Cyclic AMP- and Ca2+-Signaling in Sensory Transduction by Olfactory Receptor Neurons

这一建议的长期目标是定量详细地了解嗅觉感受器神经元(ON)在感觉转导中cAMP和Ca~(2+)信号。我们将重点放在规范 脊椎动物主要嗅觉上皮的嗅觉传导机制。此机制涉及一个 CAMP信号级联，导致Na+和Ca~(2+)通过环核苷酸门控(CNG)的非选择性阳离子通道内流，使ORN去极化到激发阈值。钙离子内流导致信号放大 通过钙离子激活的内向氯离子电流，以及通过多条钙离子激活的负反馈通路进行嗅觉适应。然而，近年来，负反馈通路的意义和表现 都陷入了怀疑和困惑之中。在目标1中，我们建议重新审查这个问题，并一次解决它。 也是为了所有人。 最近，通过使用M71-单克隆鼻小鼠Orns，我们成功地量化了 嗅纤毛膜上M71-OR分子的密度。我们还发现，当连接M71-OR时， 对于苯乙酮(M71-OR最有效的气味之一)，尽管如此，仍有很低的 激活单个下游Golf/腺苷环化酶III效应器的成功概率(名义上为~10-4) 很复杂。这种低概率与杆状光转导的情况非常不同，关于这种情况，我们 最近发现，一个光激发的视紫红质激活10-20个杆状转导蛋白/cGMP-磷酸二酯酶 效应器。因此，配体激活的GPCR途径与光激活的GPCR途径在信号放大方面可能有很大不同。在AIMS 2中，我们计划研究另一种小鼠鼻化学感受器TAAR4，它 对2-苯乙胺非常敏感，2-苯乙胺是一种讨厌老鼠的捕食者臭味剂。我们的目标是 将结果与M71-OR的结果进行比较，以确定纤毛上的TAAR4‘S分子密度 表面膜与M71-OR有很大的不同，要问扩增是否在 下游G蛋白/效应酶复合体的激活步骤与M71-OR的情况有任何不同。在……里面 目标3，作为另一个比较，我们将解决MOR256-17的相同问题，与以下其中之一进行OR 迄今已知在主嗅觉上皮细胞中含量最高，并具有异常广泛的气味 频谱。 定量阐明嗅觉转导的步骤将为了解正常 嗅觉功能，以及由于转导途径中的遗传缺陷引起的功能障碍 在视觉传导方面取得的巨大成功证明了这一点。

The long-term objective of this proposal is to understand in quantitative detail the cAMP- and Ca2+-signalings in sensory transduction by olfactory receptor neurons (ORNs). We shall focus on the canonical olfactory-transduction mechanism in the vertebrate main olfactory epithelium. This mechanism involves a cAMP-signaling cascade, leading to Na+ and Ca2+ influxes through a cyclic-nucleotide-gated (CNG), non-selective cation channel to depolarize the ORN to firing threshold. The Ca2+ influx leads to signal amplification via an inward Ca2+-activated Cl current, as well as olfactory adaptation via multiple Ca2+-activated negative-feedback pathways. Recently, however, the significance and performance of the negative-feedback pathways are thrown into doubt and confusion. In Aim 1, we propose to re-examine this question and to settle it once and for all. Most recently, by using M71-monoclonal-nose mouse ORNs, we have succeeded in quantifying the density of M71-OR molecules on the olfactory cilia membrane. We also found that an M71-OR, when liganded with acetophenone (among the most efficacious odorants for M71-OR), nonetheless still has a very low probability of success (nominally ~10-4) in activating a single downstream Golf/adenylyl cyclase III effector complex. This low probability is very different from the situation in rod phototransduction, about which we recently showed that one photoexcited rhodopsin activates 10-20 rod transducin/cGMP-phosphodiesterase effectors. Thus, a ligand-activated GPCR pathway may be quite different in signal amplification from a light-activated GPCR pathway. In Aims 2, we propose to study another mouse nasal chemoreceptor, TAAR4, which is exceedingly sensitive to 2-phenylethylamine, a predator odorant aversive to mouse. The objective is to compare the findings with those from M71-OR, to figure out whether TAAR4's molecular density on the cilia's surface membrane is very different from that of M71-OR, and to ask whether the amplification at the downstream G-protein/effector enzyme complex activation step is any different from the case of M71-OR. In Aim 3, as another comparison, we shall address the same questions for mOR256-17, an OR with one of the highest abundances known so far in the main olfactory epithelium and with an unusually broad odorant spectrum. Quantitatively elucidating the steps of olfactory transduction will provide great insight into normal olfactory functions, as well as malfunctions arising from genetic defects in the transduction pathway, as amply demonstrated by the huge success as such in the case of visual transduction.