Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia.
Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia.
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从气管上皮细胞中纯化和重建向外整流的 Cl-通道。
DOI：
10.1152/ajpcell.1998.275.2.c449
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Benos,DJ
中科院分区：
文献类型：
--
作者：
Jovov,B;Shlyonsky,VG;Berdiev,BK;Ismailov,II;Benos,DJ
We reported the identification of three outwardly rectified Cl−channel (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayers of material partly purified from bovine tracheal apical membranes with one of these antibodies as a ligand (anti-p115) resulted in the incorporation of an ORCC identical in biophysical characteristics to one we previously described. 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 combination of anion- and cation-exchange chromatography followed by immunopurification. By use of this purification scheme, 7 μg of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins. Incorporation of this highly purified material into planar lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 ± 9 pS in symmetrical Cl−solutions, halide selectivity sequence of I−> Cl−> Br−, and lack of sensitivity to protein kinase A, Ca2+, or dithiothreitol. Using anti-Gαiantibodies to precipitate Gαiprotein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Gαiprotein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.
