Cell-Based System for Identification of Olfactory Receptors - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 88

relatively rare since, with most studies limited to the assaying of Drosophila ORs
such as Or22a and Or43b [ 42 , 43 ]. ORs have also been successfully introduced
into E. coli bacteria when overexpressed as a fusion protein, where they were able
to display interactions with odorants [ 44 ]. The HeLa-Cx43 cell line has also been
used to express ORs, most notably to show that odorants can function as agonists or
antagonists for various ORs based on the type of G-protein used [ 45 ].

5.5 Do in Vitro Systems Replicate in Vivo OR Responses?

Despite the growing ability of in vitro heterologous systems to parallel odorant-
induced receptor activity in vivo, there still exist doubts concerning the ability of
in vitro systems to truly replicate unique conditions encountered within the live
olfactory system. Odors are delivered though solution in heterologous systems
whereas they travel through the air during normal mammalian olfaction. Thus, na-
sal airflow rate can alter the apparent sensitivity of certain odorants assayed through
glomerular response. For example, glomerular response in the olfactory bulb can
differ in response to passive breathing versus sniffing [ 46 ]. In addition, the presence
of pre-receptor events in vivo involving enzymes present in the nasal mucus can
affect olfactory perception. Odorants with functional groups such as aldehydes and
esters are converted into corresponding acids and alcohols when contact is made
with metabolic enzymes secreted in the mouse mucus. When an enzyme inhibitor
was used in parallel with exposure to the relevant odorants, glomerular activation
patterns and olfactory discrimination test results differed in those mice compared
to mice with the enzymes intact [ 15 ]. Studies have also shown differences in ligand
responsiveness when comparing ORs measured through calcium imaging or patch-
clamp versus ORs expressed in the heterologous system, although these studies
have also shown the effectiveness of expressing ORs in vitro as well. The receptor
mOR-EG, which responds to eugenol, shows similar patterns of activation both in
vivo and in vitro when calcium imaging is applied [ 19 ]. In addition, heterologous
cells expressing the receptor SR1, which responds to a broad panel of odorants, do
not all respond consistently to the same panel—instead, each SR1 cell responds
to different odorants at a wide variety of concentrations, although this diversity of
responses is also mirrored in vivo with patch clamp analysis [ 11 ].
The human OR OR7D4, when expressed in heterologous cells, has been shown
to mirror odor perception in humans in terms of responding to the steroids andro-
stenone and androstadienone. In humans, the OR7D4 gene exists as one of two
variants that differ by two amino acid substitutions (the most common variant “RT”,
and “WM”, which contains R88W and T133M amino acid substitutions), and it
was found that the RT variant responds to the steroids at a much higher level than
the WM variant in vitro. Likewise, human test subjects with both copies of the RT
variant found the steroids to be more intense and “sickening” then those with just
one copy of RT or two copies of WM [ 14 ].
Thus, to test for the efficacy, reliability and accuracy of our heterologous system
versus live-cell recordings of olfactory sensory neurons, we selected previous studies