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目的利用荧光共振能量转移(FRET)技术研究在细胞迁移过程中瞬时受体电位阳离子通道1(TRPC1)和蛋白激酶B(PKB/Akt)的相互作用。方法构建质粒pECFP-C1-TRPC1和pEYFP-C1-Akt,并转染非洲绿猴肾细胞(cos-7 cell),用激光共聚焦显微镜观察表皮生长因子(EGF)激活cos-7细胞前后的荧光蛋白表达和分布情况,并利用FRET检测EGF刺激cos-7细胞前后的TRPC1和Akt分子间的能量转移效率(E)和相互作用距离(R)。结果在EGF激活的cos-7细胞中,TRPC1和Akt蛋白有共定位现象;分别表达融合荧光蛋白后用EGF激活cos-7细胞,可见TRPC1由胞质转位到胞膜及近膜区,Akt未发生变化;共表达融合荧光蛋白后用EGF激活cos-7细胞,TRPC1和Akt由胞质转位到胞膜及近膜区,并共定位在细胞移动方向的前极;FRET检测结果显示其能量转移效率为40%,两个分子间的作用距离为5 nm。结论在EGF激活cos-7细胞迁移过程中,TRPC1和Akt发生了FRET,表明TRPC1和Akt之间有相互作用。
Objective To investigate the interaction between transient receptor potential cation channel 1 (TRPC1) and protein kinase B (PKB / Akt) during cell migration by fluorescence resonance energy transfer (FRET) technique. Methods The plasmids pECFP-C1-TRPC1 and pEYFP-C1-Akt were constructed and transfected into COS-7 cells. The changes of the fluorescence before and after the activation of cos-7 cells by epidermal growth factor (EGF) Protein expression and distribution, and the FRET was used to detect the energy transfer efficiency (E) and the interaction distance (R) between TRPC1 and Akt before and after EGF stimulation of cos-7 cells. Results EGF-activated cos-7 cells, TRPC1 and Akt protein co-localization phenomenon; respectively, after the expression of fusion fluorescent protein activated cos-7 cells with EGF, TRPC1 translocation from the cytoplasm to the membrane and the proximal membrane area, Akt After co-expression of fluorescent protein, COS-7 cells were activated by EGF. TRPC1 and Akt translocated from the cytoplasm to the membrane and proximal membrane area, and co-localized in the anterior pole in the direction of cell movement. FRET test results showed that The energy transfer efficiency is 40% and the distance between two molecules is 5 nm. Conclusions During the process of EGF-activated cos-7 cell migration, FRET occurred in TRPC1 and Akt, suggesting that there is interaction between TRPC1 and Akt.