Electrophysiological basis of sour taste transduction

项目摘要 拟议实验的广泛目标是确定允许哺乳动物检测基本的关键分子。 味觉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，接收一直是一个深入研究的主题， 识别苦味甜味和鲜味的感受器人们对受体的性质和功能知之甚少 酸，这种味道使我们能够在腐败的食物或柑橘类水果中发现酸。在本提案中，我们将开始 解开这个问题，因为我们测试的贡献，新发现的otopetrin质子通道在 酸性和离子味的传导。这些实验建立在上一次拨款所取得的进展之上 应用程序，其中我们使用细胞，分子和功能方法的组合来确定pH值 III型味觉受体细胞（TRCs）中的敏感离子通道，介导酸味。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组成部分。在过去 在基金资助期间，我们成功地确定了编码质子通道的基因，通过功能筛选 富含III型TRCs的基因。在检测的41个基因中，我们确定了一个，编码跨膜 蛋白Otop 1，其在表达时在爪蟾卵母细胞和HEK-293细胞中诱导质子电流。 有趣的是，Otop 1首先被确定为前庭缺陷（“倾斜”或tlt）小鼠中突变的基因，但其 前庭系统和身体其他部位的功能尚不清楚。在这些新成果的基础上， 我们提出了三个具体的目标，以测试在味觉信号的OTOP通道的作用。第一个目标是检查 Otop 1在舌和腭上皮中的功能分布，使我们能够回答这个问题 Otop 1是否是味觉系统中唯一介导质子流入的离子通道。第二个目标，我们 将测量野生型和Otop 1 KO小鼠对酸的细胞反应，以确定 Otop 1在体外对感觉反应有贡献。在第三个目标中，我们将测量 味觉神经和评估野生型和Otop 1 KO小鼠中酸检测的行为阈值， 确定Otop 1在体内介导对酸味刺激的反应的程度。我们的实验 将使我们能够确定Otop 1是否作为酸味受体发挥作用。我们对味觉机制的研究 转导可允许开发可用于增强食物的适口性的味道调节剂， 减少添加有助于糖尿病发展的甜味剂或有助于糖尿病的盐的需要， 高血压此外，所提出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，这将有助于我们了解它们对不同生物学特性的贡献。 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.