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Transmembrane segment 3 of Drosophila melanogaster odorant receptor subunit 85b contributes to ligand-receptor interactions.

ABSTRACT: The OR class insect odorant receptors are ligand-gated ion channels comprised of at least one common subunit (OR83b in Drosophila) and at least one putative odorant-binding subunit. However, little else is known about the molecular details of insect OR architecture. For example, nothing is known about how these receptors bind odorants, greatly limiting efforts to develop insect OR-targeted compounds for the control of insects involved in disease propagation and agricultural damage. Here we identify a portion of a Drosophila OR that is involved in odorant activation of the receptor. Using the substituted cysteine accessibility method, we identified residues 146-150 of OR85b, located at the predicted interface between transmembrane segment 3 (TMS3) and extracellular loop 2 (ECL2), as playing a role in odorant (2-heptanone) activation. We found that occupation of the receptor by the competitive antagonist 2-nonanone protected the receptor from methanethiosulfonate action at position 148, placing this region close to the odorant-binding site. In addition, mutations at positions 142 and 143 within TMS3 altered odorant sensitivity. Our results identify the involvement of the extracellular half of TMS3 in Drosophila OR85b in odorant activation of the receptor. This finding can serve as a starting point for future detailed analysis of the molecular basis for odorant recognition by insect ORs, a novel class of ligand-gated channel.

SUBMITTER: Nichols AS

PROVIDER: S-EPMC2852922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Flying insects have developed a remarkably sensitive olfactory system to detect faint and turbulent odor traces. This ability is linked to the olfactory receptors class of odorant receptors (ORs), occurring exclusively in winged insects. ORs form heteromeric complexes of an odorant specific receptor protein (OrX) and a highly conserved co-receptor protein (Orco). The ORs form ligand gated ion channels that are tuned by intracellular signaling systems. Repetitive subthreshold odor stimulation of olfactory sensory neurons sensitizes insect ORs. This OR sensitization process requires Orco activity. In the present study we first asked whether OR sensitization can be monitored with heterologously expressed OR proteins. Using electrophysiological and calcium imaging methods we demonstrate that D. melanogaster OR proteins expressed in CHO cells show sensitization upon repeated weak stimulation. This was found for OR channels formed by Orco as well as by Or22a or Or56a and Orco. Moreover, we show that inhibition of calmodulin (CaM) action on OR proteins, expressed in CHO cells, abolishes any sensitization. Finally, we investigated the sensitization phenomenon using an ex vivo preparation of olfactory sensory neurons (OSNs) expressing Or22a inside the fly's antenna. Using calcium imaging, we observed sensitization in the dendrites as well as in the soma. Inhibition of calmodulin with W7 disrupted the sensitization within the outer dendritic shaft, whereas the sensitization remained in the other OSN compartments. Taken together, our results suggest that CaM action is involved in sensitizing the OR complex and that this mechanisms accounts for the sensitization in the outer dendrites, whereas further mechanisms contribute to the sensitization observed in the other OSN compartments. The use of heterologously expressed OR proteins appears to be suitable for further investigations on the mechanistic basis of OR sensitization, while investigations on native neurons are required to study the presently unknown additional mechanisms involved in OSN sensitization.

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Transmembrane segment 3 of Drosophila melanogaster odorant receptor subunit 85b contributes to ligand-receptor interactions.

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