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

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.