From the Institut de Pharmacologie et de Toxicologie de l'Université, rue du Bugnon 27, CH-1005 Lausanne, Switzerland
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ABSTRACT |
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One of the characteristic features of the
structure of the epithelial sodium channel family (ENaC) is the
presence of two highly conserved cysteine-rich domains (CRD1 and CRD2)
in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat The amiloride-sensitive epithelial sodium channel
(ENaC)1 belongs to the
rapidly growing ENaC/DEG (degenerins) family of ion channels implicated
in a variety of physiological functions. The specific tissue and
species expression patterns of the channels allow us to classify them
in four subfamilies: (i) Structure-function studies performed on ENaC subunits have indicated
some specific functional roles of different domains of the proteins.
Schild et al. (7), have demonstrated that a stretch of amino
acids, preceding the second (M2) transmembrane domain (pre-M2 domain)
of each of the three subunits contributes to the formation of channel
pore and is a major site for amiloride binding. Schild et
al. (8), Snyder et al. (9), Staub et al.
(10), and Shimkets et al. (11) have identified a PY sequence
in the intracellular C termini of the To study the functional role(s) of CRD1 and CRD2 of ENaC, we replaced
the conserved cysteine residues with either serine or alanine.
Functional and cell surface expression of the mutants show that
replacement of two cysteines from the CRD1 of Site-directed Mutagenesis--
Cysteine to serine or alanine
mutations were introduced into the rat Electrophysiology and Binding Analysis--
Complementary RNAs
of each Temperature Dependence--
Under standard conditions, the
incubation temperature is kept at 19 °C. However, in our recent
study of human Cell Surface Expression of ENaC--
The cell surface expression
of ENaC was determined by specific binding of
[125I]M2IgG1 (M2Ab)
to oocytes expressing FLAG-tagged Immunoprecipitation--
Injected with wild-type or mutant ENaC
subunit oocytes were incubated in modified Bart's medium containing
0.8 mCi/ml [35S]methionine (NEN Life Science Products)
for the times indicated in the figure legends and then subjected to
different chase periods in the presence of 10 mM cold
methionine. Microsomes were prepared as described (20) and the
immunoprecipitations were performed as described (21) under
nondenaturing conditions, resolved by 5-8% gradient SDS-PAGE and
revealed by fluorography. The polyclonal antibodies used were raised
against the amino acids: 10 to 77 of the rat Functional Expression of
In the CRD2, two of the mutants ( Functional Expression of Mutant ENaC Is
Temperature-dependent--
Using the assay described under
"Experimental Procedures," we tested the temperature sensitivity of
wt and mutant channels at 30 °C versus 19 °C. At
30 °C all mutants in CRD1 (excepting
Interestingly, administration of two MTS (methanethiosulfonate)
derivatives, MTSEA (2-aminoethyl-methanethiosulfonate) and bulky
pyrene-7-MTS (2-[(3-pyrenylpropyl)carboxamido]ethyl
methanethiosulfonate) does not demonstrate a differential sensitivity
to these agents of wt and mutant channels. The affinity to amiloride is
not changed and the INa have an inhibition rate (~20%
with MTSEA and ~60% with pyrene-7-MTS) similar for the wild type and
all the 16
Finally, the studies of activity decreasing mutants indicate that the
delivery of the Functional Properties of Double Cysteine Mutants--
Various
double mutants ( Functional Effects of the C1S, C6S, C11S, and C12S Mutations in the
Protein Assembly and Stability of Mutant One of the distinct features of the ENaC/DEG gene family is its
membrane topology which predicts that more than 70% of the protein
mass is exposed to the extracellular space which is facing the external
compartment of the organism (urine in the distal nephron and feces in
the colon or airspace in the lung). This suggests that the
extracellular loop has a possible role in specific protein-protein
interaction (i.e with extracellular matrix proteins in the case of
degenerins (23)) and/or ligand-receptor interaction.
The extracellular loop of ENaC/DEG channel family proteins is
characterized by the presence of two or three cysteine-rich domains
(CRDs). It has been demonstrated that during protein folding and
maturation, extracellular cysteine residues are rapidly oxidized, and
that enzyme-catalyzed disulfide exchange continues until the most
thermodynamically stable conformation is reached. Among the transport
proteins, the formation of disulfide bonds in the extracellular loop or
N and C terminus of the protein is usually critical for proper
co-translational folding of the protein and subsequent assembly and
oligomerization in the ER compartment. For instance, mutation of
conserved cysteines forming disulfide bonds in the ectodomain of the
For the ENaC/DEG channel family, any cysteine of a given CRD could make
disulfide bonds with a cysteine located in the same CRD, with a CRD on
the same subunit (intrasubunit bonding), or with a cysteine of CRD
located on another subunit (intersubunit bonding). However, the
intersubunit bonding for these proteins is unlikely for the following
reasons: (i) immunoprecipitation of ENaC subunits under denaturing and
nonreducing conditions did not reveal intersubunit association in the
A6 cells (27), Madin-Darby canine kidney cells (13), Chinese hamster
ovary cells, and Xenopus oocytes2; (ii) FaNaC,
which is a homotetramer for which no evidence for intersubunit
disulfide bonding could be obtained (5). This indicates that the most
probable structural feature of the conserved cysteines is the formation
of the intrasubunit disulfide bonds. However, if all of the 16 cysteines are involved in disulfide bonds within the ENaC subunits, an
enormous number of permutations of disulfides is possible.
Several approaches have been used in order to determine the
exact pattern of disulfide bond formation for membrane proteins. A
possible biochemical approach uses the differential electrophoretic mobility by SDS-PAGE of the wild-type and the mutant proteins in their
nonreduced forms. However, for ENaC, the shift in the electrophoretic
mobility between the wild-type channel in its reduced and nonreduced
state was not high enough in our experiments to hope to identify as
much as eight possible disulfide bonds (data not shown). The similar
small shift between the reduced and nonreduced forms was also observed
for in vitro translated Among 16 mutated cysteines in the The important role of Cys1 in ENaC function is supported by
the recent report of a novel mutation causing PHA-1, a severe renal salt loosing syndrome caused by loss-of-function mutations of ENaC
subunits. The recently reported novel mutation is a cysteine from the
CRD1 in human Interestingly, despite of the absolute conservation of the
Cys11 and Cys12 among The absence of functional effects of other cysteine mutations (12)
could signify that they do not participate in the channel cellular
trafficking and/or in formation of protein structures necessary for
intrinsic channel activity. Therefore, a possible function of the
cysteines in formation of a ligand-binding site(s) can be considered.
In the absence of identified extracellular ligands for ENaC, the
proteins interacting with ENaC from the extracellular side can be
either the proteins of the extracellular matrix or channel modulating
proteins. The only known extracellular modulators of ENaC activity are
the serine proteases CAP1 (31) and trypsin (32). Both enzymes act
through the extracellular side and increase ENaC activity as high as 2 to 10 times without changing the number of channels expressed at the
cell surface. We tested the effect of trypsin on all the 16 To conclude, this study identifies a pair of cysteines in the CRDI of
ENaC
subunits by systematically mutating cysteine residues (singly or in
combinations) into either serine or alanine. In the Xenopus
oocyte expression system, mutations of two cysteines in CRD1 of
,
, or
ENaC subunits led to a temperature-dependent
inactivation of the channel. In CRD1, one of the cysteines of the rat
ENaC subunit (Cys158) is homologous to
Cys133 of the corresponding human subunit causing, when
mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe
salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines
in
and
but not in the
subunit also produced a
temperature-dependent inactivation of the channel. The main
features of the mutant cysteine channels are: (i) a decrease in cell
surface expression of channel molecules that parallels the decrease in
channel activity and (ii) a normal assembly or rate of degradation as
assessed by nondenaturing co-immunoprecipitation of
[35S]methionine-labeled channel protein. These data
indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite
for subunit assembly and/or intrinsic channel activity. We propose that
they play an essential role in the efficient transport of assembled
channels to the plasma membrane.
INTRODUCTION
Top
Abstract
Introduction
References
,
, and
ENaC subunits, mainly
expressed in epithelia; (ii) MDEG1, MDEG2, ASIC, and DRASIC genes,
recently identified in the nervous system of mammals; (iii) FaNaCh
involved in synaptic transmission in snail; (iv) MEC-4, MEC-10, DEG-1
(degenerins), and UNC 105 of the Caenorhabditis
elegans nematode expressed in sensory neurons and muscles,
respectively (1). All proteins of this supergene family share a common
membrane topology with two transmembrane domains, short intracellular N
and C termini and a large extracellular loop (2). An heterotetrameric
structure has been recently proposed for ENaC (3, 4), whereas an
homo-tetrameric structure has been proposed for FaNaCh (5). It is
therefore likely that this gene family of cation channel is tetrameric,
despite a study suggesting a nonameric architecture for ENaC (6).
and
subunits as possible
sites responsible for recycling of the cell surface expressed ENaC, either through the binding to an ubiquitine ligase (Nedd4), and/or by
clathrin-mediated endocytosis. In addition, mutations of the PY motif
abolishes the feedback inhibition of ENaC by intracellular Na+, an important mechanism controlling Na+
reabsorption (12). In intact cells (i.e. Xenopus
oocytes) phosphorylation studies have also demonstrated a possible role
of the C termini, as the target sequences for hormone-stimulated
protein kinases A- and C-dependent phosphorylation of the
and
ENaC subunits (13). The intracellular N terminus of the
subunit has been identified as a possible gating domain for ENaC (14).
The same portion of the
subunit protein has also been described as
participating in ENaC subunit assembly (15). Little information is,
however, available about the structural and functional role of the
extracellular loop of ENaC subunits. The extracellular loop represents
the largest domain (~70% of amino acids) and is glycosylated for
most members of the family. The most notable feature of the
extracellular loop is the presence of two cysteine-rich domains (CRD1
and CRD2) covering about 50% of the sequence. Among 15
ENaC
subunits cloned from different species, all the extracellular cysteines
(16) are conserved, suggesting that they are involved in disulfide bond formation. Most of these cysteines (14) are also conserved among the
genes expressed in mammalian nervous system (MDEG 1 and 2, ASIC, and
DRASIC) as well as among the degenerins and FaNaCh. The
degenerins family is characterized by the presence of an additional CRD
(16). Considering the number, sequence distribution, and potential
disulfide bond formation, one could suggest an important role(s) of
these cysteines in formation of mature ENaC/DEG channels. A particular
interest into the CRDs arose from the recently identified Cys133 to Tyr133 mutation in the CRD1 of human
ENaC subunit causing the pseudohypoaldosteronism type 1 (PHA-1), an
inherited disease characterized by severe neonatal salt wasting,
hyperkaliaemia, metabolic acidosis, and unresponsiveness to the
mineralocorticoid hormone aldosterone (17). In studies of the mechanism
of this mutation we have demonstrated that
C133Y replacement leads
to the temperature-sensitive decrease in functional activity of ENaC,
usually suggesting an impaired folding and/or assembly in the
endoplasmic reticulum (ER) compartment, as shown, for example, for the
CFTR
F508 mutant (18).
,
, and
ENaC
subunits and two cysteines from the CRD2 of the
and
but not the
subunit prevents the normal transport of assembled channels from
intracellular compartments to the plasma membrane, thereby decreasing
the number of ENaC molecules at the cell surface.
EXPERIMENTAL PROCEDURES
,
, and
ENaC cDNAs
by the polymerase chain reaction-based approach. The relative positions
of the conserved cysteines along the extracellular loops of
,
,
and
subunits are shown in Fig. 1.
Fig. 1 shows also an abbreviated numbering of the mutated cysteines used in this study to simplify the presentation of the data. The corresponding mutations are:
C1S is
C158S;
C2S is
C256S;
C3S is
C263S;
C4S is
C309S;
C5S is
C316S;
C6S is
C332S;
C7S is
C421S;
C8S is
C443S;
C9S is
C447S;
C10S is
C456S;
C11S is
C458S;
C12S is
C472S;
C13S
is
C483S;
C14S is
C498S;
C15S is
C502S;
C16S is
C506S;
C1S is
C98S;
C6S is
C270S;
C11S is
C399S;
C12S is
C413S;
C1S is
C100S;
C6S is
C284S;
C11S is
C410S;
C12S is
C424A. The
C424 has been mutated to an
alanine to avoid the formation of a consensus sequence for N-linked glycosylation. The rat
C158 (
C1) residue
corresponds to the human
C133 described as causing the PHA-1
phenotype when mutated to Tyr (
C133Y). All the
and
subunit
mutations were introduced into the FLAG epitope-tagged
and
cDNAs, allowing determination of the cell-surface expressing ENaC
using a binding analysis (see below). All mutations were confirmed by
the dideoxynucleotide sequencing method on both strands.
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Fig. 1.
Linear model of ENaC subunits. This
model shows two transmembrane domains M1 and M2, intracellular N and C
termini, and a large extracellular loop containing 16 cysteines
absolutely conserved among the ENaC subunits. In this study we have
classified these cysteines on two CRDs: one proximal to the M1
transmembrane segment CRDI and one distal to the M1 the CRDII. Relative
amino acid sequence positions of the conserved cysteines are depicted
as circles. The amino acid sequence numbers of these
cysteines as well as their replacements are indicated at the
top of the corresponding cysteines. The shortened numbers
(from 1 to 16) of the cysteines used in this work are indicated
under the circles. The Cys158 in rat
ENaC sequence corresponds to the Cys133 in human
ENaC
subunits. Mutation C133Y in human sequence causes the PHA-1
pathophysiology.
ENaC subunits and the corresponding mutants were
synthesized in vitro, and equal amounts of wild type (wt) or
mutated subunit cRNA at saturating concentrations for maximal channel
expression (9 ng of total cRNA) were injected into Xenopus
oocytes. Macroscopic whole oocyte Na+ currents
(INa) resulting from ENaC expression were measured using the two-electrode voltage-clamp technique and determined as the amiloride-sensitive inward current at
100 mV holding potential. For
both macroscopic current as well as binding analysis, the oocytes were
kept after injection into the low Na+ modified Barth saline
(MBS: 10 mM NaCl, 90 mM NMDG-Cl, 5 mM KCl, 0.41 mM CaCl2, 0.33 mM CaNO3, 0.82 mM
MgSO4).
ENaC C133Y mutation, this temperature has been
demonstrated to be permissive for the functional expression of the
mutant channel proteins that normally would not be expressed at the
cell membrane when the incubation temperature was raised to 30 °C
(17). To test for the temperature dependence of the cysteine mutants
activity into the rat sequence, we took advantage of the rapid
expression of ENaC in the Xenopus oocytes system, in which
significant channel activity is expressed as soon as 6-8 h after cRNA
injection. Under standard physiological temperature (19 °C), the
INa of wt
ENaC was typically between 10 and 15 µA/oocyte. Preliminary experiments showed that the
amiloride-sensitive sodium transport was
temperature-dependent and increased by 50-80% when the
temperature bath was raised to 30 °C, a maximum for a cell from a
poikilotherm animal. Temperature sensitivity was therefore tested by
comparing functional expression of wt and mutated channel protein at 19 versus 30 °C.
and
subunits as described
previously (19). Anti-FLAG M2 monoclonal antibody was
iodinated using the IODO-BEADS Iodination reagent (Pierce) and
carrier-free Na125I (Amersham), according to the Pierce
protocol. Iodinated antibody had a specific activity of 5-20 × 1017 cpm/mol. Binding of the iodinated antibody to oocytes
expressing the FLAG-containing wild-type or mutant
and
ENaC
subunits was determined 16-20 h after the cRNA injection. Twelve
oocytes in each experimental group were transferred into a 2-ml
Eppendorf tube containing the low sodium MBS supplemented with 10%
heat-inactivated calf serum and incubated for 30 min on ice. The
binding was started by the addition of 12 nM of the
iodinated antibody in a final volume of 100 µl. After 1 h
incubation on ice, the oocytes were washed eight times with 1 ml of low
sodium MBS supplemented with 5% calf serum and then transferred
individually into tubes containing 250 µl of low sodium MBS. The
samples were counted and the same oocytes utilized for two-electrode
voltage-clamp studies. Nonspecific binding was determined on oocytes
injected with untagged ENaC subunits.
ENaC subunit, 559 to
636 of the rat
ENaC subunit, and 570 to 650 of the rat
ENaC
subunit (2).
RESULTS
ENaC Cysteine Mutants in Oocytes at
19 °C--
The wild-type (wt) or
ENaC mutated subunits were
expressed with wt FLAG-tagged
and
subunits in
Xenopus oocytes to test for channel functional activity as
well as for cell-surface expression at 19 °C. As shown in Fig.
2, several cysteine mutants behave differently, both with respect to channel activity and to cell surface
expression. In the CRD1, the mutations
C1S and
C6S are characterized by a low macroscopic Na+ current
(INa) (Fig. 2A), as well as a decrease in cell
surface expression (Fig. 2B). For these mutants,
INa decreased significantly (35 ± 5%,
n = 46, p < 0.001 and 25 ± 4%,
n = 38, p < 0.001 of wt channel
activity, respectively). Binding analysis showed a corresponding
decrease in cell surface expression (21 ± 5%, n = 39, p < 0.001 and 20 ± 10%, n = 32, p < 0.001 of wt channel cell surface expression,
respectively). Mutants of other cysteines from the CRD1 (
C2S to
C5S) were indistinguishable from wild type channels (Fig. 2,
A and B).
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Fig. 2.
Effects of cysteine on serine substitutions
in the ENaC subunit on the functional activity
and cell-surface expression of ENaC at 19 °C.
Xenopus oocytes were injected with 3 ng of cRNA of each
and
FLAG-tagged ENaC subunits and 3 ng of cRNA of either wild-type
or cysteine to serine mutant
ENaC subunit. The oocytes were then
incubated 12-18 h at 19 °C. The cell surface expression of the
cysteines from the CRDI (B) or CRDII (E) was
determined by binding of 125I-labeled anti-FLAG antibody
and the same oocytes were then used for measurement of
amiloride-sensitive current INa (A and
D). The INa (µA) to cell surface binding
(fmol/oocyte) relationships (C and F) are means
of the relationships obtained in individual oocytes. Error
bars are mean ± S.E. of four experiments performed with 12 oocytes per experimental condition. Asterisk, denotes
statistical significance <0.001.
C11S and
C12S) showed a
significant decrease of INa at 19 °C (16.2 ± 3.7%, n = 27, p < 0.001 and 14.5 ± 2.6%, n = 27, p < 0.001, of wt
channel activity, respectively) (Fig. 2D). For these
mutants, a parallel decrease in cell surface expression was observed
(20.5 ± 4.8%, n = 27, p < 0.001 (
C11S) and 17.1 ± 4.1% n = 27, p < 0.001 (
C12S) of wt channel cell surface
expression) (Fig. 2E). In contrast, the other mutants
(
C7S,
C8S,
C10S,
C13S,
C15S, and
C16S) showed a
significant increase in INa with the greatest effect for
the
C7S and
C16S mutants (304 ± 27%, n = 27, p < 0.001 and 329 ± 29%, n = 29, p < 0.001, of wt channel activity, respectively) (Fig. 2D). However, the binding analysis did not reveal any
significant changes in their cell surface expression (Fig.
2E).
C4S) showed a significant
decrease in INa (Fig.
3A). The activity (INa) of
C1S and
C6S mutants further decreased to
near undetectable levels (5 ± 1% and 2.0 ± 0.5% of wt
channel activity, respectively). In CRD2, no significant activity could
be detected for the
C11S mutant (0.4 ± 0.1%,
n = 16, p < 0.001) and
C12S mutant
(0.2 ± 0.1%, n = 15, p < 0.001), respectively, of wt channel activity. For the other CRD2
mutants, a significant decrease in activity as compared with the
19 °C incubation condition was observed (Fig. 3B). Only
the
C7S mutant maintained a significant increase in channel activity
(201 ± 27%, n = 15, p < 0.005)
at 30 °C compared with the wt channel.
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Fig. 3.
Effects of cysteine on serine
substitutions in the ENaC subunit on the
functional activity of ENaC at 30 °C. Xenopus
oocytes were injected with 3 ng of cRNA of each
and
FLAG-tagged
ENaC subunits and 3 ng of cRNA of either wild-type or cysteine to
serine mutant
ENaC subunit. The oocytes were then incubated 6-8 h
at 30 °C before measurement of INa. Error
bars are mean ± S.E. of three experiments performed with 5 oocytes per experimental condition. * and # denote statistical
significance <0.001 and <0.005, respectively.
ENaC Cysteine Mutants and the Intrinsic Channel
Properties--
In Fig. 2, C and F, the
amiloride-sensitive sodium current (INa)/cell surface
expression (fmol) ratios are shown. If a mutation decreases
INa without changing the current/binding ratio, one can
deduce that channel delivery to the cell surface has been impeded with
no change in its intrinsic activity. Such was the case for
C1S and
C6S in CRD1 (Fig. 2C) and
C11S and
C12S in CRD2
(Fig. 2F). If a mutation increases the current/binding
ratio, one can deduce that the intrinsic channel activity has
increased. An example of this situation is the
C7S mutant (98.0 ± 14.3 µA/fmol, n = 27, p < 0.001)
and, to a lesser extent,
C8S (61.9 ± 8.5 µA/fmol, n = 29, p < 0.001),
C13S (44.2 ± 6.1 µA/fmol, n = 27, p < 0.001),
C15S (50.5 ± 9.5 µA/fmol, n = 29, p < 0.005), and
C16S (69.9 ± 5.3 µA/fmol,
n = 29, p < 0.001), compared with wt
channel current/binding ratio (22.5 ± 2.0 µA/fmol,
n = 30) (Fig. 2F). Changes in several biophysical properties of the channel (ion conductance, gating, ion
selectivity) could account for the differences in intrinsic channel
activity (see discussion in Ref. 22). In our experimental conditions,
it could be either the channel unitary conductance (gNa) or the mean
open probability (Po). In addition, alteration of
affinity to amiloride could influence the correct determination of
INa in loss of function mutations. To distinguish between
these possibilities, we measured the apparent inhibitory constant for
amiloride (Ki) and the unitary conductance for the
C1S,
C6S,
C11S, and
C12S as well as for the
C7S mutant. These experiments showed that neither Ki to
amiloride nor unitary conductance were affected by these mutations
(data not shown). In addition, measurements of ion selectivity did not reveal any differences between wt and mutated channels. The order of
ion permeability (Li+ > Na+
K+), characteristic of ENaC, was respected (data not shown).
ENaC mutants (data not shown). These experiments suggest
that in the three-dimensional structure of ENaC, positions of the
cysteines are distant enough from the channel pore and amino acids
determining the binding of amiloride.
C1S,
C6S,
C11S, and
C12S containing channels to the plasma membrane is severely impeded at 19 °C and even more at 30 °C. In contrast, incubation at 30 °C abolishes (
C8S,
C10S,
C13S,
C15S, and
C16S) or decreases (
C7S)
the increased activity of these mutants. We have therefore focused our
studies on the mutants where the functional effects of the mutations
were more pronounced with increased temperature.
C1/C6S,
C6/C11S,
C11/C12S,
C1/C11S,
C1/C12S,
C6/C12S) were constructed in order to verify whether these mutations affect the same functional domain of the channel (CRD1
or CRD2) or not. Additivity of the functional effect should indicate
that the two cysteines are in two distinct functional domains. Fig.
4 shows that this was not the case for
the
C1/C6S and
C11/C12S double mutants. Within CRD1 the double
mutant
C1S/C6S is not functionally different from the single
C1S
and
C6S mutants. Within CRD2, the double mutant
C11S/C12S was not
additive but rather more active than the
C11S and
C12S mutants
alone (Fig. 4). In contrast, the double mutations across CRD1 and CRD2
(
C1S/C11S,
C1S/C12S,
C6S/C11S, and
C6S/C12S) show strong
additivity and are no longer functional (Fig. 4). These data indicate
that the Cys1 and Cys6 in CRD1 on the one hand
and the Cys11 and Cys12 in CRD2 on the other
hand participate in the formation of two different functional domains
of the
ENaC subunit. Inactivation of both leads to complete loss of
channel activity.
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Fig. 4.
Effects of double cysteine mutants in
the ENaC subunit on the functional activity of
ENaC at 19 °C. Xenopus oocytes were injected with 3 ng of cRNA of each
and
FLAG-tagged ENaC subunits and 3 ng of
cRNA of either wild-type or double cysteine to serine mutant
ENaC
subunit. The oocytes were then incubated 12-18 h at 19 °C before
measurement of INa. Error bars are mean ± S.E. of four experiments performed with 5 oocytes per experimental
condition. All the groups containing mutant
ENaC subunits expressed
INa which was different from wt group with statistical
significance <0.001.
and
ENaC Subunits--
The conservation of the extracellular
cysteines among all the members of ENaC/DEG family suggests the
potential importance of the corresponding cysteine residues in the
function of
and
ENaC subunits. To verify this hypothesis, we
mutated the cysteines in the
and
ENaC subunits corresponding to
the
ENaC Cys1, Cys6,
Cys11, and Cys12 to either serine
or alanine and then expressed them in Xenopus oocytes. The
nonpermissive incubation temperature of 30 °C was chosen in order to
maximize the functional effects. Fig.
5A demonstrates that the
C1S and
C6S mutations cause similar effects compared with the
C1S and
C6S mutants. The
C1S and
C6S mutations provoked an
even more dramatic decrease in channel activity with almost no
detectable INa. The oocytes injected with triple
C1S/
C1S/
C1S and
C6S/
C6S/
C6S mutant subunits have no
detectable INa. These data indicate a similar functional
role for Cys1 and Cys6 in each of the three
ENaC subunits in CRD1. In CRD2, mutation of the Cys11 and
Cys12 in the
and
subunits revealed a different
pattern of expression. As shown in Fig. 5B, the effect of
the double
C11S/C12S mutant is similar to that of the
C11S/C12S
mutant. In contrast, the double
C11S/C12A mutant did not
significantly alter the functional activity. Under nonpermissive
condition (30 °C, 8 h of incubation) the activity of
,
C11S/C12S,
,
C11S/C12S channels was undetectable
(Fig. 5B). Mutation of the Cys11 and
Cys12 in all three
ENaC subunits led to
unfunctional channels. These data suggest an asymmetric role of
cysteines 11 and 12 in the
,
, and
subunits. The absence of
any detectable INa even after 76 h of expression of
the triple
C1S-C6S/
C1S-C6S/
C1S-C6S and
C11S-C12S/
C11S-C12S/
C11S-C12A mutants (Fig. 5C)
also shows that the cause of the impaired cell surface expression is
not due to a slow kinetic of intracellular transport but rather to a
complete blockade of routing to the cell surface.
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Fig. 5.
Effects of single
C1S,
C1S,
C1S,
C6S,
C6S,
C6S, and double
C11S/C12S,
C11S/C12S,
and
C11S/C12A mutations on the functional
activity of ENaC at 30 °C. Xenopus oocytes were
injected with 3 ng of each of the indicated cRNAs and incubated 6-8 h
at 30 °C before measurement of INa. Error
bars are mean ± S.E. of four experiments performed with 5 oocytes per experimental condition. All the groups containing mutant
ENaC subunits, except that containing
C11S/C12A mutant, expressed
INa which was different from wt group with statistical
significance <0.001.
,
, and
ENaC
Subunits--
The possible mechanisms which could explain the
decreased cell surface expression of the cysteine mutants are the
following: (i) misfolding during translation, (ii) lack of proper
oligomerization due to abnormal conformations of assembled complexes,
(iii) disruption of the normal transport of the mutant proteins from ER
to Golgi and/or from Golgi to the cell surface, or (iv) increased rate of degradation of the mutant proteins leading to a decrease of the
total channel protein pool. To test the hypothesis that misfolding by
inability to form disulfide bridges leads to the disruption of proper
assembly and oligomerization in the ER compartment (i and ii), we used
a nondenaturing co-immunoprecipitation test as described under
"Experimental Procedures." Oocytes were
[35S]methionine metabolically labeled at 30 °C for
8 h. Under this experimental condition, oocytes expressing
C1S-C6S/
C1S-C6S/
C1S-C6S and
C11S-C12S/
C11S-C12S/
C11S-C12A subunits have no detectable INa (data not shown). As shown in Fig.
6, the co-immunoprecipitation performed
with
,
, or
antibodies showed that the
C1S-C6S/
C1S-C6S/
C1S-C6S and
C11S-C12S/
C11S-C12S/
C11S-C12A mutants assembled as well as
that of
wild-type subunits. To test the degradation
hypothesis (iv), pulse-chase experiments were performed at 19 °C in
the oocytes injected either with wt
subunits or with
C1S-C6S/
C1S-C6S/
C1S-C6S and
C11S-C12S/
C11S-C12S/
C11S-C12A mutant subunits. Similar half-lives (9-11 h) were observed for both wt and mutant ENaC subunits
(Fig. 7). These results indicate
that neither subunit assembly nor the rate of degradation have
been grossly affected by the cysteine mutations.
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Fig. 6.
Co-immunoprecipitation of wild-type and
mutant ENaC subunits. Xenopus oocytes were either
injected with three wild-type subunits or three C1S/C6S,
C1S/C6S,
C1S/C6S double mutants or three
C11S/C12S,
C11S/C12S,
C11S/C12A double mutants. After 8 h of incubation at 30 °C
in the MBS solution containing 0.8 mCi/ml
[35S]methionine, the oocyte extracts under nondenaturing
conditions were prepared. Anti-
, -
, or -
polyclonal antibodies
were used to immunoprecipitate the corresponding subunits as well as
subunits associated with them. Fluorogram of 5-8% gradient SDS-PAGE
of one of the three similar experiments is shown.
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Fig. 7.
The similar rate of degradation of wild-type
and mutant ENaC subunits. Oocytes injected with 3 ng of either
wild-type or three C1S/C6S,
C1S/C6S,
C1S/C6S double mutants or
three
C11S/C12S,
C11S/C12S,
C11S/C12A double mutants were
labeled for 16 h with [35S]methionine and subjected
to the indicated chase periods. Digitonin extracts were prepared and
immunoprecipitated under nondenaturing conditions with an anti-
polyclonal antibody. Fluorogram of 5-8% gradient SDS-PAGE of one of
the three similar experiments is shown.
DISCUSSION
subunit of Na,K-ATPase prevents assembly with the
subunit
without changing the half-life of the
protein (24). Likewise,
cysteine bonds in the ectodomain of the low density lipoprotein
receptor allow the folding of this protein domain (repeated many times)
into an octahedral cage, binding of calcium molecules, required for
apoE and apoB binding and receptor recycling (25). Mutations of one or
two cysteines forming a disulfide bond in the N termini of either the
or
subunit of the acetylcholin receptor abolish channel subunit
assembly and ligand binding (26).
ENaC (28). Another broadly used
approach involves the modification of free cysteines liberated after
the mutation of their disulfide bond counterpart using the sulfhydryl
reagents. Analysis of differential sensitivity of wt and mutants to
these agents helped to identify the cysteine bond patterns in several
studies (29). However, our experiments with two MTS reagents have
demonstrated identical sensitivity of wt and mutant channels to these
reagents. Another tested approach is based on an assumption that the
effect of Cys
Ser or Cys
Ala mutations is limited to the
elimination of disulfide bonds which involved that Cys residue.
Therefore, mutation of either or both Cys involved in a given disulfide
bond results in channel mutants with the same functional properties.
For this, double cysteine mutants with mutated Cys1,
Cys6, Cys11, and Cys12 in
ENaC
were constructed. The similarity in decrease in INa, sensitivity to temperature, and absence of additivity in the
loss-of-function, suggested that the Cys1 and
Cys6, as well as Cys11 and Cys12
are pairs forming two disulfide bonds in each ENaC subunit.
ENaC subunit, four
(Cys1, Cys6 from CRD1, and Cys11
and Cys12 from CRD2) have demonstrated the critical
importance in functional expression of ENaC. The parallel decrease in
cell surface expression (Fig. 2, B and E) and the
lack of effect of the mutations on intrinsic channel properties
indicate that the primary cause of the impaired channel activity for
the mutants is the routing of the channels to the cell surface. The
temperature sensitivity of the mutants provides an additional indirect
evidence that the native protein structures formed with participation
of the cysteines are necessary for the proper accomplishment of one or
several steps in ENaC processing. In this study, we have considered two
possibilities to explain the decreased number of ENaCs containing the
mutant subunits at the cell surface: (i) affected assembly of the
channel subunits and (ii) their increased rate of degradation. However, metabolic labeling studies have demonstrated that neither assembly nor
degradation rate were affected in the mutant subunits (Figs. 6 and
7).
ENaC subunit (
C133Y) corresponding to Cys1 (Cys158) in the rat sequence. The
temperature sensitivity of
C1S mutation correlates well with the
temperature sensitivity of the human counterpart (
C158Y), described
by Gruender et al. (17) and with the severity of the
clinical phenotype.3
Similarly, the
,
, and
C6S and C11S, and
and
C12S
mutants, when incubated at 19 °C, expressed the significantly
reduced INa as compared with the wt channel (Fig.
2D), and demonstrated almost no activity when incubated at
30 °C (Fig. 3B). These results are reminiscent of a loss
of function mutation observed most frequently in cystic fibrosis
(CFTR
F508). This mutant is temperature-sensitive in vitro
and, decreasing the incubation temperature to 20 °C, rescues channel
activity to a significant level (18). The mechanism described for the
CFTR
F508 is, however, apparently distinct from that reported here.
For the CFTR
F508 mutant, the corresponding protein failed to traffic
to the plasma membrane, because it was stuck in the pre-Golgi
compartment and degraded in a ER/proteasome compartment. Loss of
function of the water channel aquaporin 2 causes nephrogenic insipidus
diabetes. Studies of the molecular cause of aquaporin 2 dysfunction in
the aquaporin 2 E258K mutant revealed that an assembled tetrameric
channel containing the mutant subunits is retained in the Golgi
complex, probably due to the introduction by this mutation of a Golgi
retention signal (30). We may have a similar situation for the C1S
mutant but, in the present study, we did not look for the precise
intracellular localization of the mutant protein. However, the correct
assembly and the normal glycosylation patterns of the mutated subunits
suggest that a similar mechanism is likely.
,
, and
ENaC
subunits, the
C11S-C12A mutant did not decrease channel function at
19 °C nor at 30 °C, while the corresponding pairs of the
and
subunits drastically decreased channel function in a
temperature-sensitive manner. This functional difference could be a
consequence of either the preferential assembly order of ENaC subunits
where assembly of the
subunit occurs in a latter step which is not
critical to complete the assembly or, of the different roles of ENaC
subunits in trafficking of assembled channels to the cell surface or,
of the differential conformational changes caused by these mutations
and allowing for the channels containing
but not
and
mutated subunits to reach the cell surface. For ENaC, the functional
differences between subunits have already been observed for (i) a
differential cell surface expression of the different subunit
combinations:
>
=
with no expression
for
,
, and
injected oocytes (19) or, (ii) the differential contribution of each subunit in functional domains such as
the amiloride binding and the sensitivity to divalent cations (7). In
other systems, such a difference has been described, for example, for
the AChR subunits, where assembly of the channel is blocked at
different steps depending on which conserved subunit the cysteine is
eliminated (26). The different behavior of the
subunit mutants
gives us an additional example of the asymmetric role of each ENaC
subunits in the function of the heterotetrameric protein.
ENaC
mutants and did not find a difference from the wild-type channel in the
level of stimulation of channel activity (data not shown). However, elimination of more than one cysteine might be necessary to abolish the
possible protein-protein interaction. Finally, we cannot exclude the
presence of an as yet unknown extracellular ENaC ligand function which
cannot be identified with the Xenopus oocyte expression system.
,
, and
ENaC subunits and a pair of cysteines in the CRDII of
and
subunits, essential for the channel routing to the plasma
membrane. It also establishes the molecular cause of human
C133S
mutation causing the pseudohypoaldosteronism type I pathophysiology,
which results in a reduced number of ENaCs at the cell surface.
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ACKNOWLEDGEMENTS |
---|
We thank Hans-Peter Gaeggeler for preparing the figures, Mark Ibberson for corrections and suggestions, and Nicole Skarda-Coderey for secretarial assistance.
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FOOTNOTES |
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* This work was supported by Swiss National Fund for Scientific Research Grants 31-43384.95 (to B. C. R.) and 31-39435.93 (to L. S.) and a National Institutes of Health SCOR Grant for hypertension.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.
Contributed equally to the present work.
§ To whom correspondence should be addressed: Institut de Pharmacologie et de Toxicologie de l'Université, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Tel.: 4121-692-5351; Fax: 4121-692-5355; E-mail: Bernard.Rossier{at}ipharm.unil.ch.
The abbreviations used are: ENaC, epithelial sodium channel family; PHA-1, pseudohypoaldosteronism type 1; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; MTS, methanethiosulfonate; CRD, cysteine-rich domains.
2 D. Firsov, unpublished data.
3 R. Lifton, personal communication.
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REFERENCES |
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