Analysis of an odorant receptor in olfactory network

嗅觉系统通过嗅觉感受器神经元启动的一系列转导事件来辨别各种气味，这一能力令人惊叹。每个嗅觉神经元预计只表达一个气味受体基因，该基因属于G蛋白偶联受体家族。然而，配体-受体的相互作用还没有得到明确的表征。本研究首次用钙离子显像法和RT-PCR法相结合的方法，从单个嗅觉神经元中证实了嗅觉受体(S)对特定气味(S)的功能鉴定。首先，从单个组织印迹的嗅觉神经元中克隆了一个候选气味受体，该神经元显示气味诱导的钙升高。接下来，以绿色荧光蛋白为标记物，建立了重组腺病毒介导的受体基因在嗅上皮细胞中的表达。当被感染的神经元暴露在最初用于识别受体基因的气味中时，会引发外部钙离子进入。为确定配基特异性而进行的实验表明，气味受体识别气味分子中的特定结构基序。气味受体介导的信号转导似乎通过两步进行重构：从单个神经元中筛选特定气味物质(S)的受体和通过重组腺病毒实现受体的功能性表达。本方法将使我们不仅能够检测气味受体的配基特异性，而且还能够检测特定气味的受体特异性和多样性。

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand-receptor interaction, however, has not been clearly characterized. This study is the first to demonstrate the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca^<2+>-imaging and RT-PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca^<2+>-increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium using green fluorescent protein as a marker. The infected neurons elicited external Ca^<2+>-entry when exposed to the odorant that was originally utilized to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two step approach : the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach will enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.