Abstracts - Association for Chemoreception Sciences

#P249 POSTER SESSION V:
HUMAN TASTE PSYCHOPHYSICS;
OLFACTION RECEPTORS; TASTE DEVELOPMENT
#P250 POSTER SESSION V:
HUMAN TASTE PSYCHOPHYSICS;
OLFACTION RECEPTORS; TASTE DEVELOPMENT
Re-Engineering of Olfactory Receptor OlfCc1 Toward
Directed Ligand Selectivity
Allison P Berke 1 , Francine Acher 2 , Hugues O. Bertrand 3 , John Ngai 4
1
Joint Graduate Group in Bioengineering, University of California
Berkeley, CA, USA, 2 Laboratoire de Chimie et Biochimie
Pharmacologiques et Toxicologiques, Unite Mixte de Recherche
8601, Centre National de la Recherche Scientifique, Universite Rene
Descartes-Paris V Paris, France, 3 Accelrys Orsay, France, 4 Department
of Molecular and Cell Biology and Helen Wills Neuroscience Institute
Berkeley, CA, USA
Fish sense food cues in their aqueous environment using family
C GPCR olfactory receptors. These C family receptors are
characterized by a large N-terminal “Venus flytrap” domain.
Zebrafish olfactory receptor OlfCc1 is a broadly-expressed
ortholog of mammalian V2R2, which shares sequence similarity
with the human Calcium-sensing Receptor (CaSR). C family
receptors are predicted to respond to amino acids, as do the
previously identified OlfCa1 and CaSR. The expression of
OlfCc1 in the entire microvillous olfactory neuron population
in the zebrafish, as well as its sequence homology with CaSR,
make it an interesting target for de-orphaning and engineering,
as it could play a generalized behavioral role in zebrafish
chemosensation. In silico modeling of OlfCc1 identified the
receptor’s binding pocket and residues likely to be directly
involved in ligand binding. OlfCc1 was then cloned into a CMVI
FLAG-tagged expression vector and expressed in HEK293
cells. Calcium imaging performed on these cells using Fluo-4
calcium-sensitive dye revealed the calcium-dependent binding
profile of OlfCc1, which includes amino acids. Amino acid
point mutations were then introduced to OlfCc1, with the
aim of broadening the receptor’s binding specificity. These
mutations succeeded in altering the sensitivity and specificity
of OlfCc1 in accordance with predictions. In conclusion, the
binding specificity and sensitivity of OlfCc1 can be selectively
engineered. Additionally, the combination of in silico homology
modeling and calcium imaging that constitute this method can
be applied to other C family GPCRs, to directly engineer ligand
binding capability. Acknowledgements: NSF GRFP and the NIH
Interactions at the olfactory receptor level contribute to the
coding of odorant mixtures
fouzia El Mountassir 1 , christine Belloir 1 , loic Briand 1 ,
thierry Thomas Danguin 1 , anne marie Le BON 1
1
Centre des Sciences du Gout et de l’Alimentation DIJON, France, 3
Numerous studies reported that the perceptual characteristics
of odorant mixtures are often different from those of their
individual compounds; e.g. the mixture intensity can be higher
or lower than the arithmetic sum of each component’s intensity.
These findings raise the question how odorants in mixtures are
detected and encoded at the peripheral level of the olfactory
system. We investigated this question through the measurement
of human olfactory receptor (OR) responses to two specific
binary mixtures of aldehydes: (i) octanal and citronellal, known
to induce a configural perception in rats (Kay et al., 2003) and
masking effects in humans (Burseg et al., 2009); (ii) octanal
and methional, known to induce masking effects in humans
(Burseg et al., 2009). We used a heterologous expression system
(HEK293T cells) in which OR (OR1G1, OR52D1, OR2W1
and OR1A1) were transfected transiently. Responses of OR to
odorants applied alone or in mixtures were measured by calcium
imaging. The results showed various interactions at the OR
level. When octanal was mixed with citronellal, the OR response
intensity was reduced thus showing subtraction, compromise or
partial addition, depending on the OR and the concentrations of
odorants. Interestingly, the mixture of octanal and methional was
found to induce mostly synergy, whatever the OR. These data
strengthen the hypothesis that interactions can occur at the OR
level and could therefore contribute significantly to the olfactory
coding of odorant mixtures. Acknowledgements: This work is
funded by the National Institute of Agricultural Research and
the region of Burgundy
#P251 POSTER SESSION V:
HUMAN TASTE PSYCHOPHYSICS;
OLFACTION RECEPTORS; TASTE DEVELOPMENT
Class-specific regulation of a zebrafish olfactory receptor gene
Stefan H. Fuss, Xalid Bayramli, Nuray Sögünmez
Bogazici University, Molecular Biology and Genetics Istanbul, Turkey
Olfactory sensory neurons (OSNs) typically express a single
allele of a single olfactory receptor (OR) gene from a much larger
genomic repertoire; a phenomenon that is not well understood.
Experimental evidence suggests that OR expression is controlled
by a combination of long- and short-range regulatory sequences.
We used promoter bashing in transgenic zebrafish to identify
proximal regulatory sites within the OR101-1 gene promoter.
Positive regulatory sites are located within the first 500 bp
upstream of the transcription start site, while more distant
sequences confer a repressive effect on transgene expression, even
in the presence of strong genomic enhancers. Interestingly, the
POSTER PRESENTATIONS
Abstracts are printed as submitted by the author(s).
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