TRP通道介导的机械力-钙信号-基因转录耦联及其在神经元发育中的作用

Living organisms could sense the mechanical forces and convert the stimuli into electrical or biochemical signals to regulate physiological processes through mechanosensitive Ca2+ channels, such as transient receptor potential (TRP) channels, Piezo channels and so on. Mechanosensitive channels are widely studied due to the broad expression and multiple functions. But the mechanism of TRP channel mediated force-Ca2+-transcription coupling is still unclear. This project is started with the research of mechanosensitive TRP channels. Firstly, by using methodology of electrophysiology, Ca2+ imaging, classical cellular mechanical stimulus and newly-designed micro-nano device of mechanical stimulus, the applicant plans to quantitatively depict the biophysical properties of force-Ca2+ transduction of mechanosensitive TRP channels. Secondly, the modulation of neuronal transcription through force-Ca2+ transduction is systematically clarified through the study of the molecular interactions between the cellular domains of TRP channels and vital signaling pathways related molecules. Finally, the physiological and pathological functions of mechanosensitive TRP channels in neural system, especially the functions of neural development in dorsal root ganglion and cerebrospinal fluid-contacting neurons, are studied through long term monitoring of Ca2+ signals by GCaMP-X, a new genetically-encoded Ca2+ sensor developed by the applicant. The proposal is aimed at deep understanding of mechanosensitive TRP channels mediated force-Ca2+-transcription coupling and the relationship between molecular biomechanics and neurophysiology/neuropathology.

生命体感受机械力刺激，进而调控相应生理过程。机械敏感离子通道(如TRP及Piezo等)是介导机械感知及下游信号的重要膜蛋白。机械敏感通道分布广泛、功能多样，尽管得到了广泛研究，但对于TRP通道介导的机械力-钙信号-转录耦联理解还相对滞后。本研究针对代表性机械敏感TRP通道，首先利用经典力学操控和新颖设计的微纳器件力学操控系统，结合电生理、钙成像等技术，定量刻画TRP介导的力-钙转导关系；其次，系统考察机械敏感通道在神经元基因转录中的作用和机制，力图从通道蛋白与信号蛋白动态互作这一视角解析机械力与信号通路间的特异性耦合；最后，利用申请人新开发的细胞无损钙探针GCaMP-X，探究TRP通道介导的力-钙耦合通路与神经元病生理的关联，着重考察其在背根神经节和脑脊液接触神经元发育中的作用。本研究旨在推动对机械敏感TRP通道及其关键信号通路的理解，以期在分子细胞水平进一步加强生物力学与神经科学的融合。

神经元可利用TRP通道等机械敏感离子通道感知力学刺激，并将力学信号转化为钙信号，进一步地，钙信号可影响转录调控及神经元发育。本项目旨在回答如下三个问题：1）神经元TRP通道的力感知机制及对神经元力-钙转导的贡献；2）神经元钙-转录-发育之间的关系；3）开发新的钙动态长期监控方法，在长时间尺度考察神经元自发钙信号与神经元发育的动态关系。利用电生理、钙成像、结构生物学等技术，我们取得的主要成果如下：1）以TRPP3为代表的TRP通道不具备直接感应机械力刺激的能力，但部分通道如TRPC5等对低渗刺激这种生物力学刺激展出出良好的感知能力。基于结构-功能研究发现，这种低渗感知能力与通道电压感受类域的钙结合口袋有关。神经元对低渗刺激这种生物力学刺激表现出良好的力-钙转导能力，这种能力由TRP通道感知并驱动，由L型电压门控钙通道放大钙信号；2）L型电压门控钙通道介导的钙信号直接调控神经元转录及发育过程。以L型电压门控钙通道为靶点，设计特异性多肽调控神经元钙信号，可直接影响神经元CREB转录因子磷酸化水平及神经元突起发育水平，说明神经元钙信号-pCREB信号通路转录调控-神经元突起发育三者是强关联；3）设计了基于细胞无损基因编码钙探针GCaMP-X的神经元长时程钙动态监控系统，揭示了神经元早期发育与自发钙动态之间的关联。在离体神经元连续监控钙信号和发育进程一个月后，我们发现神经元自发钙振荡频率、幅值、同步性等特征参数与神经元早期发育及神经网络形成具有显著关联。本项目在结构-功能水平对TRP通道的力感知机制提供了新颖视角，加深了对神经元力-钙转导及钙-转录-发育通路的理解，开发的神经元钙动态长时程监控方案具备良好的科学推广性和应用前景。