Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.

Academic Article

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.
  • Published In

    Keywords

  • 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid, Amino Acid Sequence, Animals, Calcium Channels, Cattle, Cell Line, Transformed, Chloride Channels, DNA, Complementary, Dithiothreitol, Embryo, Mammalian, Gene Library, Humans, Ionomycin, Kidney, Lung, Membrane Potentials, Mice, Molecular Sequence Data, Niflumic Acid, Patch-Clamp Techniques, Protein Biosynthesis, Recombinant Fusion Proteins, Recombinant Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Transcription, Genetic, Transfection
  • Author List

  • Gandhi R; Elble RC; Gruber AD; Schreur KD; Ji HL; Fuller CM; Pauli BU
  • Start Page

  • 32096
  • End Page

  • 32101
  • Volume

  • 273
  • Issue

  • 48