Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia.
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 microg 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-Galphai antibodies to precipitate Galphai protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Galphai protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
B
B
Jovov
V G
VG
Shlyonsky
B K
BK
Berdiev
I I
II
Ismailov
D J
DJ
Benos
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D015220:Calcium Channels / Q000096:biosynthesis / Q000302:isolation & purification / Q000502:physiology*; D002417:Cattle; D002712:Chlorides / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008970:Molecular Weight; D014132:Trachea / Q000166:cytology / Q000502:physiology*
D000906:Antibodies; D015220:Calcium Channels; D002712:Chlorides; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D019204:GTP-Binding Proteins; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid