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(Received for publication, July 16, 1996, and in revised form, September 23, 1996)
From the a Renal Division and the b Center for Cell
and Molecular Signaling, Departments of c Medicine and
i Physiology, Emory University, and d Department of
Veterans Affairs Medical Center, Atlanta, Georgia 30322, the
Departments of e Medicine and j Physiology,
University of Pennsylvania and k Department of Veterans Affairs
Medical Center, Philadelphia, Pennsylvania 19104, and
h Department of Physiology, Allegheny University,
Philadelphia, Pennsylvania 19129
Mutations in a Cl
Volume 272, Number 1,
Issue of January 3, 1997
pp. 594-600
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
channel (cystic
fibrosis transmembrane conductance regulator or CFTR) are responsible
for the cystic fibrosis (CF) phenotype. Increased Na+
transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+
channel open probability (Po). The
Xenopus renal epithelial cell line, A6, expresses both
cAMP-activated 8-picosiemen (pS) Cl channels and
amiloride-sensitive 4-pS Na+ channels, and provides a model
system for examining the interactions of CFTR and epithelial
Na+ channels. A6 cells express CFTR mRNA, as
demonstrated by reverse transcriptase-polymerase chain reaction and
partial sequence analysis. A phosphorothioate antisense
oligonucleotide, complementary to the 5
end of the open reading frame
of Xenopus CFTR, was used to inhibit functional expression
of CFTR in A6 cells. Parallel studies utilized the corresponding sense
oligonucleotide as a control. CFTR protein expression was markedly
reduced in cells incubated with the antisense oligonucleotide.
Incubation of A6 cells with the antisense oligonucleotide led to
inhibition of forskolin-activated amiloride-insensitive short circuit
current (Isc). After a 30-min exposure to 10 µM forskolin, 8-pS Cl channel activity was
detected in only 1 of 31 (3%) cell-attached patches on cells treated
with antisense oligonucleotide, compared to 5 of 19 (26%) patches from
control cells. A shift in the single-channel current-voltage
relationship derived from antisense-treated cells was also consistent
with a reduction in Cl reabsorption. Both
amiloride-sensitive Isc and Na+
channel Po were significantly increased in
antisense-treated, forskolin-stimulated A6 cells, when compared with
forskolin-stimulated controls. These data suggest that the regulation
of Na+ channels by CFTR is not limited to respiratory
epithelia and to epithelial cells in culture overexpressing CFTR and
epithelial Na+ channels.
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