Immunolocalization of chloride-transporting membrane vesicles in tracheal epithelial cells.

A membrane fraction eluted from a phenyl Sepharose column (MPS) was isolated from renal cortex and bovine tracheal epithelia that is enriched in a single type of Cl- channel [C. L. Preston, M. A. Calenzo, and W. P. Dubinsky. Am. J. Physiol. 263 (Cell Physiol. 32): C879-C887, 1992]. A 200-kDa membrane protein that copurifies with and appears to be specific to this fraction was purified and used to raise antisera for immunological characterization of these membranes. The antisera reacted in immunoblots with a 200-kDa protein in homogenates of bovine trachea, kidney, pancreas, lung, and intestine. There was also cross-reactivity with a 200-kDa protein in immunoblots in rat stomach, pancreas, and lung. There was no cross-reaction with rat skeletal muscle, cardiac muscle, or aorta. Thus this protein appears to be preferentially enriched in epithelial tissues. Examination of each major fraction during the purification of MPS membranes from trachea shows no enrichment of the 200-kDa protein in plasma, mitochondrial, or nuclear membrane fractions. The only significant enrichment was observed in MPS that is purified by hydrophobic chromatography. In frozen sections, antisera and monospecific immunoaffinity-purified antibodies localize the protein primarily to the apical domain of tracheal columnar epithelial cells with small punctate structures throughout the cytoplasmic compartment.

Isolation of a chloride channel-enriched membrane fraction from tracheal and renal epithelia.

A membrane fraction, enriched in Cl- channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The fraction (MPS) shows a 37-fold enrichment of Cl- channels over crude tracheal homogenates by net Cl- flux measurements. Alkaline phosphatase and Na(+)-K(+)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. Marker enzyme analysis for major subcellular membranes also proved negative. The MPS fraction exhibits a protein profile unlike that of other membrane fractions, with major proteins of 200 and 42 kDa, proteins of 30-35 kDa, and lesser amounts of other proteins. Reconstitution of MPS fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (H. H. Valdivia, W. P. Dubinsky, and R. Coronado. Science Wash. DC 242: 1441-1444, 1988). In addition, the voltage dependence, selectivity sequence of Cl- > Br- > or = I-, and inhibition by low concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid correspond to those of the predominant apical membrane channel. Thus, although the MPS appear to be of subcellular origin, they may be functionally related to an apical membrane Cl- permeability.