Molecular basis of differential sensitivity of insect sodium channels to DCJW, a bioactive metabolite of the oxadiazine insecticide indoxacarb

Indoxacarb (DPX-JW062) was recently developed as a new oxadiazine insecticide with high insecticidal activity and low mammalian toxicity. Previous studies showed that indoxacarb and its bioactive metabolite, N-decarbomethoxyllated JW062 (DCJW), block insect sodium channels in nerve preparations and isolated neurons. However, the molecular mechanism of indoxacarb/DCJW action on insect sodium channels is not well understood. In this study, we identified two cockroach Sodium Channel variants, BgNa(v)1-1 and BgNa(v)1-4, which differ in voltage dependence of fast and slow inactivation, and channel sensitivity to DCJW. The voltage dependence of fast inactivation and slow inactivation of BgNa(v)1-4 were shifted in the hyperpolarizing direction compared with those of BgNa(v)1-1 channels. At the holding potential of -90 mV, 20 microM of DCJW reduced the peak current of BgNa(v)1-4 by about 40%, but had no effect on BgNa(v)1-1. However, at the holding potential of -60 mV, DCJW also reduced the peak currents of BgNa(v)1-1 by about 50%. Furthermore, DCJW delayed the recovery from slow inactivation of both variants. Substitution of E1689 in segment 4 of domain four (IVS4) of BgNa(v)1-4 with a K, which is present in BgNa(v)1-1, was sufficient to shift the voltage dependence of fast and slow inactivation of BgNa(v)1-4 channels to the more depolarizing membrane potential close to that of BgNa(v)1-1 channels. The E1689K change also eliminated the DCJW inhibition of BgNa(v)1-4 at the hyperpolarizing holding potentials. These results show that the E1689K change is responsible for the difference in channel gating and sensitivity to DCJW between BgNa(v)1-4 and BgNa(v)1-1. Our results support the notion that DCJW preferably acts on the inactivated state of the Sodium Channel and demonstrate that K1689E is a major molecular determinant of the voltage-dependent inactivation and state-dependent action of DCJW.