Pesticides play a critical role in addressing global food shortages and combating vector-borne human diseases. However, the growing problem of pesticide resistance urgently requires the discovery of new compounds that target underutilized targets. Insect transient receptor potential (TRPV) channels—Nanzhong (Nan) and inactive (Iav)—can form heterologous channels (Nan-Iav) and localize to mechanosensory organs that mediate geotropism, hearing, and proprioception in insects. Some pesticides, such as aphidopyrrolidone (AP), target Nan-Iav through unknown mechanisms. AP is effective against piercing-sucking insects (hemipterans), preventing feeding by disrupting the function of the filaments. AP can bind only to Nan, but only Nan-Iav can interact with agonists, including endogenous nicotinamide (NAM), thereby exhibiting channel activity. Despite Nan-Iav’s potential as an insecticide target, little is known about its channel assembly, regulatory binding sites, and Ca2+-dependent regulation, hindering further insecticide development. In this study, cryo-electron microscopy was used to determine the structure of Nan-Iav in Hemiptera insects in the calmodulin-ligand-free state, as well as with AP and NAM at the boundary of the ankyrin repeat cytoplasmic domain (ARD). Surprisingly, we found that the Nan protein itself can form a pentamer, which is stabilized by AP-mediated ARD interactions. This study reveals molecular interactions between insecticides and agonists and Nan-Iav, highlighting the importance of ARD in channel function and assembly, and exploring the mechanism of Ca2+ regulation.
Against the backdrop of increasingly severe global climate change, deteriorating global food security is one of the major challenges of the 21st century, with cascading consequences for society. 1,2 The World Health Organization’s State of Food Security and Nutrition in the World 2023 (SOFI) report estimates that approximately 2.33 billion people worldwide suffer from moderate to severe food insecurity, a long-standing problem. 3,4 Unfortunately, an estimated 20% to 30% or more of crop yields are lost annually to pests and pathogens, and global warming is expected to exacerbate pest resistance and crop vulnerability. 4,5,6,7,8 Pesticide development is critical not only to protect crops from pests and reduce the spread of vector-borne pathogens, but also to combat vector-borne human diseases such as dengue fever, malaria, and Chagas disease, which are increasingly resistant to pesticides. 5,9,10,11
Among the major targets of neurotoxic insecticides, the heterotetrameric TRPV channel Nanchung (Nan)-Inactive (Iav) represents a class of insecticide targets discovered only in the last decade, including commercially available insecticides such as imidacloprid and pyraclostrobin. 12,13,14 The semisynthetic insecticide aphidopyrrolifen (AP) is a recently developed and commercialized product whose main component is the active insecticide Inscalis®, which binds AP at a subnanomolar activity level. 15 AP exhibits low acute toxicity to pollinators, beneficial insects, and other non-target organisms, and when used according to label instructions, it can reduce resistance pressure to other insecticides. 16,17,18 Nan and Iav are widely distributed across insect species, are co-expressed only in chordal stretch receptor neurons of the antennae and limbs, and are critical for hearing, gravity perception, and proprioception. 13,16,19,20,21,22 AP, imidacloprid, and pyraclostrobin stimulate the Nan-Iav complex through a unique mechanism, ultimately inhibiting proprioceptive signal transduction. 13,16,23 In piercing-sucking insects (hemipterans) such as aphids and whiteflies, loss of proprioception impairs their feeding ability, ultimately leading to death. 13,24 Interestingly, AP exhibits high affinity for the Nan-Iav complex and low affinity for Nan alone. Binding of AP to Nan-Iav induces an electrical current, but binding to Nan alone does not stimulate channel activity. Iav itself does not bind to AP at all. 16 This suggests that Nan and Iav may bind to form different Nan-Iav channel complexes (e.g., with different stoichiometric ratios or different arrangements within the same stoichiometric ratio) or that AP may bind to multiple sites. Furthermore, the natural agonist nicotinamide (NAM) binds to Drosophila Nan-Iav with micromolar affinity, exhibiting effects similar to those of aphids (AP) in vitro 16,25 and inhibiting aphid reproduction and feeding, ultimately leading to their death 25,26 . These data raise many questions. For example, it remains unclear how the Nan-Iav heterodimer is formed, which binding sites are used to modulate small molecules, and how these small molecules regulate channel function by suppressing proprioception. Furthermore, the reasons why Nan itself is inactive and has low affinity for AP, while the Nan-Iav heterodimer is active and binds AP with higher affinity, remain unclear. Finally, little is known about the Ca2+-dependent regulation of Nan-Iav function and how it is integrated into neuronal signaling processes . 13,21
In this study, combining cryo-electron microscopy, electrophysiology, and radioligand binding techniques, we elucidated the assembly of Nan-Iav and the mechanism of its binding to small molecule regulators. Furthermore, we detected constitutively bound calmodulin (CaM) to Iav and AP-stabilized Nan pentamers. These results provide important insights into the regulation of calcium ions in channels, channel assembly, and the factors determining ligand binding affinity. More importantly, we confirmed that ARD plays a central role in these processes. Our study of complete insect channels bound to relevant agricultural pesticides 27, 28, 29 opens up prospects for the development of the pesticide industry, improving the efficacy and specificity of pesticides, and enabling the application of TRPV-targeted compounds to other species to address global food security and the spread of vector-borne diseases.
We also found that Nan-Iav is regulated by Ca2+, and the mechanism of regulation is mediated by constitutively bound CaM. Importantly, this Ca2+-dependent regulation of Nav by CaM differs significantly from the mechanisms of regulation of other ion channels (e.g., voltage-gated Na+ channels and TRPV5/6 channels) 52,53,54,55,56,57 . In the Nav1.2 channel, the C-terminal domain of CaM helically associates with the C-terminal domain (CTD), and Ca2+ induces binding of its N-terminal domain to the distal portion of the CTD 56 . In the TRPV5/6 channel, the C-terminal domain of CaM binds to CTH, and Ca2+ induces upward extension of its N-terminal domain into the pore, thereby blocking cation permeability 53,54 . We propose a model for the Ca2+-regulated function of Nan-Iav-CaM (Fig. 4h). In this model, the N-terminal domain of CaM constitutively binds to the C-terminal domain (CTH) of Iav. In the resting state (low [Ca2+] concentration), the C-terminal domain of CaM interacts with Nan, stabilizing the ARD conformation and thereby promoting channel opening. Binding of an agonist/insecticide to the channel induces pore opening, leading to Ca2+ influx. Ca2+ then binds to CaM, causing dissociation of the C-terminal domain from the ARD of Nan. Because blocking CaM binding essentially abolishes the inhibitory effect of Ca2+, this dissociation modulates ARD mobility, thereby causing Ca2+-dependent inhibition or desensitization. The rapid recovery of channel currents after calcium ion elution (Fig. 4g) suggests that this mechanism facilitates rapid responses to Ca2+-mediated neuronal signals. Furthermore, the C-terminal region of Iav, which remains poorly understood, has been reported to play other roles in channel targeting and current regulation.21
Finally, our study presents the high-resolution structure of an insecticide-insecticide TRP channel complex of agricultural importance—a discovery previously unknown to us. Notably, we characterized the structure and function of the insect channel in human cells (HEK293S GnTi–) rather than in insect cells. In the face of growing insecticide resistance and ongoing pressure on food security and pathogens, our work provides important information that will facilitate the development of new insecticides for the benefit of human health and global food security. Studies have shown that insecticides such as AP are effective against some pests when used according to label instructions and have low acute toxicity to beneficial pollinators, demonstrating their environmental safety. 13,16 Furthermore, testing of some AP derivatives on mosquitoes has shown that they eventually lose their ability to fly. Understanding how these modulating compounds bind to Nan-Iav will facilitate the modification of existing compounds or the development of new compounds for more effective and precise pest control. Our study demonstrates that the Nan-Iav ARD interface is critical not only for regulating the activity of endogenous compounds, pesticides, and Ca2+-CaM, but also for channel assembly. We suggest that disrupting heterodimer assembly with small molecules may be a unique and promising approach for developing ion channel inhibitors.
Of the eight orthologous genes, the full-length genes of the brown beetle (Halyomorpha halys) Nanchung and Inactive were selected, displaying excellent stability in detergents. The synthesized genes were codon-optimized for human expression and cloned into the pBacMam pCMV-DEST vector (Life Technologies) using XhoI and EcoRI restriction sites. This ensured that the clones were in frame with the C-terminal GFP-FLAG-10xHis and mCherry-FLAG-10xHis tags, which are cleaved by HRC-3C protease (PPX), allowing independent expression . The primers used to clone Nanchung and Inactive into the pBacMam vector were as follows:
Microscopic images of individual particles were obtained on a Titan Krios G2 transmission electron microscope (FEI) equipped with a K3 camera and a Gatan BioQuantum energy filter. The microscope was operated at 300 keV, with an energy setting of 20 eV, a sample pixel size of 1.08 Å/pixel (nominal magnification of 81,000x), and a defocus gradient ranging from -0.8 to -2.2 μm. Video recording was performed at 40 frames per second using a Latitude S microscope (Gatan) with a nominal dose rate of 25 e–px−1 s−1, an exposure time of 2.4 s, and a total dose of approximately 60 e–Å−2.
Beam-induced motion correction and dose weighting were performed on film using MotionCor2 in RELION 4.061. Contrast transfer function (CTF) parameter estimation was performed in cryoSPARC using the patch-based CTF estimation method62. Photomicrographs with a CTF fitting resolution ≥4 Å were excluded from subsequent analysis. Typically, a subset of 500–1000 photomicrographs was used for point selection in cryoSPARC, followed by several rounds of 2D classification after filtering to obtain a clear reference image for template-based particle selection. Particles were then extracted using 64-pixel bounding boxes and 4-fold binning. Several rounds of 2D classification were performed to remove unwanted particle categories. The initial 3D model was reconstructed using ab initio reconstruction and refined using nonuniform refinement in cryoSPARC. 3D classification was performed in cryoSPARC or RELION based on ARD heterogeneity. No significant heterogeneity of membrane domains was observed. Particles were refined using the C1 and C2 methods; particles with higher C2 resolution were considered symmetric with respect to C2 and imported into RELION for Bayesian refinement. The particles were then transferred back to cryoSPARC for final nonuniform and local refinement. The final resolution and particle counts are shown in Table 1.
When processing Nan+AP pentamers, we explored various methods for improving the resolution of membrane domains (especially the pore region), such as signal subtraction and TMD masking. However, these attempts were unsuccessful due to the potentially extreme disorder in the pore region and the overall heterogeneity of the TMD. The final resolution was calculated using a mask automatically generated by the nonuniform processing method in cryoSPARC, primarily targeting the ARD region. This achieved significantly higher resolution than that of the membrane domains (especially the VSLD region).
Initial de novo models of the apo forms of the Nanchung and Inactive bugs were first generated using Coot63, and models of the Nan and Iav bugs were generated using AlphaFold264 to identify low-confidence regions. Calmodulin modeling was based on rigid-body fits of the Ca2+-binding and Ca2+-free models in PDB accessions 4JPZ56 and 1CFD65, respectively. The models were refined using spherical refinement to ensure the correct stereochemistry and good geometry. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were then modeled as well-defined lipid densities, and the NAM and AP ligands were placed in the corresponding densities in the tight junctions. Constraint files were generated from the SMILES string of the isoforms using eLBOW in PHENIX66. Finally, the models were refined in real space in PHENIX using local grid search and global minimization with secondary structure constraints. The MolProbity server was used for model refinement and structural analysis, and illustrations were performed using PyMOL and UCSF Chimera X. 67,68,69 Aperture analysis was performed using the HOLE server,70 and sequence conservation mapping was performed using the Consurf server.71
Statistical analysis was performed using Igor Pro 6.2, Excel Office 365, and GraphPad Prism 7.0. All quantitative data are presented as mean ± standard error (SEM). Student’s t-test (two-tailed, unpaired) was used to compare two groups. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used to compare multiple groups. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant depending on the data distribution. Kd, Ki values, and their asymmetric 95% confidence intervals were calculated using GraphPad Prism 10.
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The initial model was built using the calmodulin models from the PDB 4JPZ and 1CFD databases. The coordinates have been deposited in the Protein Data Bank (PDB) under accession numbers 9NVN (Nan-Iav-CaM without ligand), 9NVO (Nan-Iav-CaM bound to nicotinamide), 9NVP (Nan-Iav-CaM bound to nicotinamide and EDTA), 9NVQ (Nan-Iav-CaM bound to aphenidolpyrrolline and calcium), 9NVR (Nan-Iav-CaM bound to aphenidolpyrrolline and EDTA), and 9NVS (Nan pentamer bound to aphenidolpyrrolline). The corresponding cryo-electron microscopy images are deposited in the Electron Microscopy Database (EMDB) under the following accession numbers: EMD-49844 (Nan-Iav-CaM without ligand), EMD-49845 (Nan-Iav-CaM complex with nicotinamide), EMD-49846 (Nan-Iav-CaM complex with nicotinamide and EDTA), EMD-49847 (Nan-Iav-CaM complex with aphidopyrrolline and calcium), EMD-49848 (Nan-Iav-CaM complex with aphidopyrrolline and EDTA), and EMD-49849 (Nan pentamer complex with aphidopyrrolline). The raw data for the functional analysis are presented in this paper.
Post time: Jan-28-2026