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

嗅觉受体神经元感觉转导中的环 AMP 和 Ca2 信号传导

基本信息

批准号：
10622600
负责人：
KING-WAI YAU
金额：
$ 40.94万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-01 至 2027-04-30
项目状态：
未结题

项目摘要

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.

该提案的长期目标是定量详细了解嗅觉受体神经元 (ORN) 感觉转导中的 cAMP 和 Ca2+ 信号传导。我们将重点关注规范脊椎动物主要嗅上皮的嗅觉传导机制。该机制涉及一个 cAMP 信号级联，导致 Na+ 和 Ca2+ 通过环核苷酸门控 (CNG)、非选择性阳离子通道流入，使 ORN 去极化至放电阈值。 Ca2+ 流入导致信号放大通过内向 Ca2+ 激活的 Cl 电流，以及通过多个 Ca2+ 激活的负反馈途径进行嗅觉适应。然而，最近负反馈途径的重要性和表现陷入怀疑和困惑。在目标1中，我们建议重新审视这个问题并解决一次并为所有人。最近，通过使用 M71 单克隆鼻小鼠 ORN，我们成功地量化了嗅觉纤毛膜上 M71-OR 分子的密度。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）相比，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（通常约为 10-4）复杂的。这种低概率与视杆光转导的情况非常不同，我们对此进行了研究最近表明，一种光激发视紫红质可激活 10-20 杆转导蛋白/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途径有很大不同。在目标 2 中，我们建议研究另一种小鼠鼻化学感受器 TAAR4，它对 2-苯乙胺极其敏感，这是一种令小鼠厌恶的捕食者气味剂。目标是将结果与 M71-OR 的结果进行比较，以确定 TAAR4 的分子密度是否对纤毛的影响表面膜与M71-OR有很大不同，请问是否在下游 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，我们将为 mOR256-17 解决相同的问题，即与其中一个的 OR 迄今为止已知在主嗅上皮中的丰度最高，并且具有异常广泛的气味光谱。定量阐明嗅觉转导的步骤将为我们提供对正常嗅觉的深入了解。嗅觉功能以及转导途径中遗传缺陷引起的功能障碍，以及视觉传导方面的巨大成功就证明了这一点。