Elsevier

NeuroToxicology

Volume 26, Issue 3, June 2005, Pages 397-406
NeuroToxicology

State-Dependent Block of Rat Nav1.4 Sodium Channels Expressed in Xenopus Oocytes by Pyrazoline-Type Insecticides

https://doi.org/10.1016/j.neuro.2005.03.001Get rights and content

Abstract

Insecticidal pyrazolines inhibit voltage-sensitive sodium channels of both insect and mammalian neurons in a voltage-dependent manner. Studies on the effects of pyrazoline insecticides on mammalian sodium channels have been limited to experimentation on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channel populations of rat dorsal root ganglion (DRG) neurons. In this study, we examined the effects of the insecticidal pyrazolines indoxacarb, the N-decarbomethoxyllated metabolite of indoxacarb (DCJW), and RH 3421 on rat Nav1.4 sodium channels expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique. Both DCJW and RH 3421 were ineffective inhibitors of rat Nav1.4 sodium channels at a membrane potential of −120 mV, but depolarization to −60 mV or −30 mV during insecticide exposure resulted in substantial block. Inhibition by pyrazoline insecticides was nearly irreversible with washout, but repolarization of the membrane relieved block. DCJW and RH 3421 also caused hyperpolarizing shifts in the voltage dependence of slow inactivation without affecting the voltage dependence of activation or fast inactivation. These results suggest that DCJW and RH 3421 interact specifically with the slow inactivated state of the sodium channel. Indoxacarb did not cause block at any potential, yet it interfered with the ability of DCJW, but not RH 3421, to inhibit sodium current. Phenytoin, an anticonvulsant, reduced the efficacy of both DCJW and RH 3421. These data imply that the binding site for pyrazoline insecticides overlaps with that for therapeutic sodium channel blockers.

Section snippets

INTRODUCTION

Pyrazoline-type insecticides were identified in the 1970s as potent neurotoxic compounds causing cessation of feeding, abnormal movement, and paralysis in arthropods (Harder et al., 1996, Mulder et al., 1975, Salgado, 1990). Indoxacarb, the only commercially used compound derived from the pyrazolines, and RH 3421 are examples of pyrazoline-type insecticides (Fig. 1A and B). In insects, indoxacarb is metabolically activated by one or more hydrolases to its N-decarbomethoxyllated metabolite

MATERIALS AND METHODS

Oocytes were removed surgically from female Xenopus laevis frogs (Nasco, Ft. Atkinson, WI) anesthetized in a 0.2% tricaine (Sigma, St. Louis, MO) solution. Oocytes were digested with collagenase (type 1A, Sigma) to remove the follicle. Digestion was allowed to proceed until the oocytes were about 50% dissociated, and then aspiration through a small-bore glass pipette removed any remaining tissue. Stages IV–VI oocytes were selected and maintained at 19 °C for 2–15 h in ND-96 medium (in mM; 96

RESULTS

Perfusion with indoxacarb, DCJW or RH 3421 at 10 μM at a holding potential of −120 mV did not affect peak sodium current amplitude (Fig. 2A). Application of trains of 40-ms test pulses to −10 mV from a holding potential of −120 mV at frequencies of 1 Hz, 2 Hz or 3 Hz to oocytes treated with indoxacarb, DCJW, or RH 3421 also did not produce use-dependent block of rat Nav1.4 sodium channels (data not shown).

Prolonged depolarization of oocytes to −60 mV or −30 mV during perfusion resulted in significant

DISCUSSION

This is the first report of the action of pyrazoline-type insecticides on a single voltage-sensitive sodium channel isoform in the Xenopus oocyte expression system. We chose Nav1.4 sodium channels for these studies because they produce robust sodium currents in this system. These large currents facilitated the measurement of residual currents following more than 80% block by pyrazoline-type insecticides. The large amount of information on the effects of therapeutic sodium channel blockers on Nav

ACKNOWLEDGEMENTS

We thank G. Carlson (Rohm and Haas Company) and K. Wing (DuPont Agricultural Products) for providing us with RH 3421, and its resolved isomers, and indoxacarb and DCJW, and R.G. Kallen (University of Pennsylvania) for providing us with the rat Nav1.4 sodium channel clone. We also thank Jin Sung Choi for his assistance with electrophysiology and data analysis methods.

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