Characterization of calmodulin's regulatory role in insect odorant receptor trafficking and olfactory response

Abstract

Like most animals, insects rely on olfaction to find food and mates and avoid noxious chemicals and predators. Most insect olfactory sensory neurons (OSNs) express an odorant-specific odorant receptor (OR) along with Orco, the olfactory co-receptor. Orco binds ORs and permits their trafficking to the dendrites of antennal OSNs, where together, they are suggested to form heteromeric, ligand-gated, non-selective cation channels. Within Orco's second intracellular loop, there is an especially well-conserved sequence strongly predicted to be a calmodulin (CaM) binding site (CBS). In this study, I explored the relationship between Orco and CaM \emph{in vivo} in the olfactory neurons of Drosophila melanogaster. I first found OSN-specific knock-down of CaM at the onset of OSN development disrupts the spontaneous firing of OSNs and reduces Orco trafficking to the ciliated dendrites of OSNs. I then generated a series of Orco CBS mutant proteins and found that none of them rescue the Orco-null $Orco^1$ mutant phenotype, which is characterized by an OR protein trafficking defect that blocks spontaneous and odorant-evoked OSN activity. In contrast to an identically constructed wild-type form of Orco that does rescue the $Orco^1$ phenotype, all the Orco CBS mutants remain stuck in the OSN soma. Last, I found CaM's modulation of OR trafficking is activity-dependent. Knock-down of CaM in OSNs after OR expression is well-established has little effect on olfactory responsiveness alone. When combined with an extended exposure to odorant, however, this late-onset CaM knock-down significantly reduces both olfactory sensitivity and the trafficking of Orco only to the ciliated dendrites of OSNs that respond to the exposed odorant. Therefore, I propose a model in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odorant concentrations.