Electrophysiological basis of sour taste transduction

项目摘要 拟议中的实验的主要目标是识别使哺乳动物能够检测到基本的 并产生传导到大脑区域的电反应。味觉的分子机制 在过去的30年里，接待一直是一个密切调查的主题，在 识别苦味、甜味和鲜味的受体。对受体的性质和功能知之甚少 对于酸，这是一种味道，可以让我们在变质的食物或柑橘类水果中检测到酸。在这项提案中，我们将开始 随着我们测试新发现的奥托品质子通道在 酸味和离子味的转换。这些实验是在上次拨款取得进展的基础上进行的。 应用程序，我们使用细胞、分子和功能方法的组合来确定pH III型味觉感受器细胞(TRCs)中调节酸味的敏感离子通道。值得注意的是，我们描述了一个 新的质子选择性离子电流可能是酸味转导的关键成分。在最后一次 在资助期内，我们通过功能筛选成功地鉴定了编码质子通道的基因 富含III型TRCs的基因。在测试的41个基因中，我们确定了一个编码跨膜的基因。 Otop1蛋白，表达后在非洲爪哇卵母细胞和HEK-293细胞中都诱导出质子电流。 有趣的是，Otop1基因最初被鉴定为前庭缺陷(倾斜或TLT)小鼠的基因突变，但其 前庭系统和身体其他部位的功能尚不清楚。在这些新结果的基础上， 我们提出了三个具体目标来测试OTOP通道在味觉信号中的作用。第一个目标是检查 Otop1在舌和腭部上皮中的功能分布，使我们能够回答这个问题 Otop1是否是调节味觉系统中质子流入的唯一离子通道。在第二个目标中，我们 将测量野生型和Otop1 KO小鼠对酸的细胞反应，以确定 在体外，Otop1对感官反应有贡献。在第三个目标中，我们将衡量以下方面的反应 在野生型和Otop1 KO小鼠中评估味觉神经和酸检测的行为阈值 确定Otop1在体内调节对酸味刺激的反应的程度。一起做我们的实验 将使我们能够确定Otop1是否具有酸味感受器的功能。我们为确定味觉机制所做的努力 转导可允许开发可用于提高食物适口性的味道调节剂， 减少添加导致糖尿病的甜味剂或导致糖尿病的盐的需要 高血压。此外，拟议的实验将提供有关功能的基本信息 这一新的质子通道家族的性质将帮助我们理解它们对不同 生理过程，包括棕色脂肪代谢和前庭的发育和维持 系统。

Project Summary The broad goal of the proposed experiments is to identify key molecules that allow mammals to detect basic tastes and generate electrical responses that are conducted to brain regions. Molecular mechanisms of taste reception have been a subject of intense investigation over the last 30 years, with great strides made in identifying receptors for bitter, sweet and umami. Much less is known about the nature and function of receptors for sour, the taste that allows us to detect acids in spoiled foods or citrus fruits. In this proposal, we will begin to unravel this problem as we test the contribution of the newly discovered otopetrin proton channels in the transduction of acidic and ionic tastes. These experiments build on the progress made in the last grant application, where we used a combination of cellular, molecular and functional approaches to identify the pH sensitive ion channels in Type III taste receptor cells (TRCs) that mediate sour taste. Notably, we described a novel proton-selective ionic current that is likely to be a key component of sour taste transduction. In the last funding period, we successfully identified the gene that encodes the proton channel, through functional screening of genes enriched in Type III TRCs. Among 41 genes tested, we identified one, encoding the transmembrane protein Otop1 that upon expression induced a proton current in both Xenopus oocytes and HEK-293 cells. Interestingly, Otop1 was first identified as a gene mutated in mice with vestibular defects (“tilted” or tlt) but its function in the vestibular system and elsewhere in the body was not understood. Building on these new results, we propose three specific aims to test the role of the Otop channels in taste signaling. The first aim will examine the functional distribution of Otop1 across the tongue and palate epithelium, allowing us to answer the question of whether Otop1 is the sole ion channel mediating proton influx in the gustatory system. In the second aim, we will measure cellular responses to acids in wildtype and Otop1 KO mice in order to determine the degree to which Otop1 contributes to sensory responses, ex vivo. In the third aim, we will measure responses from gustatory nerves and assess behavioral thresholds for acid detection in wildtype and Otop1 KO mice to determine the extent to which Otop1 mediates responses to sour taste stimuli in vivo. Together our experiments will allow us to determine if Otop1 functions as a sour taste receptor. Our efforts to identify mechanisms of taste transduction may allow the development of taste modifiers that can be used to enhance palatability of food, reducing the need to add sweeteners that contribute to the development of diabetes or salts that contribute to hypertension. Moreover, the proposed experiments will provide basic information regarding the functional properties of this new family of proton channels that will help us understand their contributions to diverse physiological processes, including brown fat metabolism and the development and maintenance of the vestibular system.