内源表达的异源三聚体P2X2/3的配体识别与门控调节机制研究

P2X receptors are nonselective cation channels gated by extracellular ATP. Functional P2X receptors are mainly homomeric or heteromeric trimetric ion channels. These receptors are implicated in many physiological and pathological processes. Accordingly, they are ideal drug targets for many diseases. P2X2/3 heteromeric trimeric receptors are highly expressed in sensory neurons, including dorsal root ganglia, trigeminal ganglia and nodose ganglia. They play a key role in nociceptive transmission and mechanosensory transduction within visceral organs. However, the ligand/ATP-receptor interaction, distinct architecture of key structural motifs as well as dynamic gating mechanism in this subtype of heteromeric P2X receptors remain largely unclear, which to some extent hinders the understanding of its functions and the new drug design. To explore the special channel gating and biophysical signal transduction mechanisms of P2X2/3 heteromeric trimeric receptors, a combination of using multidisciplinary research approaches including molecular biolog, computational biology, electrophysiology and behavioral tests will be employed. We intend to further find the endogenous molecules or exogenous inhibitors specific for this unique trimeric receptor, which would favor of uncovering the molecular mechanisms of heteromeric trimeric P2X2/3 receptors in the nociceptive signaling, as well as providing new therapeutic clues for the clinical treatment of pain.

P2X受体是一类可被胞外ATP激活的以同源或异源三聚体形式存在的非选择性阳离子通道，参与诸多重要的生理病理学过程，是疾病干预的理想靶标之一。其中异源三聚体P2X2/3通道选择性高表达于感觉神经元中，在疼痛传导和内脏机械感觉传导中发挥着重要作用。虽然多种同源三聚体的结构已得到解析，但目前对P2X异源三聚体蛋白的三维构建，配体识别，动态门控以及与同源三聚体的异同等科学问题仍然不清楚，这在一定程度上阻碍了对P2X靶向药物的作用或副作用机制的理解以及进一步的新药研发。本课题拟基于已有晶体结构的同源构建，综合运用分子生物学、膜生物物理、蛋白修饰、电生理及行为学等手段，研究P2X2/3异源三聚体通道特殊的配体识别与门控机制；并以此为基础寻找内源性调节分子或外源性抑制剂，以期揭示P2X2/3异源三聚体通道介导痛觉感受的分子机制，为缓解临床疼痛提供新的策略。

P2X受体是一类可被胞外ATP激活的以三聚体形式存在的非选择性阳离子通道，P2X3同聚体和P2X2/3异聚体表达于外周初级感觉神经元，均能参与急慢性疼痛感受传导、味觉传递、化学感受性反射等生理病理过程。新上市的Gefapixant/AF219作为第一款以P2X3同聚体为靶标，用于治疗不明原因的难治性慢性咳嗽（RCC/UCC）的口服药，可同时作用于P2X2/3异聚体产生严重的味觉失调副作用。因此，探究P2X3同聚体和P2X2/3异聚体的配体识别和门控调节机制差异迫在眉睫。本课题通过建立内源性异聚体的检测体系，综合运用分子生物学、膜生物物理、电生理学等手段，确定了三聚体的其中两个口袋是ATP识别不可或缺的，而另外一个口袋仅在构象传递中起作用。在P2X2/3异聚体的Gefapixant/AF219变构调节过程中，只需要其中一个口袋，这说明针对P2X3靶向药物Gefapixant/AF219的改造和研发，面临一定的困难。以上研究结论为探究P2X2/3异聚体的小分子配体识别机制提供可行性的方法，也为以P2X同聚体和/或异聚体的高特异性靶向药物研发提供了基础。

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