Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes

Attempts to attribute ileal brush-border chloride conductance to specific p roteins 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 scree ning approaches led to the isolation of a 3.1-kb full-length sequence calle d 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. Howev er, a strong A-kinase consensus phosphorylation site in a predicted cytopla smic loop of the protein was a notable difference from the hCLCA1 gene prod uct. Several porcine exocrine epithelial tissues, including ileum, trachea, and the major salivary glands express pCLCA1 mRNA. In situ hybridization s tudies localized the expression of pCLCA1 mRNA to the crypt and villus epit helia of porcine ileum, whereas tracheal expression was observed in both su rface epithelium and submucosal glands. In situ expression of pCLCA1 in mou se 3T3 cells induces an ionomycin-dependent chloride conductance activity i n these cells.