to the mean sodium current amplitude in that oocyte obtained after 4 min of stable control recordings before insecticide application.

Fig. 5.

Voltage dependence and development of steady-state slow inactivation for Na_v1.4, Na_v1.4/F1579A and Na_v1.4/Y1586A sodium channels. A, the voltage dependence of slow inactivation was examined using the standard protocol described in Fig. 2C and shown in the inset. Amplitudes of peak transient currents measured during a 20-ms depolarization to −10 mV (Na_v1.4 and Na_v1.4/F1579A) or 0 mV (Na_v1.4/Y1586A) after a 100-s conditioning prepulse from a holding potential (V_hold) of −120 mV to potentials ranging from −90 to 0 mV and a 50-ms hyperpolarization to −120 mV were normalized to the maximum current obtained during the inactivation protocol for that oocyte and plotted as a function of the conditioning potential. Values are means ± S.E. from four to six experiments in separate oocytes; curves are fitted to the Boltzmann equation. B, the development of steady-state slow inactivation was assessed using a two-pulse protocol (see inset) in which oocytes were clamped at a V_hold of −30 mV (Na_v1.4), −35 mV (Na_v1.4/F1579A), or −50 mV (Na_v1.4/Y1586A) and stimulated every minute with a 20-ms test pulse to −10 mV (Na_v1.4 and Na_v1.4/F1579A) or 0 mV (Na_v1.4/Y1586A) that was preceded by a 2-s repolarization to −120 mV. Currents were normalized to peak test pulses elicited from a V_hold of −120 mV before depolarization; values are means ± S.E. from nine experiments in separate oocytes. Curves were fitted with a single-exponential decay function.

Fig. 6.

Effects of the F1579A and Y1586A mutations on the inhibition of Na_v1.4 sodium channels by SCI insecticides. A, time course of sodium current inhibition in oocytes expressing Na_v1.4/F1579A channels by metaflumizone, RH-4841, and DCJW. Oocytes were held at a holding potential of −35 mV and stimulated once every minute with a test pulse (20 ms) to −10 mV that was preceded by a 2-s hyperpolarization to −120 mV. After 4 min of stable control recordings at −35 mV, insecticides were perfused into the bath for 15 min; peak sodium currents were normalized to the mean sodium current amplitude obtained during the 4 min of stable control recordings before insecticide perfusion. Data were fitted using a first-order exponential decay function that yielded a single time constant (τ₁); values are means ± S.E. of four to five individual experiments in separate oocytes. B, time course of sodium current inhibition in oocytes expressing Na_v1.4/Y1586A channels by metaflumizone, RH-4841, and DCJW. Oocytes were held at a holding potential of −50 mV and stimulated once every minute with a 20-ms test pulse to 0 mV that was preceded by a 2-s hyperpolarization to −120 mV. After 4 min of stable control recordings at −50 mV, insecticides were perfused into the bath for 15 min; peak sodium currents were normalized to the mean sodium current amplitude during the 4 min of stable control recordings before insecticide perfusion. Data were fitted using a first-order exponential decay function that yielded a single time constant (τ₁); values are means ± S.E. of three or more individual experiments in separate oocytes.

Fig. 7.

Relative sensitivity of Na_v1.4, Na_v1.4/F1579A, and Na_v1.4/Y1586A sodium channels to inhibition by SCI insecticides. Values are fractional inhibition after 15 min of insecticide perfusion and are derived from data in Figs. 3 and 6. Values for Na_v1.4/F1579A and Na_v1.4/Y1586A channels marked with asterisks were significantly different from values for inhibition of Na_v1.4 channels by the same compound (one-way analysis of variance with Dunnett' post hoc analysis, P < 0.05).

Fig. 8.

Use-dependent lidocaine inhibition of sodium channels in the absence and presence of SCI insecticides. A, effects of 10 μM metaflumizone, RH-4841, or DCJW on the extent of inhibition by lidocaine (200 μM) after 0 to 20 depolarizing prepulses (25 ms) from −120 to −50 mV at 20 Hz. Values were normalized to the mean peak sodium current amplitude measured from the same oocyte before the perfusion of lidocaine. Data points are means ± S.E. of five (lidocaine, metaflumizone + lidocaine, RH-4841 + lidocaine) or four (DCJW + lidocaine) separate experiments with different oocytes; curves were fitted to mean data points using a single-exponential decay function. B, comparison of the extent of resting (after 0 prepulses) and fast-inactivated (after 10 prepulses) sodium channel inhibition by lidocaine in the absence or presence of metaflumizone, RH-4841, or DCJW. Values for use-dependent inhibition marked with an asterisk (*) are significantly (P < 0.05) different from use-dependent inhibition by lidocaine alone.