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

这个建议的长期目标是了解在定量详细的cAMP-和Ca 2 +-信号传导的嗅觉受体神经元（ORN）的感觉转导。我们将专注于规范 嗅觉传导机制在脊椎动物的主要嗅觉上皮。这一机制涉及一个 cAMP信号级联，导致Na+和Ca 2+通过环状核苷酸门控（CNG）、非选择性阳离子通道流入，以将ORN降低至放电阈值。Ca 2+内流导致信号放大 通过一个内向的Ca 2+激活的Cl电流，以及嗅觉适应通过多个Ca 2+激活的负反馈途径。然而，最近，负反馈通路的重要性和表现 都陷入了疑惑和困惑之中在目标1中，我们建议重新审查这个问题，并一次性解决它 为了所有人 最近，通过使用M71-单克隆鼻小鼠ORN，我们已经成功地定量了 M71-OR分子在嗅纤毛膜上的密度。我们还发现，当配体时，M71-OR 与苯乙酮（M71-OR最有效的气味剂之一），尽管如此，仍然具有非常低的 激活单个下游Golf/腺苷酸环化酶III效应子的成功概率（标称约10-4） 复杂.这种低概率与视杆细胞光转导的情况非常不同， 最近表明，一个光激发的视紫红质激活10-20杆转导/cGMP-磷酸二酯酶 效应器因此，配体激活的GPCR途径在信号放大方面可能与光激活的GPCR途径完全不同。在目标2中，我们建议研究另一种小鼠鼻化学感受器TAAR 4， 对2-苯乙胺敏感，2-苯乙胺是一种令小鼠厌恶的捕食性气味剂。目标是 将这些发现与来自M71-OR的结果进行比较，以弄清楚TAAR 4在纤毛上的分子密度是否 表面膜与M71-OR的表面膜非常不同，并且为了询问是否在M71-OR处扩增， 下游G蛋白/效应酶复合物激活步骤与M71-OR的情况有任何不同。在 目标3，作为另一个比较，我们将针对mOR 256 -17解决相同的问题，这是一个OR，其中一个 迄今为止在主要嗅觉上皮中已知的最高丰度，并具有异常广泛的气味 江西篇章 阐明嗅觉传导步骤的定量分析将为正常嗅觉系统的研究提供重要的参考。 嗅觉功能，以及由转导途径中的遗传缺陷引起的功能障碍， 这一点在视觉传导方面取得了巨大的成功。

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.