Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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The hypothesis that there is a highly
conserved, positively charged region distal to the second transmembrane
domain in -ENaC (epithelial sodium channel) that acts as a putative
receptor site for the negatively charged COOH-terminal
- and
-ENaC tails was tested in mutagenesis experiments. After expression
in Xenopus oocytes,
-ENaC constructs in which positively
charged arginine residues were converted into negatively charged
glutamic acids could not be inhibited by blocking peptides. These
observations provide insight into the gating machinery of ENaC.
Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region
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INTRODUCTION |
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LIDDLE'S SYNDROME is
a form of hereditary hypertension produced by mutations within the
epithelial sodium channel (ENaC) (1, 17). These mutations
result in constitutive channel activation. Both an increase in
functional channel number and an increase in single-channel open
probability (Po) have been reported
(5-9, 14, 15). The initial description of
Liddle's syndrome identified truncation mutations in the COOH-terminal
polypeptide chain of the -ENaC (and subsequently the
-ENaC)
subunit as being causative for constitutive channel activation
(3, 5-9, 14, 15). We proposed the hypothesis that the
COOH-terminal chains of
and
could act as intrinsic channel
blockers by serving as an inactivation moiety. Our evidence, obtained
in both bilayers (7, 8) and heterologous expression
systems (9), supports this type of mechanism. We tested
the hypothesis that the functional gating particle comprised the
COOH-terminal tails of both the
- and
-ENaC subunits associated
as a two-strand, antiparallel
-sheet. Support for this idea was
threefold: 1) the inhibitory effects of adding COOH-terminal
- and
-ENaC 30-amino acid residue peptides together with ENaC
comprising wild-type
- and COOH-terminally truncated
- and
-subunits produced a greater than additive inhibition of the
channel; 2) circular dichroism studies showed that the 30-mer
and
peptides formed a
-sheet; and 3) when
the isoleucines and valines within the 30-mer peptides were replaced by
the
-sheet, breaking amino acids proline or aspartic acid, the
resulting peptides were unable to affect basal-activated ENaC
(8). The paradigm that we have developed for ENaC gating
is as follows. Because
-ENaC itself forms a functional sodium
channel (4), there must be an intrinsic gating mechanism
in
-ENaC alone. We hypothesized that calcium is intimately involved
in this process and have presented evidence to this effect
(2). The overall gating properties of
-ENaC vs.
-ENaC in bilayers do not differ (7). Because the
elimination of the cytoplasmic COOH-terminal tails of either or both of
the
- and
-subunits substantially increases single-channel Po (5-9), there must be at
least two separate gating processes, one inherent to
-ENaC alone and
one conferred onto the complex by the
- and
-subunits.
To further elucidate the mechanism underlying the COOH-terminal -
and
-ENaC tail block of ENaC, we tested the hypothesis that a highly
conserved region following (or at the most distal end of) the second
transmembrane domain (M2) in
-ENaC may act as a putative receptor
region for the negatively charged COOH-terminal
- and
-ENaC tails
and, thus, facilitate their interaction with the channel. We have
identified a sequence of positively charged amino acids between
residues 586 and 591 of human
-ENaC (613-624 of the rat
-ENaC ortholog) (10) that is identical in all five mammalian
-ENaC subunits cloned to date (rat, bovine, human, mouse,
and guinea pig) and that is arginine rich (RRFRSRYWSPGR). This region
is conserved in
-ENaC (RRLRRAWFSWPR) (16) but is not
present in either the
- or
-ENaC subunit or in the ENaC-related Aplysia sodium channel that is gated by FMRF-amide
(phenylalanine-methionine-arginine-phenylalanine) (12). We
hypothesized that this concentration of positive charge may form a site
for electrostatic interaction of the negatively charged COOH-terminal
and
tails and, thus, provide an anchoring point for the
peptides somewhere near the internal channel mouth. The peptides may
bind at a site away from the actual channel mouth but exert an
inhibitory effect via steric hindrance or by inducing a conformational
change. In the present experiments, we tested this hypothesis by
mutating either two or four of the arginine residues in this sequence
to amino acids of opposite charge. Our prediction was that the
disruption of the charge distribution in this region would abolish the
ability of the COOH-terminal
- and
-ENaC peptides to inhibit the channel.
We expressed the -ENaC subunit truncated at amino acid
R564 (
R564X) and the
-ENaC truncated at
amino acid R574 (
R574X) in combination with
the wild-type rat
-ENaC subunit (i.e.,
R564X
R574X) in Xenopus
oocytes. Figure 1 shows representative amiloride-sensitive Na+ current traces obtained in
voltage-clamped oocytes before and after 1 mM peptide mixture
injection. In this series of experiments, the currents obtained from
oocytes injected with wild-type
-ENaC and truncated
- and
-ENaC subunits were greater than the corresponding currents recorded
from wild-type
-ENaC injected in oocytes obtained from the
same frogs (data not shown). The average inward current levels were
approximately twofold increased, as reported earlier (9).
Injection of a mixture of
plus
COOH-terminal peptides
(SP30
and SP30
; 1:1, 500 µM each) into
oocytes expressing the truncated ENaC constructs (i.e.,
R564X
R5764X-ENaC) decreased the
current by ~50% at negative potentials. In contrast, the
currents in oocytes expressing either of the
double mutations
(i.e.,
R586E,R587E or
R589E,R591E) or the
quadruple mutation in the
-subunit
(
R586E,R587E,R589E,R591E
R564X
R574X)
were not affected by the same concentration of peptide mixture. As
controls, the
R564X
R574X-ENaC
associated current was not affected by water injection or by injection
of SP30
plus a 30-amino acid peptide identical to
SP30
, with substitution of three prolines for one valine
and two isoleucines (see Fig. 4). Summary data for the effect of the
peptide mixture on wild-type
-subunit plus truncated
- and
-subunits, two double
arginine mutations, and the quadruple mutation (all in combination with the truncated
- and
-subunits) are shown in Fig. 2. Of these constructs,
only the channel containing wild-type
-ENaC was inhibited by 1 mM
peptide mixture. In contrast, both double mutants (namely,
R586E,R587E and
R589E,R591E) and the
quadruple mutant, in which the four positively charged arginines (R
residues) were replaced with negatively charged Glu (E residues), were
unaffected by injection of SP30
plus SP30
(Fig. 2).
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As further controls for these experiments, we made a series of mutations in the same position as the arginines, except that instead of
changing the sign from positive to negative (glutamic acid), we
maintained the same charge distribution by substitution with lysine
(K). In an additional group of oocytes, 1 mM peptide added to
R586K,R587K,R589K,R591K
R564X
R574X,
R586K,R587K
R564X
R574X, or
R589K,R591K
R564X
R574X
inhibited macroscopic currents to the same extent as when added to the
wild-type
-subunit. These data are supportive of the hypothesis that
this arginine-rich region in the most distal portion of the M2 region
plays a role in the interaction of the
-and
-subunit tails,
causing inhibition of the channel.
We used single-channel analysis to explain more precisely the results
that we obtained with these COOH-terminal peptides in macroscopic
current measurements in heterologously expressing oocytes. Figure
3 shows representative single-channel
current traces for R564X
R574X, the two
double
arginine mutants, and the quadruple mutant in combination
with the truncated
- and
-subunits. The unitary conductances of
all of these combinations of mutated and truncated ENaCs averaged 7 pS,
demonstrating that there was no effect on the single-channel
conductances by introduction of these mutations in the
-ENaC
subunit. The peptide mixture only inhibited the single-channel activity
(NPo) of the wild-type
construct (Fig. 3,
horizontal bar above traces). The peptide mixture was without effect in
either of the double Glu (R
K)
mutants or in the quadruple
Glu (R
E)
mutant. The value of NPo was decreased to 1.27 ± 0.3 by the peptide mixture from 2.67 ± 0.4 in patches isolated from oocytes expressing
R564X
R574X-ENaC. In contrast, the
value of NPo recorded from excised patches with the quadruple mutant was unchanged following peptide exposure (2.91 ± 0.4 and 2.89 ± 0.3). In contrast, the values of
NPo before and after addition of the control
peptide mixture were 1.02 ± 0.19 and 1.14 ± 0.22 (n = 3, Fig. 4).
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These direct biophysical measurements of single ENaC demonstrate
directly that the COOH-terminal regions of the - and
-ENaC subunits function as intrinsic gating particles and that the
interaction site occurs at an arginine-rich region located at the most
distal portion of the M2 segment of the
-ENaC subunit. Thus these
terminal cytoplasmic domains of
- and
-subunits play an important
role in ENaC gating.
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EXPERIMENTAL PROCEDURES |
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Construction of -ENaC mutations.
Full-length human
-ENaC cDNA was a gift from Dr. Michael J. Welsh (University of Iowa) (13), and truncated
- and
-ENaC cDNAs were a gift from Dr. Bernard Rossier (Université
et Lausanne, Switzerland) (14). Point mutations in the
-subunit were constructed by using the QuickChange mutagenesis kit
(Stratagene, La Jolla, CA). Each set of primers contained the
appropriate base changes required to code for either two or four
glutamates instead of the wild-type arginine residues. Plasmid cDNA,
PCR, in vitro transcription, and bacterial transformation were all done
as described previously (11). cDNA products containing the
specific mutations were confirmed by dideoxy sequence analysis as well.
Oocyte preparation and electrophysiological recording.
Oocytes were removed from anesthetized adult female Xenopus
laevis (Xenopus Express, Beverly Hills, FL) by standard technique (9). Follicle cells were removed in OR-2 calcium-free
medium (in mM: 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 Na2HPO4, and HEPES 5.0, pH 7.5), with the
addition of collagenase. Defolliculated oocytes were washed in both
OR-2 (calcium-free) and OR-2 (complete) medium (in mM: 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 CaCl2, 1.0 Na2HPO4, and HEPES 5.0, pH 7.4) and allowed to
recover overnight in half-strength Liebovitz's medium at 18°C. Stage
VI oocytes were injected with 50 nl (8.3 ng of the appropriate -,
-, and
-ENaC cRNA construct; all subunit mixtures were 1:1:1).
Two-electrode voltage clamp and/or single-channel measurements were
made 24-48 h postinjection as described previously
(9). Oocytes were clamped at a holding potential of 0 mV.
The current-voltage relationships were acquired by stepping the holding
potential in 10-mV increments from
100 to +100 mV. Current-voltage
data were recorded after the monitoring currents were stable, before
and after the application of 10 µM amiloride to the bath. Data were
sampled at a rate of 1 kHz and filtered at 500 kHz. Data analysis was
also as described previously (9). Analysis of
single-channel data was performed by using FETCHAN and pSTAT
programs of pCLAMP version 8.0 software (Axon Instruments,
Burlingame, CA) as previously described (9).
Peptide synthesis and purification.
Peptides were synthesized by ResGen (Huntsville, AL). After synthesis,
the peptides were subjected to reversed-phase, high-performance liquid
chromatography to increase their purity to >90%. Only a single peak
was observed on the final chromatograph. The peptides were analyzed for
amino acid composition by mass spectroscopy. The sequences for the
COOH-terminal 30-amino acid-long -ENaC (SP30
) and
-ENaC (SP30
) peptides were as follows:
SP30
, PIPGTPPPNYDSLRLQPLDVIESDSEGDAI; and
SP30
, PGTPPKYNTLRLERAFSNQLTDTQMLDEL.
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ACKNOWLEDGEMENTS |
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We thank LaToya Bishop, Susan Copeland, and Hannah Mebane for excellent technical assistance and Isabel Quinones for superb secretarial help. We also thank Dr. J. K. Bubien for helpful comment and for critically reading the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206.
Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, The Univ. of Alabama at Birmingham, 1918 University Blvd., MCLM 704, Birmingham, AL 35294-0005 (E-mail: benos{at}physiology.uab.edu).
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.
10.1152/ajpcell.00610.2001
Received 26 December 2001; accepted in final form 1 April 2002.
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