Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia

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

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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

Original languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume275
Issue number2 44-2
Publication statusPublished - Aug 1998
Externally publishedYes

Fingerprint

Purification
Epithelium
Proteins
Lipid bilayers
Lipid Bilayers
Antibodies
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Peptides
Dithiothreitol
Cyclic AMP-Dependent Protein Kinases
Chromatography
Anions
Cations
Precipitates
Membrane Proteins
Ligands
Membranes

Keywords

  • 115-kilodalton protein
  • Gα(i) proteins
  • Immunoaffinity

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

Jovov, B., Shlyonsky, V. G., Berdiev, B. K., Ismailov, I. I., & Benos, D. J. (1998). Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia. American Journal of Physiology - Cell Physiology, 275(2 44-2).

Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia. / Jovov, Biljana; Shlyonsky, Vadim G.; Berdiev, Bakhram K.; Ismailov, Iskander I.; Benos, Dale J.

In: American Journal of Physiology - Cell Physiology, Vol. 275, No. 2 44-2, 08.1998.

Research output: Contribution to journalArticle

Jovov, B, Shlyonsky, VG, Berdiev, BK, Ismailov, II & Benos, DJ 1998, 'Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia', American Journal of Physiology - Cell Physiology, vol. 275, no. 2 44-2.
Jovov, Biljana ; Shlyonsky, Vadim G. ; Berdiev, Bakhram K. ; Ismailov, Iskander I. ; Benos, Dale J. / Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia. In: American Journal of Physiology - Cell Physiology. 1998 ; Vol. 275, No. 2 44-2.
@article{b6525a0d62cb409098df7b6dcf0f16d9,
title = "Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia",
abstract = "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- Gα(i) antibodies to precipitate Gα(i) protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Gα(i) protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.",
keywords = "115-kilodalton protein, Gα(i) proteins, Immunoaffinity",
author = "Biljana Jovov and Shlyonsky, {Vadim G.} and Berdiev, {Bakhram K.} and Ismailov, {Iskander I.} and Benos, {Dale J.}",
year = "1998",
month = "8",
language = "English",
volume = "275",
journal = "American Journal of Physiology - Cell Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "2 44-2",

}

TY - JOUR

T1 - Purification and reconstitution of an outwardly rectified Cl- channel from tracheal epithelia

AU - Jovov, Biljana

AU - Shlyonsky, Vadim G.

AU - Berdiev, Bakhram K.

AU - Ismailov, Iskander I.

AU - Benos, Dale J.

PY - 1998/8

Y1 - 1998/8

N2 - 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- Gα(i) antibodies to precipitate Gα(i) protein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Gα(i) protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.

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

KW - 115-kilodalton protein

KW - Gα(i) proteins

KW - Immunoaffinity

UR - http://www.scopus.com/inward/record.url?scp=0031870578&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031870578&partnerID=8YFLogxK

M3 - Article

VL - 275

JO - American Journal of Physiology - Cell Physiology

JF - American Journal of Physiology - Cell Physiology

SN - 0363-6143

IS - 2 44-2

ER -