PURIFICATION AND RECONSTITUTION OF AN OUTWARDLY RECTIFIED CL- CHANNELFROM TRACHEAL EPITHELIA

We reported the identification of three outwardly rectified Cl- channe l (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolate d proteins. Incorporation into planar lipid bilayers of material partl y purified from bovine tracheal apical membranes with one of these ant ibodies as a ligand (anti-p115) resulted in the incorporation of an OR CC identical in biophysical characteristics to one we previously descr ibed. We developed a new purification procedure to increase the yield and purity of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combin ation of anion- and cation-exchange chromatography followed by immunop urification. By use of this purification scheme, 7 mu g of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins . Incorporation of this highly purified material into planar lipid bil ayers revealed a DIDS-inhibitable channel with the following propertie s: linear conductance of 87 +/- 9 pS in symmetrical Cl- solutions, hal ide selectivity sequence of I- > Cl- > Br-, and lack of sensitivity to protein kinase A, Ca2+, or dithiothreitol. Using anti-G alpha(i) anti bodies to precipitate G alpha(i) protein(s) from the partly purified p reparations, we demonstrated that the loss of rectification of the ORC C was due to uncoupling of Gai protein(s) from the ORCC protein and th at the 115-kDa polypeptide is an ORCC.