Identification of Amino Acid Residues in the Insect Sodium Channel Critical for Pyrethroid Binding

Abstract

The voltage-gated sodium channel is the primary target site of pyrethroids, which constitute a major class of insecticides used worldwide. Pyrethroids prolong the opening of sodium channels by inhibiting deactivation and inactivation. Despite numerous attempts to characterize pyrethroid binding to sodium channels in the past several decades, the molecular determinants of the pyrethroid binding site on the sodium channel remain elusive. Here, we show that an F-to-I substitution at 1519 (F1519I) in segment 6 of domain III (IIIS6) abolished the sensitivity of the cockroach sodium channel expressed in Xenopus laevis oocytes to all eight structurally diverse pyrethroids examined, including permethrin and deltamethrin. In contrast, substitution by tyrosine or tryptophan reduced the channel sensitivity to deltamethrin only by 3-to 10-fold, indicating that an aromatic residue at this position is critical for the interaction of pyrethroids with sodium channels. The F1519I mutation, however, did not alter the action of two other classes of sodium channel toxins, batrachotoxin (a site 2 toxin) and Lqh␣-IT (a site 3 toxin). Schild analysis using competitive interaction of pyrethroid-stereospecific isomers demonstrated that the F1519W mutation and a previously known pyrethroid-resistance mutation, L993F in IIS6, reduced the binding affinity of 1S-cispermethrin, an inactive isomer that shares the same binding site with the active isomer 1R-cis-permethrin. Our results provide the first direct proof that Leu993 and Phe1519 are part of the pyrethroid receptor site on an insect sodium channel.

Rotational Symmetry of Two Pyrethroid Receptor Sites in the Mosquito Sodium Channel

“…a References: 1 ; 2 (Du et al, 2009);3 (Du et al, 2013) and references therein; 4 (Tan et al, 2005); and 5, this study (Figs. 3B and 4).…”

“…Methods and data analysis for two-electrode voltage clamp recording of sodium currents and measurement of tail currents induced by pyrethroids were identical to those previously described (Tatebayashi and Narahashi, 1994;Tan et al, 2005). All experiments were performed at room temperature.…”

“…Procedures for preparation of oocytes and cRNA and injection were identical to those described previously (Tan et al, 2005). Methods and data analysis for two-electrode voltage clamp recording of sodium currents and measurement of tail currents induced by pyrethroids were identical to those previously described (Tatebayashi and Narahashi, 1994;Tan et al, 2005).…”

SPider Neurotoxins Targeting Voltage-Gated Sodium Channels

“…These molecular probes bind with seven identified neurotoxin binding sites, referred to as neurotoxin receptor sites 17 (Table 1 and Fig. 1B; for recent reviews see (Gordon, 1997b;1997a;Cestèle and Catterall, 2000;Tan et al, 2005). Substances are associated with a receptor site if they compete in radioligand competition binding assays, often with specific allosteric interactions with other sites, or elicit similar electrophysiological effects.…”

Batrachotoxin, Pyrethroids, and BTG 502 Share Overlapping Binding Sites on Insect Sodium Channels

“…However, recent data indicate that BTG 502, unlike BTX, acts as an antagonist, reducing the peak current of insect sodium channels expressed in Xenopus laevis oocytes (Du et al, 2011b). In addition, Phe 3i17 in IIIS6, 1 a residue important for pyrethroid activity, is also critical for the action of BTG 502 (Du et al, 2011b) but not for that of BTX (Tan et al, 2005). These results suggest that BTG 502 and BTX must have somewhat distinct binding and/or action properties that translate into distinct electrophysiological effects.…”