State-Dependent Block of Rat Nav1.4 Sodium Channels Expressed in Xenopus Oocytes by Pyrazoline-Type Insecticides
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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2014, Advances in Insect PhysiologyCitation Excerpt :Initial studies on the effects of SCBIs on mammalian sodium channels expressed in Xenopus oocytes focused on the skeletal muscle sodium channel, Nav1.4, and employed the two-electrode voltage-clamp technique. Nav1.4 channels are ideal candidates for these experiments because their electrophysiology and pharmacology is well described and they produce robust currents when heterologously expressed in Xenopus oocytes (Silver and Soderlund, 2005a, 2006, 2007; von Stein and Soderlund, 2012a,b). As expected from previous studies on nerve preparations (see above), DCJW, the insect-specific metabolite of indoxacarb (Fig. 5.2), caused state-dependent inhibition of Nav1.4 sodium channels (Silver and Soderlund, 2005b).
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2013, Pesticide Biochemistry and PhysiologyCitation Excerpt :Third, prior mutagenesis experiments identified specific amino acid determinants of SCI drug binding and slow inactivation in the Nav1.4 channel sequence that we could employ directly to probe the action of SCI insecticides. Fig. 3 illustrates the characteristic effects of SCI insecticides on Nav1.4 sodium channels under voltage clamp conditions, exemplified here with data for DCJW [35]. Perfusion with 10 μM DCJW for 15 min at a holding potential of −120 mV had no effect on sodium currents, whereas perfusion at a holding potential of −30 mV caused significant inhibition of the sodium current (Fig. 3A).