Functional identification and reconstitution of an odorant receptor in single olfactory neurons

Abstract

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca 2+ -imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca 2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca 2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Complex Transcription and Splicing of Odorant Receptor Genes

“…However, several lines of evidence argue in favor of the functionality of OR in testis: (i) promiscuous gene expression is also a feature of certain thymic medullary cells, where it serves a purpose within negative T-cell selection (50); (ii) certain OR have been detected on spermatozoa (13), proving testicular translation of OR transcripts; (iii) the expression levels of OR genes within the testis appear to be comparable with that in the main olfactory epithelium (7) 4 ; and (vi) the G-protein used by OR for signal transduction is expressed in testis (45), and spermatozoa seem to be endowed also with proteins involved in olfactory desensitization (51), implying that testicular OR expression is a meaningful event. Specific ligands are known only for very few OR (52)(53)(54)(55), and these odorants are without exception volatile, favoring their interaction with OR residing on cells of the main olfactory epithelium. However, because OR also play a major role in axonal targeting of olfactory neurons to specific glomeruli in the olfactory bulb (19, 56 -58), it may well be that OR interact with nonvolatile molecules as well, e.g.…”

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

“…One method is to use adenovirus as a vector to infect olfactory neurons. Activation of foreign ORs was measured either by electroolfactogram or calcium imaging of single cells 24,25 . Only few ORs have been expressed and analyzed using this method to date, due to low throughput.…”

Diversity and expression of odorant receptors in Drosophila

“…An alternative to genetics has been heterologous expression of ORs, either in vivo (Zhao et al, 1998;Araneda et al, 2000) or in tissue culture cells (Krautwurst et al, 1998;Touhara et al, 1999), and the use of functional imaging of isolated OSNs to determine their response profiles (Malnic et al, 1999;Leinders-Zufall et al, 2000). These studies of vertebrate chemosensory receptors have led to the conclusion that a given vertebrate OR recognizes multiple odorants and that a given odorant activates neurons expressing a number of different ORs.…”