Transduction Mechanism in Olfactory Receptor Neurons

描述（由申请人提供）： 项目摘要本提案的长期目标是定量详细了解脊椎动物嗅觉受体神经元（ORN）的转导机制。在当前的资助期间，我们已经能够解决由ORN上的单个气味剂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）都给出了类似幅度的单一反应，这表明气味结合事件下游信号传导的概率非常低。换句话说，单一的反应似乎反映了一个单一的激活高尔夫/腺苷酸环化酶复合物的效果。这种现象显然是由于气味剂在OR分子上的停留时间非常短（即，气味剂从OR中快速解离）。这种短的气味剂停留时间同样主导嗅觉转导的终止。因此，传统上认为终止G-蛋白偶联受体（GPCR）信号传导，即受体磷酸化和随后的抑制蛋白结合的决定因素，是不重要的，至少在单位响应的水平，虽然他们可能仍然是重要的强烈和长期的气味刺激。到目前为止，上述结果已经从两栖动物的ORN和低Ca 2+溶液中获得（为了提高单位响应幅度）。在本申请中，我们建议继续用两栖动物的ORN进行实验，但同时也使用小鼠的ORN，这提供了遗传操作的明显优势。目的1是获得生理条件下青蛙ORN中嗅觉单位响应幅度的最佳估计（即，正常外部Ca 2+），以表征其沿着嗅纤毛的空间分布，解剖其膜电流成分（环核苷酸门控电流与Cl电流），并估计使ORN达到放电阈值所需的单一响应的数量。目的2是启动一个类似的详细研究的单一反应，在小鼠ORN，这将作为基础，检查可用的遗传小鼠品系的嗅觉转导。目的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。目的4是研究无气味物质条件下的组成性OR活性，这在脊椎动物嗅觉信号转导中很少受到关注。我们在以前的实验过程中已经注意到这种现象。实验方法将主要涉及从青蛙和WT或基因工程小鼠的单个ORN的吸管记录。公共卫生相关性：本申请中提出的研究将增强我们对脊椎动物鼻中嗅觉受体神经元中嗅觉转导的理解。从这些研究中获得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。

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.