钙激活氯通道ANO1在小鼠心肌细胞的表达及其功能鉴定

目的:探讨钙激活氯通道蛋白anoctamin 1(ANO1)在小鼠原代培养心肌细胞中的表达及其功能特性。方法:采用胰酶与胶原酶共同消化,联合2次差速贴壁法获得C57BL/6小鼠原代心肌细胞;并用免疫荧光染色法检测α-横纹肌肌动蛋白,以鉴定心肌细胞纯度;应用RT-PCR检测小鼠心肌细胞ANO1 mRNA的表达;免疫印迹检测ANO1蛋白在小鼠心肌细胞的表达情况;应用荧光淬灭动力学实验检测ANO1钙激活氯通道的功能特性。结果:RT-PCR结果表明原代培养的小鼠心肌细胞表达ANO1 mRNA。免疫印迹实验结果显示原代培养的小鼠心肌细胞表达ANO1蛋白。荧光淬灭动力学实验证实表达于小鼠心肌细胞的ANO1具有钙激活氯通道典型的阴离子转运功能特性。结论:ANO1在小鼠心肌细胞中有明确表达,并具有钙激活氯离子通道特性,提示ANO1是钙激活氯通道的分子基础。

Expression and identification of ANO1 in mouse cardiomyocytes

AIM: To explore the expression of anoctamin 1 (ANO1), one of calcium-activated chloride channels (CaCCs), in mouse cardiomyocytes and its functional properties. METHODS: The cardiomyocytes from the myocardial tissues of C57BL/6 mice were isolated with enzyme and purified by the differential adherent method. The cells were stained with monoclonal anti-sarcomeric actin and Cy3 to evaluate the purity of the myocardial cells. RT-PCR was used to detect the mRNA expression of ANO1 in the mouse cardiomyocytes. The protein expression of ANO1 in the mouse cardiomyocytes was determined by Western blotting analysis. The fluorescence quenching kinetics experiment was used to identify the ion transport properties of ANO1 in the mouse cardiomyocytes. RESULTS: The results of RT-PCR confirmed that ANO1 was expressed in freshly isolated myocardial cells. The results of Western blotting clearly demonstrated the protein expression of ANO1 in primarily cultured myocardial cells. Fluorescence quenching kinetics experiment on freshly isolated single myocardial cell revealed a pronounced outward rectifying property of the ANO1. The functional properties were similar to the classic CaCCs. CONCLUSION: ANO1 expression was identified in the mouse myocardial cells. The function of CaCCs was generated by ANO1, suggesting that ANO1 is the molecular basis of CaCCs.