Mechanisms of sweet transduction in mammalian taste buds

描述(申请人提供)：甜味转导始于甜味化合物与味觉细胞膜上受体的结合，随后G蛋白被激活，第二信使水平发生变化。第二信使阻断K+通道，使味觉细胞去极化，增加细胞内钙离子，最终激活味觉传入神经元。虽然最近关于甜味受体的分子特性已经了解很多，但对于将受体连接到效应器离子通道的下游事件知之甚少。我们提议的实验的目标是阐明形成这种联系的分子级联。以前关于甜味转导的研究已经研究了啮齿类动物的舌味蕾，它对甜味相对不敏感。相反，我们的建议关注的是软腭的芽，这是啮齿动物对甜味最敏感的味觉区域。环状AMP、cGMP和IP3被认为是甜味信号转导中的第二信使，但没有一项研究考察这三种信使在任何一个领域中的作用，也没有评估参与这些调节的G蛋白和效应酶的作用。我们提出的证据表明，软腭中的甜味转导部分是由G蛋白Galpha-Gustducin介导的。我们将利用在味觉蛋白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。这种形态标记在野生型(GUS-GFP)和Gustducin基因敲除(GUS-Null-GFP)小鼠中都可用。记录形式、GFP标记和未标记的腭部味觉细胞和第二信使的生化测量将确定α-Gustducin在甜味转导中的作用，并将揭示甜味转导是否也存在味觉非依赖性机制。结合分子、生化和电生理技术，我们将(1)鉴定被蔗糖和合成甜味剂激活的G蛋白。(2)确定甜味细胞中存在的腺样环状病毒亚型，以及(3)确定第二信使调节甜味敏感电导的机制。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.