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J Biol Chem, Vol. 274, Issue 53, 37693-37704, December 31, 1999
From the Department of Physiology and Biophysics, University of
Alabama at Birmingham, Birmingham, Alabama 35294-0005
The hypothesis that 30-amino acid peptides
corresponding to the C-terminal portion of the
Peptide Inhibition of Constitutively Activated Epithelial
Na+ Channels Expressed in Xenopus Oocytes*
- and/or 
-rat
epithelial sodium channel (rENaC) subunits block constitutively
activated ENaC was tested by examining the effects of these peptides on
wild-type (wt) rENaC (
-rENaC), truncated Liddle's mutants
(T-, T-, and
TT-rENaC), and point mutants
(Y-, Y-rENaC) expressed in
Xenopus oocytes. The chord conductances of
T-, T-, and TT-rENaC were 2- or 3-fold greater
than for wt -rENaC. Introduction of peptides into oocytes
expressing T-, T-, and
TT-rENaC produced a
concentration-dependent inhibition of the
amiloride-sensitive Na+ conductances, with apparent
dissociation constants (Kd) ranging from 1700 to
160 µM, depending upon whether individual peptides or
their combination was used. Injection of peptides alone or in
combination into oocytes expressing wt -rENaC or single-point
mutants did not affect the amiloride-sensitive whole-cell currents. The
single channel conductances of all the mutant ENaCs were the same as
that of wild type (-). The single channel activities
(N·Po) of the mutants were
~2.2-2.6-fold greater than wt -rENaC (1.08 ± 0.24, n = 7) and were reduced to 1.09 ± 0.17 by 100 µM peptide mixture (n = 9). The peptides
were without effect on the single channel properties of either wt or
single-point mutants of rENaC. Our data demonstrate that the C-terminal
peptides blocked the Liddle's truncation mutant
(TT) expressed in Xenopus oocytes but not the single-point mutants (Y or
Y). Moreover, the blocking effect of both peptides
in combination on TT-rENaC was synergistic.
*
This work was supported by NIDDKD Grant DK37206 from the
National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, University of Alabama at Birmingham, MCLM 704, Birmingham, AL 35294-0005. Tel.: 205-934-6200; Fax: 205-934-1445; E-mail: Benos@phybio.bhs.uab.edu.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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