PERIPHERAL MECHANISMS OF OLFACTORY RECEPTION

嗅觉接收的外围机制

• 批准号: 2125810
• 负责人: JOHN H TEETER
• 金额: $ 18.61万
• 依托单位: MONELL CHEMICAL SENSES CENTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-12 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2125810
• 关键词: G protein; action potentials; alternatives to animals in research; biological signal transduction; calcium flux; calcium indicator; catfish; chemoreceptors; cilium; cyclic AMP; dendrites; electrophysiology; inositol phosphates; laboratory rat; membrane channels; membrane potentials; microscopy; neuropharmacology; olfactions; olfactory stimulus; phosphorylation; receptor binding; receptor coupling; second messengers; sensory mechanism; soma; stop flow technique; ultrafiltration; video microscopy; video recording system; voltage /patch clamp

Stimulus activation of olfactory receptor neurons is initiated by reversible interactions of odorants with receptor proteins in the membranes of cilia projecting from the dendritic ends of the cells. Stimulus- receptor interaction triggers a sequence of events leading to membrane depolarization and discharge of action potentials. It is now generally accepted that G-protein-linked second messengers are involved in coupling receptor activation with changes in membrane conductance. Particularly convincing evidence has been obtained for coupling of at least some olfactory receptors to G-protein-linked formation of cAMP, which in turn directly activates cation channels in ciliary membranes. Recent biochemical and electrophysiological evidence indicates that, in catfish and rat olfactory receptor cells, some odorants trigger a G-protein-coupled increase in inositol-1,4,5-trisphosphate (IP3), and that IP3 directly activates relatively nonselective calcium channels in cilia membranes. The major goal of the proposed research is to clarify the relative roles of cAMP and IP3 in olfactory signal transduction in two species, the catfish and the rat. Biochemical, electrophysiological and biophysical methods will be used to define the molecular mechanisms that link stimulus-receptor interaction with subsequent ionic events in olfactory neurons. Radioligand binding and second messenger formation will be measured in the millisecond time range to correlate the kinetics of second messenger formation with levels of receptor occupancy. Stimulus-induced changes in intracellular calcium levels will be monitored in real time using fura-2 and video microscopy. These changes will be correlated with the type of odorant and the second messenger pathway activated. Second messenger-regulated changes in membrane conductance will be examined in intact cells using whole-cell recording techniques and single-channel properties will be studied in excised patches and planar bilayers into which ciliary membrane fragments have been incorporated. Application of these techniques to the catfish model system, in which stimulus-receptor interaction is well characterized, will provide a comprehensive description of the mechanisms of olfactory signal transduction in this species, as well as the basis for studies in mammalian receptor neurons.

嗅觉受体神经元的刺激激活是由 气味剂与膜中受体蛋白的可逆相互作用 从细胞的树突末端突出的纤毛。 刺激- 受体相互作用触发一系列事件，导致膜 动作电位的去极化和放电。 现在一般是 承认 G 蛋白连接的第二信使参与偶联 受体激活伴随膜电导的变化。 特别 已经获得了至少一些耦合的令人信服的证据 嗅觉受体与 G 蛋白相关的 cAMP 形成，反过来 直接激活睫状膜中的阳离子通道。 最近的 生化和电生理学证据表明，在鲶鱼中 和大鼠嗅觉受体细胞中，一些气味剂会触发 G 蛋白偶联 肌醇-1,4,5-三磷酸 (IP3) 增加，并且 IP3 直接 激活纤毛膜中相对非选择性的钙通道。 这 拟议研究的主要目标是阐明以下各项的相对作用： cAMP 和 IP3 在鲶鱼这两个物种的嗅觉信号转导中的作用 和老鼠。 生物化学、电生理学和生物物理方法 将用于定义连接刺激-受体的分子机制 与嗅觉神经元中随后的离子事件的相互作用。 放射性配体 结合和第二信使的形成将以毫秒为单位进行测量 将第二信使形成动力学与相关的时间范围 受体占用水平。 刺激引起的细胞内变化 将使用 fura-2 和视频实时监测钙水平 显微镜。 这些变化将与气味剂的类型和 第二信使通路被激活。 第二信使调节的变化 将使用全细胞在完整细胞中检查膜电导 记录技术和单通道特性将在 切除的斑块和平面双层，其中睫状膜碎片 已被纳入。 这些技术在鲶鱼中的应用 模型系统，其中刺激-受体相互作用得到了很好的表征， 将提供嗅觉机制的全面描述 该物种的信号转导，以及研究的基础 哺乳动物受体神经元。