MOLECULAR MECHANISMS OF TRANSDUCTION AND TRANSMITTER RELEASE IN TASTE CELLS

当化学刺激与味觉刺激相互作用时，味觉传导就开始了 味觉感受器细胞顶膜。这种互动最终会导致 由于细胞内钙离子的增加和递质的释放 味觉细胞连接到味觉神经纤维上。在下一个资助期 转导和发射器释放的各个方面都将被研究。这个 前两个具体目标集中在甜味转导上，而第三个目标 检查味觉细胞释放的递质。 目的1：确定Gustducin在甜味转导中的作用。 在上一个资助期，有证据表明糖能激活 CAMP途径，而合成甜味剂激活一条途径，涉及 IP/3和DAG。这两条第二信使路径显然汇聚在 K+电导使味觉细胞去极化。在下一个资助期 建议进行实验以检验化学感官特异性的作用。 G蛋白，Gustducin，在这个过程中。一个转基因小鼠品系将是 从R·马戈尔斯基博士那里获得，其中Gustducin启动子已经 与绿色荧光蛋白(GFP)基因相关联。因此，味觉细胞 表达的Gustducin将是荧光的，并可以被靶向 生理记录。钙离子成像、全细胞记录和人类 心理物理研究将被用来确定味觉蛋白在 甜蜜的转导。 目标2：确定调节甜食最后一步的K+通道 转导。 将使用单通道记录来表征K+通道 对甜蜜刺激的反应是关闭的。具体地说，实验是 建议确定相同的通道是否为糖类和 合成甜味剂，以及通道是否需要磷酸化 结案了。 目的3：研究IP/3在味觉神经递质释放中的作用 细胞。 最近关于泥巴狗味觉细胞变性转导的研究结果表明 IP/3介导的细胞内钙释放可能是 足以触发从味觉细胞释放的递质。在下一个 资助期味觉细胞释放的突触小泡将被成像 直接使用荧光膜染料FM1-43。实验是 建议确定IP/3和Ca/2+在这一过程中的作用。 这些研究将提供重要的新信息，关于 味觉转导中的味觉蛋白及其作用机制 味觉细胞释放的递质。

Taste transduction is initiated when chemical stimuli interact with the apical membrane of taste receptor cells. The interaction ultimately leads to an increase in intracellular calcium and release of transmitter from the taste cell onto gustatory nerve fibers. During the next funding period aspects of both transduction and transmitter release will be examined. The first two specific aims focus on sweet transduction, while the third aim examines transmitter release from taste cells. Aim 1: To determine the role of gustducin in sweet transduction. In the previous funding period evidence was provided that sugars activate the cAMP pathway, while synthetic sweeteners activate a pathway involving IP/3 and DAG. These two second messenger pathways apparently converge on a K+ conductance to depolarize taste cells. In the next funding period experiments are proposed to examine the role of the chemosensory-specific G protein, gustducin, in this process. A transgenic line of mice will be obtained from Dr. R Margolskee in which the gustducin promoter has been linked to the gene for Green Fluorescent Protein (GFP). Thus, taste cells that express gustducin will be fluorescent and can be targeted for physiological recording. Ca2+ imaging, whole cell recording, and human psychophysical studies will be used to determine the role of gustducin in sweet transduction. Aim 2: To identify the K+ channels that mediate the final step in sweet transduction. Single channel recording will be used to characterize the K+ channels that are closed in response to sweet stimuli. Specifically, experiments are proposed to determine whether the same channels are targeted by sugars and synthetic sweeteners, and whether phosphorylation is required for channel closure. Aim 3: To examine the role of IP/3 in neurotransmitter release from taste cells. Recent results on denatonium transduction in mudpuppy taste cells suggests that IP/3-mediate release of calcium from intracellular stores may be sufficient to trigger transmitter release from taste cells. In the next funding period synaptic vesicle release from taste cells will be imaged directly using the fluorescent membrane dye FM1-43. Experiments are proposed to determine the role of IP/3 and Ca/2+ in this process. These studies will provide important new information about the role of gustducin in sweet taste transduction and mechanisms involved in the release of transmitters from taste cells.