Assembly and Conformational Dynamics of Insect Odorant Receptors

Insects rely on odorant receptors (ORs), tetrameric ligand-gated ion channels, to detect and discriminate a wide range of chemical cues. Within the insect OR family, composed of hundreds of OR subtypes, a conserved subunit termed odorant receptor co-receptor (Orco) is present in insect olfactory neurons. While Orco and some ORs can assemble into homo-tetramers, odor-activated channels are hetero-tetramers comprising Orco and an odor-specific OR. Cryogenic electron microscopy (cryo-EM) structures from heterologous expression systems revealed that Orco and OR assembled with a 3:1 (Orco:OR) stoichiometry. In these complexes, the OR subunit is solely responsible for ligand-recognition, whereas the Orco subunits form the majority of the ion conduction pathway and are key for assembly and membrane trafficking. Because Orco and OR can assemble in diverse stoichiometries, of which Orco(4) and OR(4) homo-tetramers, as well as Orco(3)/OR(1) hetero-tetramers, have been experimentally documented, we sought to investigate assembly and conformational dynamics of insect Orco/OR complexes. Here, using high-speed atomic force microscopy (HS-AFM), unique in providing simultaneous structural and temporal resolution, we find that Orco/OR complexes can reversibly transition into stable non-tetrameric oligomeric states, such as trimers and pentamers, through membrane-diffusive oligomer exchange. Furthermore, while all cryo-EM structures showed a fourfold symmetric or pseudo-symmetric conformation, we observed that tetrameric assemblies frequently adopt a symmetry-broken 'pinched' configuration, in addition to the canonical symmetric 'square' arrangement. Notably, ligand-binding modulates the 'pinching' kinetics in Orco/OR hetero-tetramers. Overall, these findings reveal key aspects of assembly and conformational dynamics of the insect OR family.