Transduction Mechanism in Olfactory Receptor Neurons

嗅觉受体神经元的转导机制

• 批准号: 7873021
• 负责人: KING-WAI YAU
• 金额: $ 34.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873021
• 关键词: Action Potentials; Adenylate Cyclase; Affect; Amphibia; Arrestins; Attention; Binding; Brain; Cilia; Complex; Cyclic Nucleotides; Detection; Disabled Persons; Disease; Dissociation; Event; Feedback; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Genetic; Genetically Engineered Mouse; Golf; Grant; Health; Human; Individual; Insecta; Membrane; Mus; Nose; Odorant Receptors; Olfactory Receptor Neurons; Pathway interactions; Phosphorylation; Phototransduction; Physiological; Probability; Rana; Relative (related person); Reporting; Retinal Pigments; Shapes; Signal Transduction; Smell Perception; Solutions; Stimulus; Suction; System; Time; Vertebrate Photoreceptors; genetic manipulation; insight; public health relevance; receptor; research study; response; retinal rods

DESCRIPTION (provided by applicant): PROJECT SUMMARY The long-term objective of this proposal is to understand in quantitative detail the transduction mechanism in vertebrate olfactory receptor neurons (ORNs). During the current grant period, we have been able to resolve the response triggered by a single odorant-binding event on an ORN (the so- called "unitary response"). Surprisingly, we found that randomly encountered ORNs (and therefore randomly encountered odorant receptors, or ORs) all gave unitary responses of similar amplitudes, suggesting that an odorant-binding event has a very low probability of signaling downstream. In other words, the unitary response appears to reflect the effect of a single activated Golf/adenylyl cyclase complex. This phenomenon is apparently due to a very short odorant dwell-time on the OR molecule (i.e., rapid dissociation of the odorant from the OR). This short odorant dwell-time likewise dominates the termination of olfactory transduction. Hence, the traditionally believed determinants for terminating G-protein-coupled receptor (GPCR) signaling, namely, receptor phosphorylation and subsequent arrestin binding, are unimportant at least at the level of the unitary response, although they may still be important with intense and prolonged odorant stimulation. So far, the above results have been obtained from amphibian ORNs and in low-Ca2+ solution (in order to boost the unitary-response amplitude). In this application, we propose to continue experiments with amphibian ORNs but at the same time to use also mouse ORNs, which offer the distinct advantage of genetic manipulations. Aim 1 is to obtain the best estimate of the olfactory unitary-response amplitude in frog ORNs in physiological conditions (i.e., normal external Ca2+), to characterize its spatial spread along the olfactory cilium, to dissect its membrane-current components (cyclic-nucleotide-gated current versus Cl current), and to estimate the number of unitary responses required for bringing the ORN to firing threshold. Aim 2 is to launch a similar detailed study of the unitary response in mouse ORNs, which will serve as the groundwork for examining available genetic mouse lines for olfactory transduction. Aim 3 is to study/dissect Ca-dependent and Ca-independent adaptation by ORNs in more detail, largely by making use of genetic mouse lines. Aim 4 is to study constitutive OR activity in the absence of odorants, which so far has received little attention in vertebrate olfactory transduction. We have noticed this phenomenon in the course of previous experimentation. The experimental approach will involve predominantly suction-pipette recording from single ORNs of frog and WT or genetically engineered mice. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE The studies proposed in this application will enhance our understanding of olfactory transduction in vertebrate olfactory receptor neurons in the nose. Any new information derived from these studies will also be highly relevant to disease states affecting odorant detection by the nose.

描述（由申请人提供）： 项目摘要 该提案的长期目标是定量详细地了解脊椎动物嗅觉受体神经元 (ORN) 的转导机制。在当前资助期内，我们已经能够解决 ORN 上单个气味结合事件触发的响应（所谓的“单一响应”）。令人惊讶的是，我们发现随机遇到的 ORN（因此随机遇到的气味受体，或 OR）都给出了相似幅度的单一响应，这表明气味结合事件向下游发出信号的概率非常低。换句话说，单一响应似乎反映了单个激活的高尔夫/腺苷酸环化酶复合物的作用。这种现象显然是由于加味剂在 OR 分子上的停留时间非常短（即加味剂从 OR 上快速解离）造成的。这种短暂的气味停留时间同样决定了嗅觉转导的终止。因此，传统上认为终止 G 蛋白偶联受体 (GPCR) 信号传导的决定因素，即受体磷酸化和随后的视紫红质抑制蛋白结合，至少在单一反应水平上并不重要，尽管它们对于强烈和持久的反应可能仍然很重要。气味刺激。到目前为止，上述结果是在两栖动物 ORN 和低 Ca2+ 溶液中获得的（为了提高单一响应幅度）。在此应用中，我们建议继续使用两栖动物 ORN 进行实验，但同时也使用小鼠 ORN，这提供了遗传操作的独特优势。目标 1 是获得生理条件下（即正常外部 Ca2+）青蛙 ORN 嗅觉单一响应幅度的最佳估计，以表征其沿嗅纤毛的空间分布，剖析其膜电流成分（环核苷酸） -门控电流与 Cl 电流），并估计使 ORN 达到激发阈值所需的单一响应数量。目标 2 是针对小鼠 ORN 的单一反应开展类似的详细研究，这将作为检查可用的嗅觉转导基因小鼠系的基础。目标 3 是更详细地研究/剖析 ORN 的 Ca 依赖性和 Ca 非依赖性适应，主要是通过利用遗传小鼠品系。目标 4 是研究在没有气味的情况下的组成型 OR 活性，迄今为止，这一点在脊椎动物嗅觉转导中很少受到关注。我们在之前的实验过程中就注意到了这个现象。实验方法主要涉及用吸管记录青蛙和野生型或基因工程小鼠的单个 ORN。公共卫生相关性：项目叙述本申请中提出的研究将增强我们对脊椎动物鼻子嗅觉受体神经元嗅觉转导的理解。从这些研究中得出的任何新信息也将与影响鼻子气味检测的疾病状态高度相关。