TRPV channel nanchung and TRPA channel water witch form insecticide-activated complexes

We have previously shown that insect vanilloid-type transient receptor potential (TRPV) channels Nanchung (Nan) and Inactive (Iav) form complexes, which can be over-stimulated and eventually silenced by commercial insecticides, afidopyropen, pymetrozine and pyrifluquinazon. Silencing of the TRPV channels by the insecticides perturbs function of the mechano-sensory organs, chordotonal organs, disrupting sound perception, gravitaxis, and feeding. In addition to TRPV channels, chordotonal organs express an ankyrin-type transient receptor po-tential (TRPA) channel, Water witch (Wtrw). Genetic data implicate Wtrw in sound and humidity sensing, although the signaling pathway, which links Wtrw to these functions has not been clearly defined. Here we show that, in heterologous system, Nan and Wtrw form calcium channels, which can be activated by afidopyropen, pymetrozine and an endogenous agonist, nicotinamide. Analogous to Nan-Iav heteromers, Nan forms the main binding interface for afidopyropen, whereas co-expression of Wtrw dramatically increases its binding affinity. Pymetrozine competes with afidopyropen for binding to Nan-Wtrw complexes, suggesting that these compounds have overlapping binding sites. Analysis of Drosophila single-nucleus transcriptomic atlas revealed co-expression of nan and wtrw in audio-and mechanosensory neurons. The observation that Nan can form insecticide-sensitive heteromers with more than one type of TRP channels, raises a possibility that Nan may partner with some other TRP channel(s). In addition, we show that Wtrw can be activated by plant-derived reactive electrophiles, allyl isothiocyanate and cinnamaldehyde, defining new molecular target for these repellents.