TRPV1内化的机制及其在炎症热痛觉敏化中的作用

Transient receptor potential vanilloid 1 (TRPV1) plays an important role in the development and maintenance of inflammatory pain. TRPV1 function can be regulated by many kinases through phosphorylation which changes the channel characteristics and influences the membrane trafficking, leading to the redistribution of surface TRPV1. Our previous results have shown that the phosphorylation of rat TRPV1 at threonine-406 by cyclin dependent kinase 5 (Cdk5) increases the cell membrane distribution of TRPV1. We also prove that Cdk5 promotes the interaction between KIF13B and TRPV1 through phosphorylating kinesin motor protein KIF13B, leading to an increase of surface TRPV1. The above results indicate that Cdk5 is involved in the regulation of inflammatory thermal hyperalgesia. The ion channel in the cell surface is in the dynamic balance between trafficking to cell surface and the reverse internalization. However, the mechanism of TRPV1 internalization is largely unknown. We will perform a series of existing experiments with molecular biology, cell biology, immunohistochemistry, in vitro kinase assay, mass spectrometry, calcium imaging, and animal behavior tests and initiate a new more suitable imaging technology for researching internalization to for the first time clear TRPV1 internalization and its cell biological mechanism, and explore the regulation of Cdk5 on TRPV1 internalization and its mechanism. We also will construct and apply the specific membrane penetrating fusion peptide to detect the effect of TRPV1 internalization and its regulation by Cdk5 on inflammatory thermal hyperalgesia in rats, which will provide new insights for seeking the potential drug targets for the treatment of pain.

TRPV1是炎症痛发生和维持的关键分子，其功能受多种激酶的磷酸化调控，一方面改变其通道特性，另一方面影响其膜转运。我们前期研究结果表明，Cdk5可通过磷酸化驱动蛋白KIF13B，增加TRPV1向细胞膜迁移或直接磷酸化TRPV1使其在细胞膜分布增多，参与炎症热痛觉敏化。离子通道向膜迁移与逆向内化处于动态平衡的过程，但迄今为止TRPV1的内化作用鲜有报道，机制尚不清楚。我们拟通过利用分子生物学、细胞生物学、免疫组化、同位素体外激酶实验、质谱检测、钙成像和大鼠炎症痛模型等现有实验技术和开创新的适合观测内化作用的样品制备及高分辨成像技术，首次揭示TRPV1的组成型内化作用及机制，并探究Cdk5对TRPV1内化的调控及机制；同时通过特异性融合肽的应用，探讨TRPV1的内化及其调控在大鼠炎症热痛觉敏化中的作用，为寻找临床治疗疼痛的潜在药物作用靶点提供新线索。

瞬时受体电位香草酸亚型1（transient receptor potential vanilloid 1, TRPV1）是炎症痛发生和维持的关键分子，其功能受多种激酶的磷酸化调控，一方面改变其通道特性，另一方面影响其膜转运。我们前期研究结果表明，细胞周期素依赖性激酶 5（cyclin-dependent kinase 5, Cdk5）可通过磷酸化驱动蛋白家族成员KIF13B，增加TRPV1向细胞膜迁移；Cdk5也可直接磷酸化TRPV1第407位苏氨酸使其在细胞膜分布增多，参与炎症热痛觉敏化。离子通道向膜迁移与逆向内化处于动态平衡的过程，但迄今为止TRPV1的内化作用鲜有报道，机制尚不清楚。我们通过利用分子生物学、细胞生物学、同位素体外激酶实验、钙成像和大鼠炎症痛模型等现有实验技术和开创新的适合观测内化作用的免疫荧光染色及成像技术，首次揭示TRPV1的组成型内化作用为网格蛋白Clathrin、缢断蛋白Dynamin及衔接蛋白AP2复合体依赖的途径，在这一过程中TRPV1与AP2的μ2亚基存在直接相互作用；Cdk5通过磷酸化AP2μ2第45位丝氨酸（S45）负性调控TRPV1的内化；应用可穿膜的融合肽TAT-S45特异性干扰Cdk5对S45的磷酸化，可使TRPV1与AP2μ2的结合增加，TRPV1的内吞增多，细胞膜上TRPV1分布减少，对辣椒素（capsaicin）刺激的钙反应减弱；大鼠鞘内注射TAT-S45干扰肽，可明显减缓完全弗氏佐剂（complete Freund’s adjuvant, CFA）诱导的自发痛及炎症热痛觉敏化，急性分离该组大鼠背根神经节（dorsal root ganglion, DRG）神经元，细胞对capsaicin反应性降低。综上所述，TRPV1存在clathrin/dymain/AP2μ2依赖的内化作用，Cdk5通过磷酸化AP2μ2的S45干扰TRPV1的内吞，使其膜上分布增多功能增强，调控大鼠炎症热痛觉敏化的形成。本研究 首次揭示了TRPV1的内化机制及Cdk5对这一过程的调控作用，进一步完善了Cdk5通过TRPV1调控炎症热痛觉敏化的分子机制，为寻找临床治疗疼痛的潜在药物作用靶点提供了新的线索。