Biomedical Engineering Reference
In-Depth Information
11.2.1
Intracellular Ca
2+
Imaging-based Odorant Sensing
Measuring the change in intracellular Ca
2+
levels via olfactory signal transduction
is a very useful tool for examining the function of the OR proteins expressed in
olfactory neuron and heterologous cell systems [
45
,
48
]. In this regard, Ca
2+
imag-
ing is a basic strategy for detecting physiological odorant responses by measuring
the temporal and spatial properties of Ca
2+
changes caused by odorant stimuli. Ca
2+
signals in the cytosol and organelles are important for cellular signal transduction
and are usually measured using a synthetic fluorescent chelator, such as Fura-2.
Odorant stimulation causes Ca
2+
entry through CNG channels in individual respon-
sive neurons, which is then regulated by a series of signal transductions. Essentially,
if an OR specifically binds with a ligand, a signal cascade induces an influx of
Ca
2+
ions. Intracellular Ca
2+
ions then bind to Fura-2, which was previously loaded
into cells. The excitation wavelength of Fura-2 changes from 380 to 340 nm when
Fura-2 binds to the Ca
2+
ions. As a result, the increase in the amount of intracel-
lular Ca
2+
ions can be estimated by the ratio of fluorescence emissions (excitation
at 340/380 nm).
Identification and functional characterization of human OR (hOR)17-4 by the
ratio-fluorometric Ca
2+
imaging was reported by Spehr et al. [
49
]. This expression
of hOR17-4 protein in a heterologous HEK-293 cell line allowed for the identifica-
tion and structure-function analysis of cognate receptor-odorant pairs using single
cell Ca
2+
recording as shown in Fig.
11.3
. An
in situ
Ca
2+
-imaging technique was
adopted to monitor odorant responses of more than several hundreds of neurons
simultaneously through an intact coronal slice of the olfactory epithelium. The sen-
sitivity and resolution of Ca
2+
-imaging were high enough to distinguish between
olfactory neurons with threshold concentrations for a particular odorant at the single
cell level. Increasing odorant concentrations resulted in increases in the numbers of
odorant-responsive neurons. This methodology is powerful tool to visualize spatial
distributions of odorant responsive neurons at a cellular resolution, and to construct
odor maps in a coronal view of the olfactory epithelium [
50
].
Ca
2+
imaging technique can be used for identification of OR-odorant pair and
structure-activity relationship. In a study from Wetzel and co-workers, an odorant
screening strategy in heterologous cells was demonstrated using Ca
2+
imaging [
51
].
A mixture of 100 different odorants (Henkel 100) elicited a transient increase in
intracellular Ca
2+
, and a specific single odorant component to human olfactory re-
ceptor (hOR) 17-40 protein was identified by subdividing the odorant mixture into
progressively smaller groups. Ca
2+
imaging was also applied for de-orphaning of
the OR through high-throughput screening using fluorescence imaging plate reader
(FLIPR) experiments [
43
]. Ca
2+
imaging and HeLa/OR heterologous cell systems
were applied to a large-scale odorant-receptor that screened and established recep-
tor-specific odorant profiles, resulting in the de-orphaning of two ORs for the odor-
ants from an expression library of 93 receptors. Unfortunately, the de-orphaning of
OR is still complicated by its combinatorial odorant coding and lack of efficient
assay tools. Although ORs are efficiently expressed in various mammalian cell lines
(e.g., HEK-293, COS-7, Hana3A,
X. laevis oocyte
and CHO-K1 cells) [
51
-
55
],
Search WWH ::
Custom Search