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#P35
POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
Deorphanization of human olfactory receptors by Luciferase
and Ca-imaging methods: Differences & similarities
Kaveh Ashti Baghaei, Günter Gisselmann, Hanns Hatt
Ruhr-Universität Bochum/Cell Physiology Department
Bochum, Germany
Olfactory receptors (ORs) expressed at the cell surface of olfactory
sensory neurons lining the olfactory epithelium and act as first
actors for perception and recognition of odorants. Up to now,
few human ORs have been deorphanized, mainly because the
difficulties encountered to express ORs functionally in a
recombinant system. Within this work, our aims were 1) to
deorphanize human ORs and 2) to compare outcome of the
2 techniques mostly used for deorphanization. For this, we used
two tests for deorphanization of some several ORs and analyzed the
responses of them to a number of alcohol & ketone compounds.
We found that some of these ORs are able to induce cellular
responses by the use of Ca-imaging and CRE-luciferase assay.
Our experiments indicated that in Ca-imaging assays OR4X2 was
activated by ketones and OR5D13, OR1N1 & OR8D2 by alcohols.
In the CRE-luciferase assay, only cells transfected with OR5D13 or
OR8D2 were significantly activated by alcohols. Our results
represent, OR4X2 is receptor for ketones and OR5D13, OR1N1
and OR8D2 are activated by alcohols. Different results by
CRE-luciferase and Ca-imaging method could reflect some
technical reasons like stimulation or transfection time. Finally,
according to these results it could be suggested, that using of two
complementary tests in parallel can benefit deorphanization.
#P36
POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
Sequencing the olfactory receptor repertoire
Joel D. Mainland
1,2
, Jason Willer
2
, Anna Lindstrand
2
, Andreas
Keller
3
, Leslie Vosshall
3,4
, Nicholas Katsanis
2
, Hiroaki Matsunami
2
1
Monell Chemical Senses Center Philadelphia, PA, USA,
2
Duke University Durham, NC, USA,
3
The Rockefeller Univeristy
New York, NY, USA,
4
Howard Hughes Medical Institute
New York, NY, USA
Humans have approximately 400 functional olfactory receptors,
but among this set there are a large number of variations between
individuals. In some cases, these variations cause a receptor to be
nonfunctional in a subset of the population. These variations
likely underlie inter-individual variation in olfactory perception.
We identified ligands for 19 odorant receptors and, along with
previously published ligands for 9 additional receptors,
characterized polymorphic variation in an ethnically diverse
population. We related the genotypic variation to functional
variation using a heterologous system. In our population, 86% of
the odorant receptors we examined had polymorphisms that caused
functional changes. On average, two individuals differ functionally
at 42% of their odorant receptor alleles. This high level of
functional variability in the primary receptors transducing olfactory
information is strongly suggestive of high interindividual
variability in odorant detection at the receptor level. Here we set
out to translate this variability at the receptor level to a human
subject by applying a targeted capture method to enrich the odorant
receptor repertoire for next-generation sequencing. We demonstrate
that we are able to fully sequence an individual’s OR repertoire
with enough coverage to call heterozygous sites. This method can
be used to identify the functional role of a single odorant receptor
in olfactory perception. Acknowledgements: R01 DC005782 R03
DC011373 F32 DC008932
#P37
POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
Exploring the variation in molecular receptive range among
mammalian olfactory receptors
Selvan Bavan, Jingyi Li, Vanessa Santos, Jakub Bartkowiak,
Charles W. Luetje
Molecular and Cellular Pharmacology, University of Miami,
Miller School of Medicine Miami, FL, USA
The recognition of odorant molecules by odorant receptors (ORs) is
a critical early step in olfaction. Mammalian ORs are a large family
of G-protein coupled receptors located on the cilia of olfactory
sensory neurons. The molecular receptive ranges (MRRs) of ORs
are thought to vary in breadth, from narrowly tuned ORs activated
by few odorants, to broadly-tuned ORs recognizing a large variety
of odorant molecules, all contributing to a combinatorial
mechanism of odorant recognition. To explore MRR variation, we
screened 55 mouse ORs (MORs) that were representative of many
MOR subfamilies with a panel of 155 odorants chosen to represent
a broad portion of odor space. MORs were expressed in Xenopus
oocytes, together with Gαolf and the human cystic fibrosis
transmembrane regulator, to allow assay of odorant responses by
two-electrode voltage clamp electrophysiology. Of the 55 MORs
screened, 9 gave significant responses to at least one odorant in our
panel. The MRRs of these receptors varied in breadth from
receptors responding to a single odorant (for example, MOR283-3
responded only to 2-ethylpyrazine) to MORs responding to a wide
variety of odorants (for example, MOR208-3 responded to about a
third of the odorants). To explore how MRRs overlap and diverge
among members of the MOR family, we cloned and are screening
an additional series of receptors related to MOR260-3 (a receptor
responding to cyclic alcohol and ester structures). These additional
receptors were chosen based on a phylogenetic organization of the
MORs, as well as an alternate organisational scheme (Man et al.,
2007, PLoS One 2: e682). This work should help distinguish
among different organisations of MORs into groups that exhibit
similar surveillance of odor space. Acknowledgements: NIH
RO1DC008119
#P38
POSTER SESSION I:
MULTIMODAL RECEPTION;
CHEMOSENSATION & DISEASE;
TASTE PERIPHERY; OLFACTION PERIPHERY
First agonists of a human trace-amine associated receptor
(TAAR) expressed in the human olfactory epithelium
Ivonne Wallrabenstein, Sandra Zborala, Lea Weber,
Markus Werner, Günter Gisselmann, Hanns Hatt
Ruhr University Bochum/Cellphysiology Bochum, Germany
Trace-amine associated receptors (TAAR) belong to the family of
G-protein coupled receptors and can be activated by amines. All
TAAR-subtypes except TAAR1 were exclusively expressed in the
main OE of mice and can be activated by different volatile amines,
40 | AChemS Abstracts 2012
Abstracts are printed as submitted by the author(s)
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