CHEMPLUSCHEM
FULL PAPERS
nounced lily-of-the-valley character, but instead green, anisic,
and slightly balsamic facets are perceivable.
Essentially, except rac-1d and 2d, all other derivatives of
rac-1a and 2a possess a typical lily-of-the-valley note of more
or less pronounced green character; compound 2a is most
green-aldehydic in character and rac-1c is the most pro-
nounced floral one. The fattiness increases in both series from
carbon!silicon!germanium!tin, independently of the floral
character and the lily-of-the-valley note. In terms of odor
threshold, compounds rac-1a and 2a display the lowest
values, and thus, show the best performance.
Generation of a HEK293 cell line with stable tetracycline-
regulated expression of hOR17-4
To analyze recombinant hOR17-4 activation by lily-of-the-valley
odorants, we generated T-REx-293 cells that stably expressed
rho-tagged hOR17-4 under the control of a tetracycline-regu-
lated promoter. To investigate the integration of the targeted
sequence into the genome of T-REx-293 cells, we analyzed
hOR17-4 expression at the RNA and protein levels. Reverse
transcription polymerase chain reaction (RT-PCR) analyses with
primers specific for hOR17-4 showed that expression of hOR17-
4 could be induced in T-REx-293-hOR17-4 cells, but was absent
in parental T-REx-293 cells (Figure S1 in the Supporting Infor-
mation). By western blot analysis with a monoclonal antibody
against the C-terminal rho tag of recombinant hOR17-4, we
found that hOR17-4 protein was expressed in induced T-REx-
293-hOR17-4 cells (Figure S2 in the Supporting Information).
Immunocytochemical staining revealed hOR17-4 protein ex-
pression in approximately 80% of induced T-REx-293-hOR17-4
cells, but not in parental T-REx-293 cells (Figure S3 in the Sup-
porting Information). These results indicate stable integration
of the hOR17-4-rho construct into the genome of T-REx-293
cells. Experimental details of the studies described in this para-
graph are given in the Supporting Information.
Figure 1. Activation of heterologously expressed hOR17-4 by rac-1a–d and
2a–d. A) Representative Ca2+ imaging measurements of HEK293 cells stably
expressing hOR17-4 (T-REx-293-hOR17-4 cells). The upper panel shows pseu-
docolor images of fura-2-loaded, induced T-REx-293-hOR17-4 cells that were
captured during the measurements. Relative cytosolic Ca2+ levels are shown
in pseudocolor, which indicates changes in the cytosolic Ca2+ concentration.
In a randomly selected field of view, compound 2a induces transient in-
creases in the cytosolic Ca2+ concentration in individual fura-2-loaded cells
(e.g., white circles). Compound 2a (250 mm) was applied for 10 s, and 20 mm
ATP served to control cell excitability. Cytosolic Ca2+ levels were monitored
as integrated f340/f380 fluorescence ratios expressed as a function of time.
Traces are shown in greyish colors (lower panel). B) Dose–response relation-
ships of heterologously expressed hOR17-4 and rac-1a–d and 2a–d, respec-
tively. As an indirect measure for hOR17-4 activation, the response probabili-
ties to rac-1a–d and 2a–d were determined in Ca2+ measurements of in-
duced T-REx-293-hOR17-4 cells. The data were normalized to the response
probability of 20 mm ATP in the same experiment. The means were calculat-
ed from 3 to 11 independent experiments (each with 160–900 cells) for each
tested concentration; error bars indicate the standard error of the mean
(SEM).
Functional characterization of hOR17-4
We analyzed the responsiveness of recombinant hOR17-4 to
rac-1a–d and 2a–d in detail by ratiofluorometric Ca2+ imaging
measurements of induced T-REx-293-hOR17-4 cells. Application
of activating odorants led to a robust transient increase in cy-
tosolic Ca2+ concentration owing to hOR17-4 activation and
signaling (Figure 1). As an indirect measure of the receptor’s
responsiveness to an odorant, we quantified the response
probability in respect to ATP (positive control) and established
dose–response relationships for rac-1a–d and 2a–d. No tested
compound elicited Ca2+ signals in control cells (here T-REx-293
cells) that were higher than background cellular activity (Fig-
ure S4 in the Supporting Information). The maximal tested
odorant concentration did not exceed 1 mm, because higher
concentrations induced nonspecific cellular activation. In Ca2+
imaging analysis of heterologously expressed hOR17-4, com-
pounds rac-1a, 2a, and 2b activated the receptor. Coapplica-
tion of the hOR17-4 blocker undecanal[11] inhibited odorant-
evoked Ca2+ responses of induced T-REx-293-hOR17-4 cells
(Figure S5 in the Supporting Information); this indicated that
the Ca2+ signals depended on hOR17-4 activation. Compound
2a showed the highest activation potency on recombinant
hOR17-4 (Emax =25% of ATP response), whereas rac-1a and 2b
exhibited lower activation potencies (Emax =12 and 10% of ATP
response, respectively). The EC50 values were calculated to be
in the same range for all three odorants (rac-1a, EC50 =
125 mm; 2a, EC50 =130 mm; 2b, EC50 =200 mm). These results
indicate that the agonists bind with comparable affinities to
the receptor, but differ in their abilities to activate Ca2+ signal-
ing. Notably, it cannot be excluded that the hOR17-4 activation
properties of the single enantiomers of rac-1a–d differ from
those determined for the racemates. Compounds rac-1b, rac-
1c, and rac-1d, as well as 2c and 2d, did not activate hetero-
logously expressed hOR17-4 at sub-millimolar concentrations
(Figure 1). These data only partially overlap with results of
a previous study that compared Ca2+ signal amplitudes of
a few transiently hOR17-4 expressing HEK293 cells that re-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2014, 79, 1747 – 1752 1750
D?rrich, Steffen
