Project Summary Insects are the primary vectors for many deadly diseases. Mosquitoes and other biting insects rely on their exquisite sense of smell to identify and hone in on their human hosts. Consequently, one strategy to control the transmission of insect-borne diseases is to target insect olfactory receptors, disrupt the initial detection of human volatiles, and prevent insects from locating humans. The goal of developing potent insect repellents that cripple host-seeking behavior is greatly facilitated by the unique molecular nature of insect olfactory receptors. Insect olfactory receptors form a novel class of heteromeric ion channels comprised of two distinct subunits—a highly divergent odorant receptor (OR) subunit that confers odorant specificity and a common Orco subunit, that is virtually invariant amongst diverse insect species, reflecting its essential role in olfactory transduction. Given the conserved and critical role that Orco plays in odor detection, it forms an ideal molecular target for a much-needed new generation of insect repellents with the potential to halt the transmission of insect-borne diseases. Unfortunately, as these receptors represent a unique specialization of insects and lack structural homology to any other ion channel family, many of their most elementary structural and functional properties have remained elusive, precluding sufficient mechanistic understanding to guide repellent design. To fill this important void in the field, my lab recently determined the structure of an Orco homomeric channel using cryo- electron microscopy, providing the first structural snapshot of an insect olfactory receptor. Building on this advance and our expertise in odorant receptor biochemistry, we propose to elucidate the structure of Orco in conditions that replicate its native environment: embedded within a lipid membrane (Aim 1) and assembled with an OR to form a heteromeric channel (Aim 2), alone and in complex with synthetic agonists and odorants. Revealing multiple structures of insect olfactory receptors in both apo and ligand bound states will provide direct insight into the structural and mechanistic basis for their allosteric modulation. Together, the proposed studies offer a unique and powerful inroad to the rational design of small-molecule repellents that disrupt odor detection and host-seeking behavior in insect vectors of human disease.