Functional identification and reconstitution of an odorant receptor in single olfactory neurons.
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 Ca2+-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 Ca2+ 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 Ca2+ 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.
Touhara
K
K
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan. ktouhara@mail.ecc.u-tokyo.ac.jp
Sengoku
S
S
Inaki
K
K
Tsuboi
A
A
Hirono
J
J
Sato
T
T
Sakano
H
H
Haga
T
T
D000256:Adenoviridae; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000502:physiology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009812:Odorants; D009831:Olfactory Mucosa / Q000166:cytology; D018034:Olfactory Receptor Neurons / Q000166:cytology / Q000502:physiology*; D018035:Receptors, Odorant / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D008164:Luminescent Proteins; C118713:Olfr16 protein, mouse; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D002118:Calcium; D016257:Fura-2