Decarbomethoxylated JW062 (DCJW), the active component of the oxadiazine insecticide (S)-methyl 7-chloro-2,5-dihydro-2-[[(methoxycarbonyl)[4-(trifluoromethoxy)phenyl] amino]carbonyl] indeno[1,2-e][1,3,4]oxadiazine-4a(3H)-carboxylate (DPX-JW062)(indoxacarb), was tested on 2 inward voltage-dependent sodium currents (named INa1 and INa2) expressed in shortterm cultured dorsal unpaired median neurons of the cockroach Periplaneta americana. Under whole-cell voltageclamp conditions, application of DCJW resulted in a biphasic dose-dependent inhibition of the global sodium current amplitude illustrating the differing sensitivity of sodium channels to DCJW. INa2 was less sensitive to DCJW [half-maximal inhibitory concentration (IC 50 ) ϭ 1.6 M] compared with INa1 (IC 50 ϭ 1.7 nM). Although a previous study demonstrated that INa1 was regulated by the cAMP/protein kinase A cascade, we showed that INa2 was mainly regulated in an opposite way by the activation of calcium-calmodulin-dependent protein phosphatase 2B (PP2B) and calcium-calmodulin-dependent protein kinase II (CaM-kinase II). Furthermore, we demonstrated that activation of CaM-kinase II by intracellular calcium via the calcium-calmodulin complex affected the sensitivity of INa2 channels to DCJW. By increasing the intracellular calcium concentration and/or using 1,2-bis(oaminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) (a calcium chelator), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) (a calmodulin inhibitor), cyclosporine A (a PP2B inhibitor), and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) (a CaM-kinase II inhibitor), we revealed that activation of CaMkinase II was involved in the modulation of the voltage dependence of steady-state inactivation and that the CaMkinase II pathway activated by elevation of the intracellular calcium concentration might render INa2 channels approximately 3000-fold more sensitive to DCJW. These results indicated that manipulating specific intracellular signaling pathways involved in the regulation of sodium channels might have fundamental consequences for the sensitivity of insects to insecticides. This finding reveals an exciting research area that could lead to improvement in the efficiency of insecticides.Voltage-dependent sodium channels are critical elements of electrical activity in excitable cells and thus are important targets of diverse neuroactive compounds, including pyrethroids and pyrazoline-type insecticides (Dong, 2007;Soderlund, 2008;Silver et al., 2009). Synthetic pyrethroid insecticides, which are derived from natural compounds (pyrethrins), have been used for more than 40 years and account for 25% of the worldwide insecticide market, because of their potent and rapid insect intoxication, low toxicity for mammals, and lim-B.M. was supported by a doctoral fellowship from the Région Pays de la Loire.Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
Insect voltage-gated sodium (Nav) channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation “temperature-induced-paralysis locus E.” The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1) strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3′UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1) co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280) in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280). PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be modulated by an intron retention process in the transcription of the neuronal TEH1-like ancillary subunits of P. americana.
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