Abstract

Olfactory receptors (Ors) convert chemical signals—the binding of odors and pheromones—to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K+ selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex.

Highlights

  • The detection of odorants and pheromones is essential for insects to find food, avoid predators and noxious agents in the environment, and find appropriate mating partners

  • BmOrco Y464A expressed with Or47a showed normal spontaneous activity but a strong increase in odor-evoked activity (Figure 4E,F). These results suggest that the TM7 Tyr residue in Orco (Y478 in Drosophila and Y464 in Bombyx) contributes to the function of the insect odorant receptors (Ors)-Orco complex in vivo, the function of this Tyr residue may not be identical between DmOrco and BmOrco

  • We carried out comprehensive site-directed mutagenesis of all conserved Glu, Asp, and Tyr residues in the silkmoth bombykol receptor to probe the structure-function relationships of the Or-Orco complex. 13 of the 83 residues caused functional alterations in odor-evoked cation channel activity

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Summary

Introduction

The detection of odorants and pheromones is essential for insects to find food, avoid predators and noxious agents in the environment, and find appropriate mating partners. Insects sense odorants and pheromones via specialized olfactory sensory neurons (OSNs) located on two sensory appendages on the head, the antennae and maxillary palps. Both of these appendages are covered with specialized sensory hairs called sensilla that house one to four OSNs in the vinegar fly Drosophila melanogaster and the silkmoth Bombyx mori. The third functional class is a single member of the Or family called Orco, which is highly conserved in all known insect species and functions as an obligate chaperoning co-receptor in complex with ligand-selective Ors [19,20,21,22]. Most OSNs co-express one of the canonical Ors and the Orco family, and these two types of receptors comprise a heteromultimeric complex of unknown stoichiometry [12,22,23]

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