Biomedical Engineering Reference
In-Depth Information
mined in heterologous cells really reflects
in vivo
responsiveness of the target OR.
As such, caution must be prioritized when evaluating structure-activity relationship
of ORs
in vitro
expression systems [
44
].
11.1.3
Olfactory Signal Transduction
Odorant stimulation elicits a signal transduction cascade mediated by OR expres-
sion on the surface of the olfactory neuronal cilia. The olfactory signal cascade is
generated through two pathways: cAMP and IP
3
. The cAMP cascade is dominant in
transmitting odorant signals in the olfactory neurons, whereas the role of the inosi-
tol-1,4,5-triphosphate (IP
3
)-mediated pathway remains unclear [
45
]. The initial step
in the cAMP pathway is binding of odorant molecules to specific OR proteins on the
cilia surface [
8
,
34
,
46
]. Ligand binding triggers a change in the OR structural con-
formation via the activation of a membrane-bound type III adenylyl cyclase (ACIII)
in an olfactory-specific G protein subunit (G
αolf
). ACIII leads to the generation of
cyclic adenosine monophosphate (cAMP), which directly opens a heteromeric cy-
clic nucleotide-gated (CNG) in the cilia membrane. The opened CNG-channel leads
to an influx of Ca
2+
and Na
+
, and subsequent opening of the Ca
2+
-activated Cl
−
chan-
nel results in the depolarization of olfactory cells through cation influx as shown in
Fig.
11.1
(c). The secondary mechanism in olfactory signal transduction involves
the generation of IP
3
from phosphatidyl inositol 4,5-bisphosphate (PIP
2
) by phos-
pholipase C [
4
,
47
]. For example, the specific binding of diacetyl to ODR-10 is the
OR protein of the nematode
Caenorhabditis elegans
(
C. elegans
), which triggers
signal transduction through the IP
3
pathway. Activated OR subsequently activates
phospholipase C (PLC), which converts the phosphatidyl inositol 4,5-bisphosphate
(PIP
2
) into inositol 1,4,5-triphosphate (IP
3
). The IP
3
opens the Ca
2+
channel on the
surface of endoplasmic reticulum (ER), which increases cytosolic Ca
2+
ion. Ulti-
mately, both the cAMP- and IP
3−
pathway can be analyzed by simply measuring the
influx or efflux of Ca
2+
ions upon ligand stimulation.
11.2
Labeled Optical Methods for Odorant Binding Assay
Fluorescence or bioluminescence measurements are some of the techniques used in
standard optical assays developed for monitoring cellular activation events. For the
characterization of ORs, these basic optical methods include Ca
2+
imaging, cAMP-
reporter assay, fluorescent resonant energy transfer (FRET), bio-luminescence reso-
nant energy transfer (BRET), and so on, used to measure the interaction between
the OR and odorant in the cellular level that result from signal transduction. These
cell-based assays, which use cells as the sensing elements, have many advantages
over other techniques in molecular interaction study and can be used to derive func-
tional information of biologically active analytes. This section provides a general
overview of the most common types of optical detection technique.
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