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Molecular and Functional Characterization of a Calcium-sensitive Chloride Channel from Mouse Lung
- Source :
- Journal of Biological Chemistry. 273:32096-32101
- Publication Year :
- 1998
- Publisher :
- Elsevier BV, 1998.
-
Abstract
- A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.
- Subjects :
- DNA, Complementary
Patch-Clamp Techniques
Transcription, Genetic
Recombinant Fusion Proteins
Molecular Sequence Data
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Biology
Kidney
Transfection
Biochemistry
Membrane Potentials
Mice
Chloride Channels
HSPA2
medicine
Animals
Humans
Amino Acid Sequence
Lung
Molecular Biology
Cell Line, Transformed
Gene Library
Gel electrophoresis
Messenger RNA
Sequence Homology, Amino Acid
cDNA library
Ionomycin
Niflumic acid
Niflumic Acid
Cell Biology
Embryo, Mammalian
Molecular biology
Recombinant Proteins
Dithiothreitol
Open reading frame
Transmembrane domain
Protein Biosynthesis
Chloride channel
Cattle
Calcium Channels
Sequence Alignment
medicine.drug
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 273
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....80a6cc7af43da92bbdad52c707c71708