Transduction Mechanism in Olfactory Receptor Neurons

项目总结 这一建议的长期目标是对信号转导的定量细节进行了解 脊椎动物嗅觉感受器神经元的机制。在目前的授权期内，我们有 能够解决由ORN上的单个气味绑定事件触发的响应(SO- 称为“统一响应”)。令人惊讶的是，我们发现随机遇到的兽人(因此 随机遇到的气味感受器，或OR)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。在其他 换言之，单一反应似乎反映了单个激活的高尔夫/腺苷环化酶的影响 很复杂。这种现象显然是由于气味在OR分子上的停留时间很短 (即，气味从OR快速解离)。这种短的气味停留时间同样占主导地位。 嗅觉传导的终止。因此，传统上认为的终止的决定因素 G蛋白偶联受体(GPCR)信号转导，即受体磷酸化及随后 Arrestin结合，至少在单一反应水平上是不重要的，尽管它们可能仍然 重要的是强烈和持久的气味刺激。到目前为止，上述结果是 取自两栖动物的Orns和在低钙溶液中(为了增强单一反应 幅度)。在本申请中，我们建议继续进行两栖兽角的实验，但在 同时，还可以使用老鼠角，这提供了遗传操作的明显优势。 目的1是获得青蛙嗅觉单一反应波幅的最佳估计值 生理条件(即正常的外部钙离子)，以表征其沿 嗅觉纤毛，解剖其膜电流成分(环核苷酸门控电流与氯 当前)，并估计使ORN发射所需的单位响应的数量 临界点。目标2是在小鼠的角上发起类似的单一反应的详细研究， 将作为检测可用于嗅觉转导的遗传小鼠品系的基础。 目标3是由Orns更详细地研究/剖析钙依赖和非钙依赖的适应，主要是 通过利用遗传小鼠品系。目标4是在不存在的情况下研究构成或活性 气味物质，到目前为止在脊椎动物的嗅觉传导中几乎没有受到关注。我们已经注意到了 在以前的实验过程中出现了这种现象。 实验方法将主要涉及吸移器记录从单次 青蛙和WT或转基因小鼠的卵。

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.