Cloning of an epithelial chloride channel from bovine trachea

Sonia A. Cunningham, Mouhamed S. Awayda, James K. Bubien, Iskander I. Ismailov, M. Pia Arrate, Bakhram K. Berdiev, Dale J. Benos, Catherine M. Fuller

Research output: Contribution to journalArticle

181 Citations (Scopus)

Abstract

We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (αp38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)- sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903- amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock- transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by αp38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the αp38 antibody.

Original languageEnglish
Pages (from-to)31016-31026
Number of pages11
JournalJournal of Biological Chemistry
Volume270
Issue number52
DOIs
Publication statusPublished - Dec 29 1995
Externally publishedYes

Fingerprint

Chloride Channels
Cloning
Trachea
Organism Cloning
Anions
Dithiothreitol
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Complementary DNA
Clone Cells
COS Cells
Proteins
Base Pairing
Oocytes
Antibodies
Calcium-Calmodulin-Dependent Protein Kinases
Ionomycin
Lipid bilayers
Clamping devices
Lipid Bilayers
Xenopus

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cunningham, S. A., Awayda, M. S., Bubien, J. K., Ismailov, I. I., Arrate, M. P., Berdiev, B. K., ... Fuller, C. M. (1995). Cloning of an epithelial chloride channel from bovine trachea. Journal of Biological Chemistry, 270(52), 31016-31026. https://doi.org/10.1074/jbc.270.52.31016

Cloning of an epithelial chloride channel from bovine trachea. / Cunningham, Sonia A.; Awayda, Mouhamed S.; Bubien, James K.; Ismailov, Iskander I.; Arrate, M. Pia; Berdiev, Bakhram K.; Benos, Dale J.; Fuller, Catherine M.

In: Journal of Biological Chemistry, Vol. 270, No. 52, 29.12.1995, p. 31016-31026.

Research output: Contribution to journalArticle

Cunningham, SA, Awayda, MS, Bubien, JK, Ismailov, II, Arrate, MP, Berdiev, BK, Benos, DJ & Fuller, CM 1995, 'Cloning of an epithelial chloride channel from bovine trachea', Journal of Biological Chemistry, vol. 270, no. 52, pp. 31016-31026. https://doi.org/10.1074/jbc.270.52.31016
Cunningham SA, Awayda MS, Bubien JK, Ismailov II, Arrate MP, Berdiev BK et al. Cloning of an epithelial chloride channel from bovine trachea. Journal of Biological Chemistry. 1995 Dec 29;270(52):31016-31026. https://doi.org/10.1074/jbc.270.52.31016
Cunningham, Sonia A. ; Awayda, Mouhamed S. ; Bubien, James K. ; Ismailov, Iskander I. ; Arrate, M. Pia ; Berdiev, Bakhram K. ; Benos, Dale J. ; Fuller, Catherine M. / Cloning of an epithelial chloride channel from bovine trachea. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 52. pp. 31016-31026.
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AU - Berdiev, Bakhram K.

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