2 In vitro olfactory receptor assay
2.4 Results
2.4.3 dORs expressed in HEK293T cells are functional…
The results above indicated that the HEK cells express and properly traffic dORs in case of co-expression of the co-receptor Orco. In order to test whether these proteins are functional we measured odor-induced calcium changes in transfected cells.
Cells were imaged for 250 s at 0.2 Hz (5 s interval, total no. of frames is 50; stimulus protocol is shown in Fig 2.4A). First ten frames were imaged prior to addition of odorant or solvent (quantified as basal fluorescence intensity - Bo). Bo varied a lot within the experiments (Fig 2.4B, C, E, F). A possible reason for such variations is the difference in the level of uptake of the dye by the cells or the general heterogeneity of cell culture (different cell cycle phases, chromosomal aberrations, etc.). After odor addition cells were imaged for 150 s (odor is present throughout). In average, more than 30% of cells responded to odor by increasing the levels of calcium in them above the threshold value of 1.5 (see Materials and Methods). Relative increase in fluorescence intensity was individual for each cell and reached up to 8 times of basal (Fig 2.4B, E).
Most of the responder cells showed increased calcium levels within 5–15 s after odor addition (frames 11–13; examples traces are shown in Fig 2.4C). In general the maximum calcium increase mediated by odors peaked at about 30 s after odor addition (16th frame; Fig 2.4D) and then the response decreased slowly and in most cases it reached their basal fluorescence level. Though the cells were exposed to odorants for 150 s the responses were transient (as it was shown for other receptors (Smart et al., 2008)). Addition of ionomycin elicited the calcium increase in almost all of the cells regardless of they have previously responded or not to the odor (Fig 2.4B, C, F).
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Figure 2.4. Calcium imaging of HEK293T cells expressing dORs.
(A) Schematic representation of the stimulation protocol. (B) False color coded picture of HEK293T cells transfected with dOr22a and Orco before (basal: left image) and after addition of 10 mM EtBE (middle image) and ionomycin (right image). (C) Calcium response of selected cells (shown as mean fluorescence intensity) over the time-course of the assay (as shown in panel A). Dark-red, green, blue and cyan colored traces indicate responder cells and black-colored trace indicates nonresponder. (D) Violin plot (combination of boxplot (red-colored) and kernel density distribution plot; grey color indicates the probability density of the data) depicts the time at which the peak calcium response is obtained after odor addition (10-2 M EtBE; nonresponder cells are excluded) to HEK293T cells transfected with dOr22a and Orco. n = 2089 cells. (E) Plot of odor response (Ro/Bo, Y-axis) against basal fluorescence (Bo, X-axis) of HEK293T cells transfected with dOr22a and Orco. Red circles and gray triangles indicate responder and non responder group of cells, respectively. Color coding is maintained in panel E and F (F) Plot of ionomycin response (Ri/Bi, Y-axis) against basal fluorescence (Bi, X-axis) of HEK293T cells transfected with dORs. n = 6,663 cells, from 10–50 different experiments of 4–20 independent transfections for panel E and F.
Addition of ethyl butyrate (EtBE) or of 2,3-butanediol (BDOL) to the final concentration of 10-2 or 10-4 M elicited calcium response in about 30% of cells transfected with dOr22a and Orco (Fig 2.5A) or dOr92a and Orco respectively (Fig 2.5B). In contrast, upon addition of the assay buffer or 0.1% DMSO (solvent) the calcium response was observed in about 0.6 – 1.7% of cells which is negligible when compared to odor response (Fig 2.5A, B). Further, addition of odorants or solvent to control cells (mock transfected by pCDNA3.1(+)) elicited insignificant calcium response varying between 1 – 2.5% and 0.4 – 1.9% respectively (Fig 2.5A, B). These results indicate that heterologous expression of dORs is functional and that the expressed dORs
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respond to well-described ligands as reported by the in vivo studies (de Bruyne et al., 2001; Hallem et al., 2004).
Figure 2.5. Calcium response of HEK293 cells upon odor addition.
(A, B) Barplot shows the percentage of cells responded to odor (10-2 M EtBE or 10-4 M BDOL) or solvent (assay buffer or 0.1% DMSO). Green and red color indicates control (mock-transfected by pCDNA3.1(+)) and transfected (dOr22a and Orco or dOr92a and Orco) group of cells, respectively.
Asterisks indicate statistical significance (chi-squared test; p-value < 2.2e-16). n = 10–50 different experiments of 4–20 independent transfections.
As a next step we tested the sensitivity of the receptors by measuring dose-dependent odor responses. Cells transfected by combinations of dOr22a and Orco or dOr92a and Orco were tested with concentrations of EtBE or BDOL ranging from 10-2 to 10-7 M or 10-3 to 10-9 M respectively. Mock-transfected cells elicited statistically insignificant calcium response to all concentrations of EtBE or BDOL tested, whereas dOR-transfected (dOr22a and Orco or dOr92a and Orco) cells elicited significant responses to most of the concentrations tested (Fig. 2.6A, B). Mock-transfected cells elicited negligible responses to the solvent (assay buffer or 0.1% DMSO), while the ORs transfected cells (dOr92a and Orco) elicited a slightly but significantly higher response to 0.1% DMSO than mock-transfected cells (Fig. 2.6B); the reason for this difference is unknown. We speculate that that DMSO may interact with dOr92a or Orco or both that leads to a slight calcium increase in these cells. dOr22a and Orco or dOr92a and Orco cells elicited highest response to 10-2 M or 10-4 M dilution respectively. dOr22a and Orco cells elicited higher response than control to 10-2 to 10-4 M dilution, but the response was statistically significant for 10-2 dilution alone (Fig. 2.6A). dOr92a and Orco transfected cells gave statistically significant response to 10-3 to 10-9 M dilution when compared to
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odor control, whereas 10-3 to 10-6 Mdilution alone gave statistically significant response when compared to solvent control (Fig. 2.6B; asterisks indicate the comparisons between odor control).
Figure 2.6. Odor responses are concentration dependent.
(A) Violin plot of calcium response (log (odor response) = log (Ro/Bo)) of control (green colored boxplots) and ORs transfected (dOr22a and Orco; red colored boxplots) cells to solvent (assay buffer) or to varied concentrations of the odorant (EtBE). (B) Similar to panel A but the cells were transfected with dOr92a and Orco (red colored boxplots) and the odor tested is BDOL and the solvent used is 0.1%
DMSO. Asterisks indicates statistical significance between the odor control and test group for a particular
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concentration, two way ANOVA (concentration and plasmid are used as factors; significance codes; ‘***’:
p<0.001). n = 350-6,663 cells from 8–50 different experiments of 4–20 independent transfections.
2.4.4 Addition of increasing odor concentrations elicited significant responses