Transduction Mechanism in Olfactory Receptor Neurons

描述(由申请人提供)： 本研究的长期目标是对脊椎动物嗅觉感受器神经元(ONs)的转导机制进行详细的定量研究。在目前的授权期内，我们已经能够解决由ORN上的单个气味结合事件引发的响应(所谓的“单一响应”)。令人惊讶的是，我们发现随机遇到的Orn(因此随机遇到气味受体或ORs)都给出了类似幅度的单一反应，这表明气味结合事件向下游发出信号的可能性非常低。换句话说，单一反应似乎反映了单个激活的Golf/腺苷环化酶复合体的影响。这种现象显然是由于气味在OR分子上停留的时间非常短(即气味从OR快速解离)。这种短的气味停留时间同样支配着嗅觉转导的终止。因此，传统上认为的终止G蛋白偶联受体信号的决定因素，即受体磷酸化和随后的arrestin结合，至少在单一反应水平上是不重要的，尽管它们在强烈和长期的气味刺激中仍然是重要的。到目前为止，上述结果已经在两栖动物和低钙溶液中获得(为了提高单位响应幅度)。在这一应用中，我们建议继续使用两栖兽角进行实验，但同时也使用鼠角，这提供了遗传操作的明显优势。目的1是获得青蛙眼球在生理条件下(即正常的外钙)嗅觉单一反应幅度的最佳估计值，描述其沿嗅觉纤毛的空间分布，解剖其膜电流成分(环核苷酸门控电流与氯电流)，并估计使嗅觉神经元达到放电阈值所需的单一反应的数目。目标2是在小鼠Orns中启动类似的单一反应的详细研究，这将作为检查可用于嗅觉转导的遗传小鼠系的基础。目标3是更详细地研究/解剖Orns的钙依赖和非钙依赖适应，主要是利用遗传小鼠品系。目标4是研究在没有气味的情况下的结构性OR活性，到目前为止，这在脊椎动物的嗅觉转导中还很少受到关注。在之前的实验过程中，我们注意到了这一现象。实验方法将主要涉及吸移管记录青蛙和WT或基因工程小鼠的单卵。公共卫生相关性：项目描述本申请中提出的研究将增强我们对脊椎动物鼻子中嗅觉感受器神经元的嗅觉传导的理解。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.