Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes.
Attempts to attribute ileal brush-border chloride conductance to specific proteins were pursued by screening a porcine intestinal cDNA library. A 0.94-kb clone was identified on expression screening with a monoclonal antibody that inhibited enterocyte brush-border chloride conductance. Further screening approaches led to the isolation of a 3.1-kb full-length sequence called pCLCA1, consistent with the identification of a 2.9-kb transcript through Northern analysis. This sequence had significant homology to the CLCA gene family of calcium-regulated chloride channels, especially to hCLCA1. However, a strong A-kinase consensus phosphorylation site in a predicted cytoplasmic loop of the protein was a notable difference from the hCLCA1 gene product. Several porcine exocrine epithelial tissues, including ileum, trachea, and the major salivary glands express pCLCA1 mRNA. In situ hybridization studies localized the expression of pCLCA1 mRNA to the crypt and villus epithelia of porcine ileum, whereas tracheal expression was observed in both surface epithelium and submucosal glands. In situ expression of pCLCA1 in mouse 3T3 cells induces an ionomycin-dependent chloride conductance activity in these cells.
Gaspar
K J
KJ
Department of Veterinary Physiological Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4.
Racette
K J
KJ
Gordon
J R
JR
Loewen
M E
ME
Forsyth
G W
GW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D016764:Cell Polarity; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D003001:Cloning, Molecular; D004553:Electric Conductivity; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020869:Gene Expression Profiling; D006031:Glycosylation; D006801:Humans; D007082:Ileum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017403:In Situ Hybridization; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D016415:Sequence Alignment; D013552:Swine / Q000235:genetics*; D014162:Transfection
C492109:CLCA1 protein, human; D018118:Chloride Channels; D002712:Chlorides; C116464:Clca3a1 protein, mouse; D008565:Membrane Proteins; D012333:RNA, Messenger; D015759:Ionomycin