Mechanisms of sweet transduction in mammalian taste buds

描述（由申请人提供）：甜味转导开始于甜味化合物与味觉总线细胞上的膜受体结合，随后是G蛋白的活化和第二信使水平的变化。第二信使阻断K+通道以使味觉细胞去兴奋，增加细胞内Ca 2+，并最终激活味觉传入神经元。 虽然最近已经了解了很多关于甜味受体的分子特性，但对连接受体与效应离子通道的下游事件知之甚少。 我们提出的实验的目标是阐明形成这种联系的分子级联。 以往关于甜味传导的研究主要针对啮齿类动物对甜味相对不敏感的舌味蕾。相反，我们的建议集中在软腭的芽上，在啮齿动物中，软腭是最敏感的味觉区域。 环AMP、cGMP和IP 3被认为是甜味传导中的第二信使，但没有一项研究在任何一个领域检查了所有三种信使，也没有评估G蛋白和效应酶在这些调节中的作用。 我们目前的证据表明，甜的软腭转导介导的部分G蛋白，Galpha-gustducin。 我们将利用转基因小鼠表达绿色荧光蛋白（GFP）的味蛋白谱系细胞选择味觉细胞的电生理记录。 形态学标记在野生型（Gus-GFP）和gustducin敲除（gus-null-GFP）小鼠中均可用。 记录形式GFP标记和未标记的腭味觉细胞和第二信使的生化测量将定义α-gustducin在甜味转导中的作用，并将揭示甜味转导是否也存在不依赖于gustducin的机制。 我们将结合分子、生物化学和电生理技术，（1）鉴定蔗糖和合成甜味剂激活的G蛋白。(2)确定存在于甜味敏感味觉细胞中的腺苷酸环化酶的亚型;（3）确定第二信使调节甜味敏感传导的机制。 这些研究结果将为甜味信号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.