PERIPHERAL MECHANISMS OF OLFACTORY RECEPTION

嗅觉感受器神经元的刺激激活是由 气味物质与膜上受体蛋白的可逆相互作用 纤毛从细胞的树突末端伸出。刺激方案-- 受体相互作用触发一系列事件导致细胞膜 动作电位的去极化和放电。现在一般都是 接受G蛋白连接的第二信使参与偶联 随着膜电导的变化，受体被激活。尤其是 已经获得了令人信服的证据，证明至少有一些 嗅觉受体与G蛋白连接的cAMP的形成，进而 直接激活睫状膜上的阳离子通道。近期 生化和电生理证据表明，在鲶鱼中 和大鼠嗅觉感受器细胞，一些气味触发G蛋白偶联 增加肌醇-1，4，5-三磷酸(IP3)，且IP3直接 激活纤毛膜上相对非选择性的钙通道。这个 拟议研究的主要目标是澄清 CAMP和IP3在两种鱼类嗅觉信号转导中的作用 还有那只老鼠。生化、电生理和生物物理方法 将被用来定义连接刺激-受体的分子机制 嗅觉神经元与后续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测量 与第二信使形成动力学相关的时间范围 受体占有率水平。刺激引起的细胞内变化 钙水平将使用Fura-2和视频进行实时监测 显微镜。这些变化将与气味的类型和 第二个信使通路被激活。第二信使调节的变化 用全细胞技术检测完整细胞的膜内电导 记录技术和单声道属性将在 睫状膜碎片形成的切除的斑块和平面双层 已经被合并了。这些技术在鲶鱼养殖中的应用 模型系统，其中刺激-感受器相互作用被很好地描述， 将全面描述嗅觉的机制 在该物种中的信号转导，以及研究的基础 哺乳动物感受器神经元。

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.