气味受体调谐宽度、基础活性及其机械敏感性的关联性研究

Smell depends on the olfactory sensory neurons (OSNs) located on olfactory epithelium (OE) and expressing odorant receptors (ORs). It is the consensus that odor information is encoded by combinatorial scheme of odorant receptors. Most odorant receptors recognize one or a few odorants with similar structures. By contrast, a small number of receptors are broadly tuned, responsive to a wide range of odorants with totally different structures. Our previous work indicated that the tuning breadth of odorant receptors depends on both activation threshold and binding cavity. We also found that OR type determines mechanosensitivity of OSNs. Based on these findings, this proposal aims to establish 3D atomic model of binding cavity and disclose the key amino acid residues responsible for receptor activation and ligand binding in MOR256 family through 3D model, luciferase assay and calcium imaging. Meanwhile, we will elucidate the correlation between basal activity and tuning breadth, as well as interaction between mechanosensitivity and spontaneous firing rate in OSNs through patch clamp recording. This work will reveal the mechanism underlying ligand-OR binding and spontaneous activation. Furthermore, it will provide the theoretical evidence to the hypothesis that high spontaneous firing rate leads to the mechanosensitivity in OSNs. These findings will lay a solid and innovative foundation for better understanding the sense of smell.

嗅觉发生依赖于嗅上皮中表达气味受体的嗅感觉神经元。普遍认为气味信号是由不同受体的组合进行编码。其中大多数气味受体只能识别一种或几种结构相似的气味分子（称为窄谱性受体），但有小部分受体能够识别多种不同结构的气味分子（称为宽谱性受体）。课题组前期的工作结果表明气味受体的调谐宽度受到受体激活阈值和结合腔的双重调控。此外，表达不同气味受体的嗅感觉神经元对机械刺激表现出不同的敏感性。基于以上研究结果，本项目拟构建MOR256受体家族结合腔三维模型，结合荧光素酶分析、钙成像、膜片钳等技术进一步揭示影响受体激活与配体结合的关键氨基酸残基位点，随之阐明气味受体基础活性和调谐宽度，以及嗅感觉神经元机械敏感性和自发放电频率之间的关联。因此，本研究课题将揭示气味受体配体结合与自身激活的机制，并且探讨嗅感觉神经元的高自发放电频率引起机械敏感性的发生，进而为更好的了解嗅觉发生过程提供了创新性的理论基础。

嗅觉感知的发生依赖于气味受体与气味分子的结合，但受体与气味结合的调控因素目前仍不十分清楚，受体机械敏感性与基础活性及调谐宽度的关联也值得深入研究。通过此项目，我们构建了MOR256-22的三维结构模型，鉴定出控制其调谐宽度的氨基酸残基；通过突变体构建和受体功能分析明确调控MOR256-8对气味响应的关键氨基酸残基；利用气味受体库的大规模筛选，发现受体基础活性与调谐宽度的正相关性；通过钙成像检测不同MOR256-3突变体的机械敏感性，证明受体的机械敏感性与基础活性、调谐宽度呈正相关；MOR256-3与β2肾上腺素能受体等的嵌合体研究阐明胞外结构域 2（ECL2）在受体识别气味分子中的关键作用；我们以MOR256-31三维结构模型为基础，构建出机器学习模型预测受体与气味分子的结合，鉴定出54对新的受体-气味配对，并脱孤了20个气味受体。这些研究成果从多方面揭示了受体识别气味分子的关键因素，为推动气味受体的脱孤与更好地理解嗅觉编码机制提供了扎实的基础。