PERIPHERAL MECHANISMS OF OLFACTORY RECEPTION

嗅觉受体神经元的刺激激活是由 气味物质与膜中受体蛋白的可逆相互作用 从细胞树突末端伸出的纤毛。 刺激- 受体相互作用引发一系列事件，导致膜 动作电位的去极化和放电。 现在人们普遍 接受G蛋白连接的第二信使参与偶联 受体激活与膜电导的变化。 特别 已经获得了令人信服的证据，至少有一些耦合 嗅觉受体与G蛋白连接的cAMP形成， 直接激活睫状膜中的阳离子通道。 最近 生物化学和电生理学证据表明，在鲶鱼中， 和大鼠嗅觉受体细胞，一些气味触发G蛋白偶联 肌醇-1，4，5-三磷酸（IP3）增加，IP3直接 激活纤毛膜中相对非选择性的钙通道。 的 拟议研究的主要目标是澄清的相对作用， cAMP和IP3在两种鲶鱼嗅觉信号转导中的作用 还有老鼠 生物化学、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 与嗅觉神经元中随后的离子事件的相互作用。 放射配 结合和第二信使的形成将在毫秒内测量 将第二信使形成的动力学与 受体占有水平。 刺激诱导的细胞内 将使用Fura-2和视频对钙水平进行真实的实时监测 显微镜 这些变化将与气味剂的类型相关， 第二信使通路被激活 第二次信使调节变化 在完整细胞中，将使用全细胞 记录技术和单通道特性将在 切除的贴片和平面双层，其中睫状膜碎片 已被纳入。 将这些技术应用于鲶鱼 模型系统，其中刺激-受体相互作用被很好地表征， 将提供一个全面的描述机制的嗅觉 信号转导在这个物种，以及研究的基础， 哺乳动物受体神经元。

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.