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Tesi etd-04142015-112841

Thesis type
Tesi di laurea magistrale
Cloning TRPC1 to enhance Calcium signal in a model of bitter taste transduction.
Corso di studi
relatore Dal Monte, Massimo
relatore Marchiori, Alessandro
Parole chiave
  • TRPC1
  • TAS2R
  • taste transduction
  • Calcium signal
Data inizio appello
Riassunto analitico
The sense of taste is used to evaluate the quality of food. In particular, animals detect at least five basic tastes: sweet, bitter, salty, umami and sour. Each one is associated to a given food’s property, for instance bitter taste has been thought to be necessary to detect toxins.
Bitter taste transduction starts with the binding of bitter tastant with one or more bitter taste receptors (TAS2Rs). They are G protein-coupled receptors (GPCRs) and the binding with tastants leads to the activation of G protein α-gustducin. In particular, βγ subunits activate phospholipase PLCβ2. This event causes the production of inositol 1,4,5-thriphosphate (IP3) and release of Ca2+ from internal stores. The elevation of Ca2+ activates transient receptor potential channel M5 (TRPM5) and this allows the depolarization of the cell.
The elevation of [Ca2+]i is the studied event to understand the behavior of taste receptors in presence of different tastants. Indeed, although some receptors give a strong signal with one tastant, the same receptors could give a low signal with other molecules, or some receptors may give low signal per se.
Unfortunately TRPM5 does not allow Ca2+ entry and it could not be used in our experiments. Hence, since the mechanism of activation by elevation of intracellular Ca2+ is common between TRPM5 and TRPC1, another TRP channel, and since the latter is permeable also for Ca2+ , further increasing the [Ca2+]i , we chose to clone TRPC1 in a cellular model of bitter taste transduction, trying to enhance the calcium signal.
We started from an extract of fetal human brain to isolate the cDNA of TRPC1, then we included the cDNA in an expression vector and transiently transfected HEK293 cells, with both TRPC1 and a TAS2R. These cells already stably express a chimeric G-protein α subunit G16gust44, involved in the signal pathway of taste. Thanks to the fluorescence given from Fluo-4 when it binds Ca2+, we studied the Ca2+ signals obtaining different profiles in presence or not of TRPC1. For example, in cells expressing TRPC1 and TAS2R43 or TAS2R14 the signal was higher than in cells transfected with TAS2R and mock (plasmid without any insert, used as negative control). On the contrary, with TAS2R10 we had the opposite result, with a higher signal in cells expressing only the receptor.
Improving Ca2+ signal could be possible to deorphanize receptors whose tastants are still not known just because of low signal, or to find other molecules that activate a given TAS2R and so extent the range of activators of that receptor. Moreover, it could be possible to study inhibitors in those receptors that have strong signal with a tastant and low signal with other(s) substance(s).