Am J Physiol Cell Physiol AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 275: C449-C458, 1998;
0363-6143/98 $5.00
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jovov, B.
Right arrow Articles by Benos, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jovov, B.
Right arrow Articles by Benos, D. J.
Vol. 275, Issue 2, C449-C458, August 1998

Purification and reconstitution of an outwardly rectified Clminus channel from tracheal epithelia

Biljana Jovov, Vadim G. Shlyonsky, Bakhram K. Berdiev, Iskander I. Ismailov, and Dale J. Benos

Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

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-Galpha i antibodies to precipitate Galpha i protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Galpha i protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.

115-kilodalton protein; immunoaffinity; Galpha i proteins


This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Spiegel, M. Phillipper, H. Rossmann, B. Riederer, M. Gregor, and U. Seidler
Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion
Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G887 - G897.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Jovov, A. Tousson, H.-L. Ji, D. Keeton, V. Shlyonsky, P.-J. Ripoll, C. M. Fuller, and D. J. Benos
Regulation of Epithelial Na+ Channels by Actin in Planar Lipid Bilayers and in the Xenopus Oocyte Expression System
J. Biol. Chem., December 31, 1999; 274(53): 37845 - 37854.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Cuppoletti and D. H. Malinowska
Ca2+-activated Cl- channels Focus on "Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland"
Am J Physiol Cell Physiol, June 1, 1999; 276(6): C1259 - C1260.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. D. Gruber, K. D. Schreur, H.-L. Ji, C. M. Fuller, and B. U. Pauli
Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland
Am J Physiol Cell Physiol, June 1, 1999; 276(6): C1261 - C1270.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H.-L. Ji, M. L. Chalfant, B. Jovov, J. P. Lockhart, S. B. Parker, C. M. Fuller, B. A. Stanton, and D. J. Benos
The Cytosolic Termini of the beta - and gamma -ENaC Subunits Are Involved in the Functional Interactions between Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel
J. Biol. Chem., September 1, 2000; 275(36): 27947 - 27956.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
X. C. Sun and J. A. Bonanno
Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells
Am J Physiol Cell Physiol, April 1, 2002; 282(4): C673 - C683.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online