Cloning and characterization of a cDNA involved in porcine exocrine chloride conductance
Gaspar, Kimberly Jayne
A heterogeneous population of apical membrane chloride channels is responsible for transepithelial secretion of chloride ions in the small intestine, thereby providing a driving force for fluid secretion into the intestinal lumen. Previous experimental characterization of one of these pathways led to the selection of a monoclonal antibody capable of significantly inhibiting conductive chloride uptake into ileal apical membrane vesicles. This antibody was used to isolate a 0.94 kb cDNA clone, PG33, from a porcine intestinal expression library. A labeled probe constructed from this sequence identified a 2.7 kb band in a Northern blot of porcine parotid gland mRNA. Further clones were isolated through conventional oligonucleotide screening of the porcine library, as well as PCR-based screening of the porcine library and a human intestinal cDNA library. The total sequence cloned to date includes 2.3 kb, encoding a 679-amino acid translation product. The primary sequence bears significant homology to only two known genes: Ca-CC, a calcium-regulated chloride channel expressed in bovine tracheal epithelium, and Lu-ECAM-1, a lung endothelial cell adhesion molecule. Expression of PG33 mRNA was detected by reverse transcriptase PCR in several porcine secretory epithelial tissues, including ileum, trachea, and the major salivary glands (parotid, submandibular, and sublingual). In situ hybridization studies confirmed the ileal and tracheal expression. In porcine ileal mucosa, PG33 mRNA was detected in both the crypt and villus epithelial regions, while in the trachea, expression was observed both in the surface epithelium and the submucosal glands. Reverse transcriptase PCR studies demonstrated that the tissue expression of PG33 mRNA is distinct from that of the bovine chloride channel Ca-CC. Expression of Ca-CC appears restricted to bovine tracheal epithelium, and could not be detected in bovine ileum or parotid gland, nor in porcine trachea, ileum, or parotid gland. PG33 gene expression could not be detected in bovine tissues, though both PG33 and Ca-CC mRNA were detected in the human intestinal cell line T84 and the human airway cell line HBE-1. Expression of the PG33 and Ca-CC genes in these two lines may depend upon the differentiation status of the cell.