Expression of TMEM16 A as a calcium-activated chloride channel in Fis-cher rat thyroid follicular epithelial cells and its electrophysiologic pro-perties
10.3969/j.issn.1000-4718.2014.09.015
- VernacularTitle:TMEM16A钙激活氯离子通道在Fisher大鼠甲状腺滤泡上皮细胞的表达及其电生理特性研究
- Author:
Feng HAO
;
Xuesong BAI
;
Xiaohong JU
;
Fang FANG
;
Yuxuan ZANG
;
Hangfei ZHU
;
Guoyan XIANG
;
Yunqiao ZHANG
;
Zhonghai YUAN
- Publication Type:Journal Article
- Keywords:
Transmembrane protein 16A;
Calcium-activated chloride channels;
Patch-clamp techniques;
Fis-cher rat thyroid follicular epithelial cells
- From:
Chinese Journal of Pathophysiology
2014;(9):1633-1639
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thy-roid 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 ob-tain 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 TMEM 16A in the FRT cells were observed under an in-verted 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 se-quencing 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 TMEM 16A 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 .
