Expression Clone of TMEM16A as a Calcium-Activated Chloride Channels in CHO Cells

Abstract

There is compelling evidence that TMEM16A fuctions as calcium-activated chloride channels (CaCCS), which was discovered by three independent labs in 2008 after Calcium-activated chloride channel current was first recorded in the 1980s. CaCCs are involved in many physiological processes, including transepithelial fluid secretion, smooth muscle contraction , sensory signal transduction and others. CaCCs are considers as potential drug therapy of hypertension, secretoy diarrheas, neuropathic pain, asthma, cystic fibrosis and certain tumors. In our previous study, TMEM16A with green fluorescence protein (GFP) fusion protein were subcloned into pcDNA3.1/Zeo. In this study, TMEM16A transient transfection conditon of Chinese hamster ovary (CHO) cells were optimized through liposome transfection and CHO cells expressing TMEM16A were got by stable transfection in which the classical calcium-activated chloride channels current was recorded by whole cell patch clamp technique. By a comparison between the results in this study and the results in previous study, both CHO and FRT cells are suitable for TMEM16A-pcDNA3.1 expression through liposome transfection and currents were recorded in both FRT and CHO cells by whole cell patch clamp technique, but our results indicated different purposes should require different cell lines and methods. These results were beneficial for the delving into study of TMEM16A-CaCCs by patch clamp technique which is the gold standard for real-time investigation of ion channels and their effectors.