Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties |
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Authors: | HAO Feng BAI Xue-song JU Xiao-hong FANG Fang ZANG Yu-xuan ZHU Hang-fei XIANG Guo-yan ZHANG Yun-qiao YUAN Zhong-hai |
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Institution: | 1.Department of Biochemistry Laboratory, 2Department of Nutrition, 3Department of Pathogen, Jilin Medical College, Jilin 132013, China. |
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Abstract: | AIM:To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS:The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS:The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION:The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels. |
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Keywords: | Transmembrane protein 16A Calcium-activated chloride channels Patch-clamp techniques Fischer rat thyroid follicular epithelial cells |
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