The ultimate goal of future work built on the present proposal is to gain insight into how taste information is coded in taste cells. Activation of many G protein coupled receptors (GPCRs), including taste receptors, initiates a signaling cascade which leads to an increase in intracellular calcium. Calcium mobilization in taste cells in response to various tastants is a sensitive and specific indicator of activation of taste receptors. We will monitor in situ the cellular responses of defined subsets of taste cells to tastants using transgenic mice that selectively express GFP-aequorin under taste specific promoters. GFP-aequorin is a minimally-intrusive bioluminescent Ca++ sensor that is suitable for monitoring calcium mobilization in single cells. It is capable of detecting minute variations in calcium levels due to its very high dynamic range. We will generate transgenic mice expressing GFP-aequorin under control of the Trpm5 promoter so that we may directly measure cell- specific responses to taste stimuli in selected population of taste cells in situ. To validate this technical approach we will first evaluate GFP-aequorin as a monitor of Ca++ responses to bitter tastants in a cell-line. We will use mouse STC-1 cells, a gut-derived cell line that expresses many T2r taste receptors, Ga-gustducin and other taste transduction elements and has been shown to respond to bitter compounds with Ca++ mobilization. In the next phase of this project we will prepare GFP-aequorin constructs that can be expressed specifically in taste cells using TrpmS and T2r5 promoters, and generate a transgenic mouse expressing GFP- aequorin under control of the TrpmS promoter. We will then analyze expression of GFP-aequorin and monitor taste responses to bitter, sweet and umami compounds in isolated or in situ Trpm5+ taste cells from transgenic animals. Results of this initial work will produce important information on taste coding and taste transduction in taste-receptor cells and preliminary data for future R01 grants in which we will generate transgenic lines expressing GFP-aequorin from Tlr3 and T2r5 promoters and to address fundamental questions on selectivity of taste detection in specific subpopulations of taste cells. ? ?
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