Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels

Abstract

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.

Keywords: DCJW; RH3421; indoxacarb; local anesthetic receptor; metaflumizone; sodium channel.

Fig. 1

Structures of SCI insecticides.

Fig. 2

The common core structure of SCI insecticides identified by Takagi et al [4] (bold) exemplified in the structures of RH3421, indoxacarb and metaflumizone.

Fig. 3

Voltage-dependent inhibition of rat Na_v1.4 sodium channels expressed in Xenopus oocytes by DCJW. (A) Example current traces recorded before (0 min) or after perfusion for 15 min with 10 µM DCJW at holding potential of either −120 mV (top) or −30 mV (bottom). Currents were recorded during a 40-ms test pulse to −10 mV; oocytes held at −30 mV were given a 2-s repolarization pulse to −120 mV prior to the test pulse to convert fast-inactivated channels to resting channels. (B) Voltage-dependent inhibition and recovery of sodium currents in the presence of DCJW. Inset: pulse protocol employed to sample sodium currents once per minute at each holding potential. The figure is redrawn from the data of Silver and Soderlund [35].

Fig. 4

Conceptual model illustrating state transitions between closed (C), open (O), fast-inactivated (I_fast) and slow-inactivated (I_slow) sodium channels, the selective interaction of SCI insecticides (SCI) with slow-inactivated channels, and the unique interactions of metaflumizone (MF) with closed and fast-inactivated channels. Further explanation is provided in the text.

Fig. 5

Relative sensitivity of four rat sodium channel isoforms expressed in Xenopus oocytes to inhibition by indoxacarb, DCJW and RH3421. The figure is re-drawn from the data of Silver and Soderlund [37].

Fig. 6

Antagonism by metaflumizone (10 µM) of the use-dependent inhibition of rat Na_v1.4 sodium channels expressed in Xenopus oocytes by lidocaine (200 µM). Left inset: pulse protocol used. Right inset: Boltzmann plots of the voltage dependence of activation and fast inactivation of rat Na_v1.4 sodium channels; dashed line indicates the prepulse potential (−50 mV) used to induce fast inactivation without channel activation. The figure is re-drawn from the data of von Stein and Soderlund [21].

Fig. 7

Helical wheel representation of the amino acid sequences of the four S6 transmembrane domains arranged in relation to the central ion pore as proposed by Mike and Lukacs [23]. Residues shown by site-directed mutagenesis are shown in boldface; The Phe1579 residue thought to be a critical determinant of binding for all SCI drugs is boxed.

Fig. 8

Voltage dependence of slow inactivation of unmodified and specifically mutated rat Na_v1.4 sodium channels expressed in Xenopus oocytes. The holding potentials (V_h) and prepulse durations employed for each channel variant are indicated. The figure is redrawn from the data of von Stein and Soderlund [22].

Fig. 9

Inhibition of rat Na_v1.4 and Na_v1.4/V787K sodium channels expressed in Xenopus ooyctes. (A) Example current traces illustrating the approximately equivalent induction of slow inactivation of both variants. Top: traces recorded from Na_v1.4 channels at holding potentials of either −120 mV or −30 mV. Bottom: traces recorded from Na_v1.4/V787K channels at holding potentials of −140 mV or −110 mV. Currents from both channels held at depolarized potentials were recorded during 20-ms test pulses to −10 mV that was preceded by a 2-s repolarizing pulse to the holding potential (−120 or −140 mV depending on the variant examined). (B) Fractional inhibition of Na_v1.4 and Na_v1.4/V787K channels measured using the pulse protocols illustrated in Panel A 15 min after perfusion with SCI insecticides at 10 µM. The figure is re-drawn from the data of von Stein and Soderlund [22].

Fig. 10

Fractional inhibition of native and specifically mutated rat Na_v1.4 sodium channels expressed in Xenopus oocytes by SCI insecticides. The extent of inhibition was determined following perfusion with insecticide (10 µM) as described in the legend to Fig. 9. The Na_v1.4/V787A and Na_v1.4/V787C variants were assayed using the pulse protocol described in Fig. 9 for Na_v1.4 channels. Asterisks indicate inhibition of mutated channels that is significantly different from that measured for the same compound assayed against native Na_v1.4 channels. The extent of inhibition (top) is correlated with the relative hydrophobicity of amino acid residues (bottom). The amino acids at position 787 in the four channel variants employed are shown in boldface type. The figure is drawn from the data of von Stein and Soderlund [22].

Fig. 11

Helical wheel representation of the amino acid sequences of the four S6 transmembrane domains showing putative binding interactions of metaflumizone and the proposed conformational rotation of DII-S6 (arrow) in slow-inactivated channels required to expose Val787 to the channel pore.

Fig. 12

Structures of mibefradil, lacosamide and Z123212.