Bringing light to taste coding

描述(由申请人提供)：在本提案的基础上，未来工作的最终目标是深入了解味觉细胞中味觉信息是如何编码的。许多G蛋白偶联受体(GPCRs)的激活，包括味觉受体，启动了一系列信号级联，导致细胞内钙离子的增加。味觉细胞对各种味觉反应的钙动员是味觉感受器激活的一个敏感而特异的指标。我们将使用在味觉特异性启动子下选择性表达GFP-aequorin的转基因小鼠，原位监测特定味觉细胞亚群对味觉的细胞反应。GFP-Aequorin是一种侵入性最小的生物发光钙离子传感器，适用于监测单细胞中的钙动员。由于其非常高的动态范围，它能够检测到钙水平的微小变化。我们将在Trpm5启动子的控制下产生表达GFP-aequorin的转基因小鼠，这样我们就可以直接在选定的味觉细胞群中原位测量细胞对味觉刺激的特异性反应。为了验证这一技术方法，我们将首先评估GFP-aequorin作为细胞系中钙离子对苦味反应的监测器。我们将使用小鼠STC-1细胞，这是一种肠道来源的细胞系，表达许多T2R味觉受体、GA-Gustducin和其他味觉转导元件，并已被证明通过钙动员对苦味化合物做出反应。在这个项目的下一阶段，我们将利用TrpmS和T2r5启动子制备能够在味觉细胞中特异性表达的GFP-aequorin载体，并在TrpmS启动子的控制下产生表达GFP-aequorin的转基因小鼠。然后，我们将分析GFP-aequorin的表达，并监测转基因动物分离或原位Trpm5+味觉细胞对苦味、甜味和鲜味化合物的味觉反应。这项初步工作的结果将为味觉感受器细胞的味觉编码和味觉转导提供重要的信息，并为未来的R01资助提供初步数据，我们将从TLR3和T2r5启动子获得表达GFP-aequorin的转基因系，并解决在特定味觉细胞亚群中味觉检测的选择性的基本问题。

DESCRIPTION (provided by applicant): 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.