基于钙激活氯离子通道检测胞质内第二信使Ca2+

目的 建立一种基于钙激活氯离子通道(CaCC)可敏感检测胞质内第二信使Ca2+的细胞模型.方法 构建氯离子通道蛋白1(ANO1)和YFP-H148Q/I152 L真核表达载体,应用脂质体转染法构建共表达ANO1和YFP-H148Q/I152 L的FRT细胞,倒置荧光显微镜观察其表达情况,流式细胞仪检测细胞纯度;应用膜片...

Establishment of calcium-activated chloride channel-based second messenger Ca2+detection method

Objective To establish a cell model based on calcium-activated chloride channel（CaCC）that could sensitively detect the second messenger Ca²⁺ in the cytoplasm. Methods The eukaryotic expression vectors of anoctamin 1(ANO1) and YF-H148Q/I152 L were constructed respectively. FRT cells co-expressing ANO1 and YFP-H148Q/I152 L were obtained by liposome transfection. The expression of ANO1 and YFP-H148Q/I152 L in FRT cells was observed by an inverted fluorescence microscope, and flow cytometry was used to detect the purity of cells. Patch clamp was applied to study physiological characteristics of CaCC. The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CaCC modulators was verified by the fluorescence quenching kinetics experiments. The fluorescent probe was used to detect the calcium concentration in cytoplasm after adding CaCC activator. Results The result of the inverted fluorescence microscope showed that ANO1 was expressed in the cell membrane of FRT, and YFP-H148Q/I152 L was expressed in the cytoplasm of FRT cells. The cell model had the physiological characteristics of classical calcium-activated chloride channels. The FRT cell model stably co-expressing ANO1 and YFP-H148Q/I152 L was successfully constructed. The model could screen CaCC modulators, and the slope of fluorescence change and the concentration of CaCC modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the calcium concentration in the cytoplasm. The cell model can sensitively detect intracellular calcium concentration. Conclusion The cell can efficiently and sensitively detect the second messenger Ca²⁺ concentration in the cytoplasm, and it provides a simple and efficient method for the study of other targets associated Ca²⁺ signal.