AJP - Cell Physiology, Vol 263, Issue 4 C879-C887, Copyright © 1992 by American Physiological Society
Isolation of a chloride channel-enriched membrane fraction from tracheal and renal epithelia
C. L. Preston, M. A. Calenzo and W. P. Dubinsky
Department of Physiology and Cell Biology, University of Texas Health Science Center, Houston 77225.
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.
