Cloning of an Epithelial Chloride Channel from Bovine Trachea

Abstract

We have isolated and cloned a novel epithelial Cl ؊ channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (␣p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca 2؉ /calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl ؊ channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by ␣p38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the ␣p38 antibody.

Regulation of a Human Chloride Channel

“…Earlier molecular studies have distinguished two members of the human Cl Ca channel family, hCLCA1 and hCLCA2 (14,16,17,36), but these all have larger unitary conductances (13-30-pS) than the 1-pS Cl Ca channel that we have studied. We also used pharmacological criteria to distinguish the 1-pS channel from hCLCA1 and hCLCA2; the latter are blocked by adding 2 mM dithiothreitol outside the cell (16,17), whereas the Cl Ca channel in CFPAC-1 cells was not significantly affected by dithiothreitol.…”

Re-expression of Detachment-inducible Chloride Channel mCLCA5 Suppresses Growth of Metastatic Breast Cancer Cells

“…Another common feature is a symmetrical multiple-cysteine motif, CX 12 CX 4 CX 4 CX 12 C, in the amino-terminal tail. Potential phosphorylation sites for calmodulin kinase II and protein kinase C are consistent with calcium regulation, and ectopic expression of CLCA proteins leads to a novel chloride current that is activated by calcium and blocked by typical chloride channel inhibitors such as DIDS (19,23,26,28). However, it has not been firmly established whether CLCAs conduct chloride directly or are instead channel regulators.…”

Association of the hCLCA1 gene with childhood and adult asthma

“…The hCLCA1 enhances mucus secretion by mediating the active transport of chloride ions. 10 Currently, the family includes two bovine homologues (bCLCA1 and bCLCA2), [17][18][19] four murine homologues (mCLCA1, mCLCA2, mCLCA3 (gob-5), mCLCA4), [20][21][22][23][24] and four human homologues (hCLCA1, hCLCA2, hCLCA3, hCLCA4 (hCaCC2)). 15,16,25,26 All human CLCA genes identified are clustered on the short arm of chromosome 1 (1p22-31).…”