Epitope tagging permits cell surface detection of functional cftr
.
Howard, Marybeth, Michael D. Duvall, Daniel C. Devor, Jian-Yun Dong,
Karlin Henze, and Raymond A. Frizzell.
Department of Physiology and Biophysics and Gregory Fleming James
Cystic Fibrosis Research Center, University of Alabama at Birmingham,
Birmingham, Alabama 35294-0005
APStracts 2:0249C, 1995.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a
phosphorylation-activated chloride channel responsible for cAMP
-induced Cl secretion across the apical membranes of epithelial cells.
To optimize its detection for membrane localization studies, we
tagged CFTR with epitope sequences at the carboxy terminus or in the
fourth external loop. When epitopes were added to the fourth external
loop, the N-linked glycosylation sites in that loop were either
preserved or they were mutated to produce a deglycosylated CFTR
(dgCFTR). Tagged-CFTRs were expressed in HeLa cells and their cAMP
-sensitive Cl permeability was assayed using the halide-sensitive
fluorophore, SPQ. CFTRs containing the M2 epitope showed halide
permeability responses to cAMP, whereas cells expressing CFTR with
the hemagglutinin HA tag showed little or no cAMP response. Xenopus
oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl
conductance responses that were 20% of the wt response, whereas M2
-tagged constructs retaining the glycosylation sites responded like wt
CFTR. External M2-tagged CFTR was detected in the surface membrane of
non-permeabilized cells. The surface expression of the mutant M2
-tagged CFTRs correlated with processing of these mutants (Gregory et
al., 1993). M2-901/CFTR is a useful reporter for the trafficking of
wt and mutant CFTRs to the cell surface.
Received 11 April 1995; accepted in final form 14 June 1995
APS Manuscript Number C0206-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 18 July 1995.