In insects, olfaction is mediated by a large family of integral membrane proteins, called olfactory receptors (ORs), that mediate the transduction of odorant binding into a neuronal signal. A functional assay for insect ORs was developed utilising calcium imaging in Sf9 cells. The Drosophila melanogaster OR, Or22a, was expressed using transient transfection, and its activity measured by monitoring increased intracellular calcium levels using a calcium–sensitive dye. The interaction of the odorants ethyl butyrate, pentyl acetate and ethyl acetate with Or22a were both dose–dependent and sensitive, with EC50 values of 1.53 x 10−11 M, 5.61 x 10−10 M and 3.72 x 10−9 M, respectively. Furthermore, Or22a expressed in Sf9 cells has a similar response profile to a range of odorants previously tested in vivo. This assay system will provide a useful tool for the investigation of insect olfactory receptor structure and function. A consensus of eleven transmembrane (TM) domain prediction algorithms suggested a model for Or22a that contains seven TM domains, reminiscent of GPCRs. To test this model empirically, the membrane topology of Or22a was determined using epitope–tagging of predicted loops followed by immunochemistry. These experiments revealed that Or22a has seven TM domains but that its orientation in the membrane is opposite to that of GPCRs, having a cytoplasmic N–terminus. This orientation was also observed for Epiphyas postvittana Or1, which suggests that this inverted topology may be common to all insect ORs. To test whether Or22a forms higher order structures, fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins inserted into the intracellular loops of Or22a was employed. The third intracellular loop interacts strongly with itself in homo–multimers, with interactions between the first and first loops and first and third loops also observed. These experiments show that ligand binding ORs can form multimeric structures in heterologous cells. The co–transfection of Or83b into S2 cells had no impact on these interactions, however Or83b is likely expressed in this cell line. Finally, models of how a ligand binding OR interacts physically with the ion channel Or83b are presented, and approaches that could be used to distinguish between these models are discussed.
| Identifer | oai:union.ndltd.org:ADTP/247679 |
| Date | January 2008 |
| Creators | Kiely, Aidan |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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