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.
Preston
C L
CL
Department of Physiology and Cell Biology, University of Texas Health Science Center, Houston 77225.
Calenzo
M A
MA
Dubinsky
W P
WP
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D002847:Chromatography, Agarose; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology; D008566:Membranes / Q000378:metabolism; D008854:Microscopy, Electron; D011189:Potassium Chloride / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure; D014132:Trachea / Q000378:metabolism*
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D011189:Potassium Chloride; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid