Insect odorant receptor trafficking requires calmodulin.

Suhyoung Bahk,Walton D Jones

doi:10.1186/s12915-016-0306-x

Suhyoung Bahk, Walton D Jones

Open Access

https://doi.org/10.1186/s12915-016-0306-x

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Abstract

BackgroundLike most animals, insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators. Most insect olfactory neurons 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 olfactory sensory neurons (OSNs), where together, they are suggested to form heteromeric ligand-gated non-selective cation channels. While most amino acid residues in Orco are well conserved across insect orders, one especially well-conserved region in Orco’s second intracellular loop is a putative calmodulin (CaM) binding site (CBS). In this study, we explore the relationship between Orco and CaM in vivo in the olfactory neurons of Drosophila melanogaster.ResultsWe 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 without affecting their morphology. We then generated a series of Orco CBS mutant proteins and found that none of them rescue the Orco-null Orco1 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 Orco1 phenotype, all the Orco CBS mutants remain stuck in the OSN soma, preventing even the smallest odorant-evoked response. Last, we found CaM’s modulation of OR trafficking is dependent on activity. Knock-down of CaM in all Orco-positive 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.ConclusionsIn this study, we show CaM regulates OR trafficking and olfactory responses in vivo in Drosophila olfactory neurons via a well-conserved binding site on the olfactory co-receptor Orco. As CaM’s modulation of Orco seems to be dependent on activity, we propose a model in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odorant concentrations.

Highlights

Like most animals, insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators
We combined a upstream activating sequence (UAS)-CaM-IR transgene with the peripheral sensory neuron driver Pebbled-GAL4. It is expressed in larvae, Pebbled-GAL4 expression begins in nascent Olfactory sensory neuron (OSN) 12–18 hours after puparium formation (APF) [23], long before the earliest odorant receptor (OR) expression begins 50–60 hours APF [1]
While flies kept at 18 °C (CaM-IR OFF) show normal levels of Orco in both the soma and inner and outer dendrites, flies kept at 29 °C (CaM-IR ON) show reduced Orco staining in many but not all soma and in both the inner and outer ciliated dendrites (Fig. 1b, c)

Summary

Introduction

Insects rely on their olfactory systems for finding food and mates and in avoiding noxious chemicals and predators. Orco binds ORs and permits their trafficking to the dendrites of antennal olfactory sensory neurons (OSNs), where together, they are suggested to form heteromeric ligand-gated non-selective cation channels. In Drosophila, volatile odorants are detected by odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) [1, 2]. These OSNs are housed, most often in pairs, within sensory hairs called sensilla that cover the maxillary palps and third antennal segments [3]. Each OSN sends a single dendrite comprising an inner segment, a ciliary constriction, and a branched outer segment into the sensory lymph-filled lumen of an olfactory sensillum [7]. Insect ORs have seven transmembrane domains and appear superficially

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