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

The olfactory system is remarkable in its capacity to discriminate a wide r ange of odorants through a series of transduction events initiated in olfac tory 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 cha racterized. This study demonstrates the functional identification of olfact ory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription-coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printe d olfactory neuron that displayed odorant-induced Ca2+ increase. Nest, reco mbinant adenovirus-mediated expression of the isolated receptor gene was es tablished in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed t o the odorant that originally was used to identify the receptor gene. Exper iments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. Th e odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) fro m single neurons and the functional expression of the receptor via recombin ant adenovirus, The present approach should enable us to examine not only l igand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.