Insects detect specific chemicals in the environment with olfactory receptors (ORs), which represent a novel class of ligand-gated ion channel. Insect ORs are comprised of at least one common subunit (OR83b in Drosophila) and at least one odorant-binding subunit. However, the molecular details of insect OR architecture, such as how they bind odorants, are unknown. This lack of knowledge hinders the development of compounds that may modulate OR function and potentially control insects involved in disease propagation and agricultural damage. The intent of this project is to investigate the structure and function of insect ORs. To this end, the utility of the Xenopus oocyte heterologous expression system was explored. Assay optimization, accuracy, and investigations on functional requirements were first performed using the Drosophila OR (DmOR) 35a/83b. The utility of the assay system was also demonstrated by identification of the honey bee (Apis mellifera) OR 11/2 as a receptor for the queen pheromone, 9-oxo-2-decenoic acid. A series of DmORs was cloned and expressed in Xenopus oocytes and individual receptors were selected for further study. DmOR85a/83b was shown to possess an incredibly high degree of enantioselectivity for the odorant ethyl 3-hydroxybutyrate. The receptive range of DmOR67a/83b was explored and observations were made on potential features of the odorant-binding site and a ligand odorophore. DmORs were also used to investigate the contributions of individual subunits toward the odorant-binding site and pore structure. Also, evidence for receptor antagonism by odorants was revealed. DmORs were screened with methanethiosulfonate reagents and the substituted cysteine accessibility method to identify residues 146-150 of DmOR85b as functionally important in receptor activation. This region, located at the predicted interface between transmembrane segment 3 (TMS3) and extracellular loop 2, was shown to be physically adjacent to the odorant-binding site itself. Finally, residues within the extracellular half of TMS3 in DmOR85b were implicated in odorant-induced activation by screening DmOR85b mutants for altered ligand preferences. Therefore, this project provides the first identification of insect OR subunit components involved in odorant recognition, and represents an important starting point for detailed analysis of the molecular basis for insect OR activation by odorants.
| Identifer | oai:union.ndltd.org:UMIAMI/oai:scholarlyrepository.miami.edu:oa_dissertations-1360 |
| Date | 04 February 2010 |
| Creators | Nichols, Andrew S. |
| Publisher | Scholarly Repository |
| Source Sets | University of Miami |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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