Taste perception - Signal transduction and assaying the potency of tastants
13 important questions on Taste perception - Signal transduction and assaying the potency of tastants
How does the bitter signal transduction go for a T2R and for T1R?
- When the bitter compound is recognised by the GPCR, the receptor adopts a different conformation and adopts the so-called G-protein
- The beta gamma subunit is enzymically cleaved from the alpha subunit
- The beta gamma subunits activate a phospholipase, resulting in the production of inositol 1,4,5-triphosphate (IP3), which triggers influx of Ca2+ into the cell (depolarisation of taste cell), followed by neurotransmitter release
- The neurotransmitters are transported to the brain, ultimately leading to taste perception
T1R similar, but the tasting binds into the VFTD of the receptor
What are the drawbacks of the use of human taste panels?
- Bitterness is disliked by Westerners
- Expensive
- Require training of panelists
- Time-consuming
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Are cell-based assays more advantageous than human based assays?
How does a cell-based assay work?
- A yeast cell or human embryonic kidneys (HEK) cells (host cells) are genetically modified that the taste receptors are accumulated at their membranes
- Bitter: 1 receptor gene
- Sweet: T1R2/T1R3
- Umami: T1R1/T1R3
- Upon stimulation by a tasting, the intracellular Ca2+ increases, which can be visualised with a fluorescent dye, e.g. Fluo-4. By itself, this dye is not fluorescent, but when it binds to Ca2+ it is.
- The fluorescence signal is measured and is linearly related to the Ca2+
How is the potency of molecules tested?
What dose the plateau in signal intensity mean?
Which 2 values are representative for the potency of the tastant?
- Threshold
- EC50
What is the EC50 value?
How is EC50 and potency of molecules related?
The EC50 is the preferred manner to express the potency of tastants. However, in some cases one has to rely on the determination of the threshold value for 2 reasons.
- If the water solubility of the tastant is low (the assays are always performed in aqueous environment), one cannot dissolve sufficiently high concentrations to measure the plateau value, and hence the EC50 cannot be extrapolated
- The tastants might be toxic to the test or HEK cells, and consequently the cells can die at higher concentrations.
What are mock transfected cells?
- They are applied as negative control. They do not contain the introduced genes for bitter, sweet or umami receptors, but express only the receptors which are naturally present in the host cell (endogenous receptors).
- In principle, these endogenous receptors might also react to the tastant, resulting in a fluorescent response. By testing the negative control, one can ensure that the fluorescent increase is only caused by activation of the taste receptors and not by activation of one of the endogenous receptors.
Why is a negative control used (mock-transfected cells)?
- To decrease the change of false-positive results
- One can ensure that the fluorescent increase is only caused by activation of the taste receptor and not by activation of one of the endogenous receptors
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