Mechanisms of sweet transduction in mammalian taste buds

描述（由申请人提供）：甜味转导始于甜味化合物与味觉细胞上膜受体的结合，随后是G蛋白的激活和第二信使水平的变化。第二信使阻断K+通道，使味觉细胞去极化，增加细胞内Ca2+，最终激活味觉传入神经元。尽管最近人们对甜味受体的分子特性有了很多了解，但对连接受体和效应通道的下游事件知之甚少。我们提出的实验目标是阐明形成这种联系的分子级联。先前关于甜味转导的研究已经检测了舌味蕾，在啮齿动物中舌味蕾对甜味相对不敏感。相反，我们的建议集中在软腭的味蕾上，这是啮齿动物对甜味最敏感的味觉区域。环状AMP、cGMP和IP3被认为是甜转导的第二信使，但没有一项研究在任何一个领域检查了这三种信使，也没有评估G蛋白和效应酶在这些调节中的作用。我们提出的证据表明，软腭的甜味转导部分是由G蛋白介导的。我们将利用在gustducin谱系细胞中表达绿色荧光蛋白（GFP）的转基因小鼠来选择味觉细胞进行电生理记录。形态学标记在野生型（Gus-GFP）和gustducin敲除（gus-null-GFP）小鼠中均可用。记录gfp标记和未标记的腭味觉细胞以及第二信使的生化测量将确定α -味觉传导素在甜味转导中的作用，并揭示味觉传导是否存在不依赖于味觉传导素的机制。结合分子、生化和电生理技术，我们将(1)鉴定由蔗糖和合成甜味剂激活的G蛋白。(2)明确了甜敏感味觉细胞中腺苷环壳体的同工型；(3)确定了第二信使调节甜敏感传导的机制。这些研究结果将为甜味转导的下游信号机制提供重要信息。

DESCRIPTION (provided by applicant): Sweet taste transduction begins with the binding of sweet tasting compounds to membrane receptors on taste bus cells, followed by activation of G proteins and change in the levels of 2nd messengers. The 2nd messengers block a K+ channel to depolarize taste cells, increase intracellular Ca2+, and eventually, activate gustatory afferent neurons. Although much has been learned recently about the molecular identities of sweet taste receptors, comparatively little is know about the downstream events linking the receptors to the effector ion channels. Our goal for the proposed experiments is to elucidate the molecular cascade forming this link. Previous studies on sweet taste transduction have examined lingual taste buds, which are relatively insensitive to sweet in rodents. Our proposal focuses instead on that buds of the soft palate, which, in rodents is most sweet-sensitive taste field. Cyclic AMP cGMP and IP3 have been implicated as 2nd messengers in sweet transduction, but no single study has examined all three messengers in any one field, nor have the roles of the G proteins and effector enzymes involved in these modulation been assessed. We Present evidence that sweet transduction in the soft palate is mediated in part by the G protein, Galpha-gustducin. We will utilize transgenic mice expressing green fluorescent protein (GFP) in gustducin-lineage cells to select taste cells for electrophysiological recordings. The morphological marker is available in both wild type (Gus-GFP) and gustducin knockout ( gus-null-GFP) mice. Recording form GFP-labeled and unlabeled palatal taste cells and biochemical measurements of 2nd messengers will define the roles of alpha-gustducin in sweet transduction and will reveal whether gustducin-independent mechanisms also exist for sweet transduction. Using a combination of molecular, biochemical, and electrophysiological techniques, we will (1) identiy the G proteins activated by sucrose and synthetic sweeterners. (2) defne the isoforms of adenyly cylcase present in sweet-sensitive taste cells, and (3) determine the mechanisms by which second messengers modulate the sweet-sensitive conductance. Results from these studies will provide important information about downstream signaling mechanisms in sweet taste transduction.