Compound-specific effects of mutations at Val787 in DII-S6 of Nav1.4 sodium channels on the action of sodium channel inhibitor insecticides

Abstract

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding.

Molecular Mechanism of Action and Selectivity of Sodium Ch annel Blocker Insecticides

“…A valine at position 787 (V 2i18 ) is critical for determining the voltage dependence of slow inactivation, and mutation V 2i18 K shifts the voltage dependence of slow inactivation in the hyperpolarizing direction, whereas mutations V 2i18 C or V 2i18 A shift it in the depolarizing direction, resulting in incomplete slow inactivation [56, 84]. Previous publications suggest that hyperpolarizing shifts in the voltage dependence of slow inactivation enhance sensitivity to SCBIs as was observed in cockroach sodium channels at a different residue [59], whereas depolarizing shifts should decrease channel sensitivity.…”

“…Previous publications suggest that hyperpolarizing shifts in the voltage dependence of slow inactivation enhance sensitivity to SCBIs as was observed in cockroach sodium channels at a different residue [59], whereas depolarizing shifts should decrease channel sensitivity. The results from these experiments, however, showed varying effects of these three mutations on the ability of indoxacarb or DCJW to cause inhibition of Na + current with no correlation to their effects on the voltage dependence of slow inactivation, indicating that V 2i18 does not interact with indoxacarb or DCJW [56]. In contrast, all three mutations reduced the sensitivity of Na v 1.4 channels to inhibition by metaflumizone, implying that V 2i18 is involved in the binding of metaflumizone to VGSCs.…”

“…However, specific SCBIs clearly interact with residues that are not involved in the binding of other SCBI compounds or LAs ( e.g. V 2i18 A affects channel sensitivity to metaflumizone, but not indoxacarb or DCJW [56]). As such, we propose that the SCBI receptor includes residues in IVS6, which are critical to the action of LAs.…”

Voltage-Gated Sodium Channels as Insecticide Targets

“…Na v 1.4 channels are ideal candidates for these experiments because their electro-physiology 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). …”

“…However, some evidence from mutagenesis experiments, which substituted different amino acids at this site, indicate that this residue may not participate directly in the binding of certain LAs or merely affects their binding allosterically (Li et al, 1999; Mike and Lukacs, 2010). The few studies that have attempted to localize the SCBI receptor site on voltage-gated sodium channels have focused on these two residues as likely participants in SCBI binding (Silver and Soderlund, 2007; von Stein and Soderlund, 2012a,b). …”