Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor

Abstract

The insecticidal effects of ω‐hexatoxin‐Hv1a, κ‐hexatoxin‐Hv1c and ω/κ‐hexatoxin‐Hv1h are currently attributed to action at calcium and potassium channels. By characterizing the binding of these toxins to neuronal membranes, we show that they have more potent effects as positive allosteric modulators (PAMs) of insect nicotinic acetylcholine receptors (nAChRs), consistent with their neuroexcitatory toxicology. Alanine scanning analysis of ω‐hexatoxin‐Hv1a reveals a structure–activity relationship for binding that mirrors that for insecticidal activity. Spinosyn A does not compete with ω‐hexatoxin‐Hv‐1a for binding, and we show that these two PAMs have distinct pharmacology of binding indicating that they act at different receptor populations. These toxins represent valuable tools for the characterization of insect nAChRs and for the development of more selective agrochemicals.

Selective actions of insecticides on desensitizing and non‐desensitizing nicotinic acetylcholine receptors in cockroach (Periplaneta americana) neurons

“…Hexatoxins produced by an Australian funnel web spider are potent modulators of insect nAChRs in patch‐clamped cockroach neurons, 25 but the subtype selectivity has not been determined. Specific binding of [ 3 H]‐ω‐hxtx‐Hv1a was enhanced by nanomolar imidacloprid concentrations, whereas specific [ 3 H]‐dihydrospinosyn A binding was enhanced by imidacloprid only at micromolar concentrations and was not affected by ω‐hxtx‐Hv1a, which, consistent with the known allosteric modulation of nAChN receptors by spinosyns, indicates that the hexatoxin binds allosterically to nAChD receptors 21 . GS‐ω/κ‐hxtx‐Hv1a, is selectively toxic to insects by contact or feeding and has been commercialized by Vestaron Corporation for control of a broad spectrum of agricultural pests.…”

“…The effect was rapidly reversible and appears to be a true antagonist effect due to binding of TFMP to the resting state of the channel, because the effect on resting channels is rapid and the inhibition decreases during the ACh pulse, indicating that ACh replaces bound TFMP to activate the channels, which could not occur if the channels were desensitized. Furthermore, while other Group 4 insecticides, which bind selectively to nAChD receptors in the desensitized state, enhanced [ 3 H]‐ω‐hxtx‐Hv1a binding to Myzus persicae membranes, TFMP and other nicotinic antagonists did not affect binding but did antagonize the ability of nicotine to enhance binding 21 …”

“…The biphasic effect of GS‐ω/k‐hxtx‐Hv1a on the nAChD current suggests the possibility of two toxin binding sites per nAChD receptor. Binding of the toxin to the first site would lead to inhibition of current and presumably promote receptor desensitization, since high affinity toxin binding is enhanced by agonists and inhibited by antagonists 21 . Binding to the second site appears to partially reverse the effect of the first ligand and allows the receptors to be activated by ACh.…”

“…The spinosyns, represented in the market by spinosad and spinetoram, have been shown to act selectively at an allosteric site on nAChN receptors 5,19,20 . Recently, radioligand binding assays have been used to show that a novel spider‐toxin‐based insecticide, SPEAR® insecticide (GS‐ω/k‐hexatoxin‐Hv1a or GS‐ω/k‐hxtx‐Hv1a), binds to a novel allosteric site on insect nAChRs, 21 which was designated by IRAC as allosteric site II, in contrast to the spinosyn site, which is now designated allosteric site I.…”

A Taxon-Specific and High-Throughput Method for Measuring Ligand Binding to Nicotinic Acetylcholine Receptors

“…α-nAChR subunits are targeted by toxins within venoms from a diverse range of animals including cone snails, scorpions, snakes, and spiders, as well as poisonous organisms such as dart frogs and cyanobacteria [4,5,6,7,8]. The targeting of α-1 nAChR by these toxins allows effective immobilisation of prey through the flaccid paralysis of voluntary muscles, leading to death by respiratory failure.…”