From the Departments of Medicine and Physiology,
University of Pennsylvania and Veterans Administration Medical Center,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The epithelial Na+ Channel
(ENaC) mediates Na+ reabsorption in a variety of epithelial
tissues. ENaC is composed of three homologous subunits, termed ,
, and
. All three subunits participate in channel formation as
the absence of any one subunit results in a significant reduction or
complete abrogation of Na+ current expression in
Xenopus oocytes. To determine the subunit stoichiometry, a
biophysical assay was employed utilizing mutant subunits that display
significant differences in sensitivity to channel blockers from the
wild type channel. Our results indicate that ENaC is a tetrameric
channel with an
2
stoichiometry, similar to that
reported for other cation selective channels, such as Kv,
Kir, as well as voltage-gated Na+ and
Ca2+ channels that have 4-fold internal symmetry.
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INTRODUCTION |
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Epithelial Na+ channels are expressed in apical plasma membranes of principal cells in the distal nephron, airway and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, and other tissues including ducts of salivary and sweat glands (1-3). These channels mediate reabsorptive Na+ transport across epithelial cell layers (2-5) and are selectively inhibited by submicromolar concentrations of the diuretic amiloride (6). Epithelial Na+ channels have a key role in the regulation of urinary Na+ reabsorption, extracellular fluid volume homeostasis, and control of blood pressure, and are a major site of action of volume regulatory hormones, including aldosterone (2, 7, 8). The role of Na+ channels in blood pressure regulation has been illustrated in recent studies that have identified mutations in ENaC as the basis of the pathogenesis of Liddle's disease, a disorder characterized by volume expansion and hypertension (9, 10); as well as type I pseudohypoaldosteronism, a disorder characterized by volume depletion and hypotension (11).
The epithelial Na+ channel consists of at least three
structurally related subunits, termed
-ENaC,1
-ENaC, and
-ENaC (epithelial Na+ channel) (12). The primary and
predicted secondary structures of these ENaCs have been described
(12-15). Each subunit has two predicted
-helical membrane spanning
regions separated by a large extracellular domain. Significant amino
acid sequence similarities across species have been observed for
individual subunits (on the order of ~60% to greater than 90% amino
acid homology), although regions are present that are more highly
conserved. A family of genes identified in Caenorhabditis
elegans based on mutations that result in mechanosensation defects
(mecs) and degeneration of selected neuronal cells (degs) are
structurally related to ENaCs (16-18). Several of these genes,
including mec-4, mec-6, and mec-10,
are thought to form an ion channel in a manner analogous to the three
ENaC subunits (16, 19). These observations suggest that ENaCs and mecs
(and degs) are members of a new gene superfamily. Members of this
family include ENaCs, mecs and degs, FaNaCh (a peptide-gated channel
cloned from the marine snail Helix aspers),
-ENaC, and
BNaC (cloned from human brain)), ASIC (an acid-sensing ionic channel),
DRASIC (dorsal root ganglia acid-sensing ion channel) (20-24), and
likely includes mechanosensitive cation channels present on cochlear
hair cells and oocytes (2, 3, 5, 18). Epithelial Na+
channels are composed of at least three structurally distinct subunits
(12). We have used a biophysical approach to assess number of each ENaC
subunit that assembles to form the Na+ channel complex
(i.e. subunit stoichiometry). Our results suggest that ENaC
has a tetrameric structure and is composed of two
-, one
-, and
one
-subunit.
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EXPERIMENTAL PROCEDURES |
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Materials-- Reagents were purchased from vendors listed below or from Sigma.
Preparation of Mouse ENaC (mENaC) Mutants-- All three mutants were made using a polymerase chain reaction (PCR)-based mutagenesis technique (25). For each mutant, two rounds of PCR amplification were performed using Pfu DNA polymerase (Stratagene Corp., La Jolla, CA) and an MJ Research thermal cycler (Watertown, MA). Two overlapping fragments were generated in the first round of PCR amplification using the internal mutagenic forward primer with the 3' end external reverse primer and, in a separate reaction, using the internal mutagenic reverse primer with the 5' end external forward primer. In the second round of PCR amplification, the two overlapping fragments generated in the first PCR were first spliced together by two rounds of thermocycling, followed by 22 rounds of amplification with added external primers. The PCR product from the second step was ligated into the original mENaC clone, using unique restriction sites at the two ends. All sequences were confirmed by automated DNA sequence analysis performed at the University of Pennsylvania's DNA sequencing facility.
Expression of Na+ Channels in Xenopus
Oocytes--
The mutant and the wild type cRNAs were prepared using a
cRNA synthesis kit (m-MESSAGE mMACHINE, Ambion Inc., Austin, TX). cRNA
concentration was measured spectroscopically. For optimum ENaC
expression, equal amounts (3 ng) of each of the three subunits were
injected into oocytes and electrophysiological measurements were
performed 1 to 5 days after injection. Two-electrode voltage clamp
experiments were performed by clamping oocytes to 100 mV (with
reference to bath) for 500 ms and 0 mV for 450 ms. For all measurements, the current difference between the two command potentials was used for data analysis. The difference in current measured in the
presence and absence of amiloride (100 µM for wild type mENaC and 1 or 5 mM amiloride for mutant mENaCs) was used
to define the amiloride-sensitive current. Amiloride titration
measurements were performed under continuous flow (~4 ml/min) of
buffers containing varying concentrations of amiloride. Single channel
recordings were performed in the cell attached configuration. All data
were collected at room temperature and were filtered at 100 Hz. The applied voltage to the membrane patch represents the voltage deflection from the resting membrane potential. Inward Na+ current is
represented by a downward deflection in single channel recordings.
Solutions-- Following injection, oocytes were incubated in modified Barth's saline solution containing (in mM): 88 NaCl, 1 KCl, 2.4 NaHCO3, 15 HEPES, 0.3 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, pH 7.2. The buffer was supplemented with 10 µg/ml penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamycin sulfate, and 10 µg/ml nystatin.
The bath media for two-electrode voltage clamp experiments contained in mM: 100 sodium gluconate, 2 KCl, 1.8 CaCl2 ,10 HEPES, 5 BaCl2, and 10 TEA-Cl, pH 7.2. Measurements of single channel conductance were performed with a buffer containing (in mM): 100 NaCl, 1.8 CaCl2, 2 KCl, 10 HEPES, pH 7.2, in the pipette and in the bath. In patch-clamp studies performed to examine the effects of (2-aminoethyl)methanethiosulfonate (MTSEA, Toronto Research Chemicals Inc., Toronto, Ontario, Canada), the bath solution contained (in mM): 100 potassium gluconate, 2 KCl, 1.8 CaCl2 10 HEPES, 5 BaCl2, 10 TEA-Cl, pH 7.2; the pipette solution in these experiments contained (in mM): 100 LiCl, 1.8 CaCl2, 2 KCl, 10 HEPES, pH 7.2. To investigate the effects of MTSEA on single channels, a 5 mM MTSEA solution was freshly prepared for each patch-clamp, and was perfused into the pipette (26) following single channel recordings in the absence of the drug.Data Analysis-- Curve fitting and statistical analysis were performed using Matlab software package (The MathWorks, Inc., Natick, MA). The inhibition constant, Ki, and the pseudo Hill coefficient, n', described by the Langmuir inhibition isotherm (Equation 1) were determined by nonlinear regression,
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(Eq. 1) |
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RESULTS |
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Wild type and mutant mENaC subunits that express different sensitivities to channel blockers were used to determine mENaC stoichiometry. The injection of oocytes with varying ratios of wild type and mutant mENaC cRNAs results in expression of oligomeric channels, each containing either inhibitor-sensitive and/or inhibitor-insensitive subunits. Analyses of the effects of inhibitors on the expressed channels allows for determination of subunit stoichiometry (27-29). mENaC is blocked by amiloride in the submicromolar range (IC50 of 0.1 µM).2 All three subunits appear to participate in amiloride binding, as single point mutations in the putative pore forming regions of any of these subunits results in altered amiloride binding (30).
The mutants S583C,
G525C, and
G542C were used for these
studies, as they differ from wild type ENaC in their sensitivities to
channel blockers (30).
,
G525C,
-mENaC and
,
,
G542C-mENaC are inhibited by amiloride with IC50
values of 73 and 43 µM, respectively (Fig.
1 and Table
I).
S583C,
,
-mENaC is blocked by
amiloride with an IC50 of 0.6 µM (Table I),
sufficiently close to the IC50 of wild type mENaC to
preclude the use of amiloride in the determination of the stoichiometry
of the
-subunit. However, wild type mENaCs are insensitive to the
sulfhydryl reagent MTSEA (0.5 mM), whereas
S583C,
,
-ENaC is blocked by MTSEA (Fig.
2A).
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The analysis of subunit stoichiometry assumes that the functional
properties of mutant and wild type channels are similar, other than
differential sensitivities to inhibitors. We examined the single
channel conductances of the mutant mENaCs (S583C,
,
-mENaC;
,
G525C,
-mENaC; and
,
,
G542C-mENaC) expressed in
Xenopus oocytes (Fig. 3). The
slope conductance of
S583C,
,
-mENaC was 4.7 pS, essentially
indistinguishable from wild type mENaC.2 In contrast, the
slope conductance for
,
G525C,
-mENaC was 2.8 pS, and for
,
,
G542C-mENaC was 3.2 pS. These slope conductances are in
reasonable agreement with previous observations (30). As the single
channel conductances of
,
G525C,
-mENaC and
,
,
G542C-mENaC differ from wild type
,
,
-mENaC, these
differences were taken into account when determining
- and
-subunit stoichiometry.
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-Subunit Stoichiometry--
The differential sensitivities of
wild type
,
,
-mENaC and
S583C,
,
-mENaC to MTSEA were
used to determine
-subunit stoichiometry. Fig. 2 illustrates the
response to MTSEA (0.5 mM) of oocytes injected with a 1:1
(Fig. 2B) or 4:1 (Fig. 2C) mixture of wild type
and
mutant (
S583C) cRNAs. This figure also illustrates the
predicted responses to MTSEA for
-subunit stoichiometries (N) of 1, 2, or 3; assuming, 1) a binomial distribution of wild type and mutant
-subunits in the expressed mENaCs; and 2) that a single
MTSEA-sensitive subunit (i.e.
S583C) confers blocker
sensitivity to the heteroligomeric channel. The responses of oocytes
injected with either 1:1 or 4:1 mixtures of wild type and mutant cRNAs
were consistent with an
-subunit stoichiometry of two. The maximum
likelihood of N to match all data is attained with an n = 2. An n = 2 is e59 times more
likely than n = 1, and e23 times
more likely than n = 3 to produce the experimental
results.
|
-Subunit Stoichiometry--
The differential
sensitivities of wild type mENaC and
,
G525C,
-mENaC to
amiloride were used to determine
-subunit stoichiometry. The
responses to amiloride of oocytes injected with a 1:1 or a 1:4 mixture
of wild type (
,
,
) and mutant (
,
G525C,
) mENaC cRNAs
are illustrated in Fig. 5. This figure
also illustrates the predicted responses of oocytes injected with a 1:1
or 1:4 mixture of wild type and mutant cRNAs, for
-subunit
stoichiometries (n) of 1 or 2. A random assembly resulting
in a binomial distribution of wild type and mutant
-subunits
expressed in channels was assumed. In generating the predicted
responses, the conductance difference between the mutant and the wild
type channels has been taken into account (see Fig. 5, legend). If
channels are composed of 2
-subunit, three distinct populations of
channel will be present: (i) wild type channels, (ii) fully mutant
channels, and (iii) channels that have both a wild type and a mutant
-subunit (i.e. hybrid channels). The values for the
Ki for amiloride, Hill coefficient, and single
channel conductance for the hybrid channels were obtained by minimizing
the
2 error of the predicted response for a
stoichiometry of 2 to the experimental data. Optimized parameters from
nonlinear regression were utilized in likelihood ratio analysis. This
analysis indicated that n = 1 is
e25 times more likely to produce the
experimental results than n = 2. Therefore, the
experimental data is most consistent with the a
-subunit
stoichiometry of one.
|
-Subunit Stoichiometry--
A similar approach was
adopted to determine the stoichiometry of
-subunit. The responses to
amiloride of oocytes injected with a 1:1 or a 1:4 mixture of wild type
(
,
,
) and mutant (
,
,
G542C) mENaC cRNAs is
illustrated in Fig. 6A. The
predicted responses for
-subunit stoichiometries of 1 and 2 are also
plotted in the figure. In generating the predicted response for a
stoichiometry of 2, the Hill coefficient, Ki for
amiloride and single channel conductance for the hybrid species
(channels containing both wild type
and the
G542C mutant) were
inferred from minimizing
2 error by nonlinear
regression. Likelihood analysis indicate that n = 1 fits the experimental data e61 times better than
n = 2.
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DISCUSSION |
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Our analyses of inhibition of wild type and mutant mENaCs suggest
that ENaCs are composed of two -subunits, one
-subunit, and one
-subunit. This channel has a quaternary structure, similar to that
reported for Kv, Kir, and voltage-gated
Na+ and Ca2+ channels (Na+ and
Ca2+ channels are composed of a large polypeptide with
4-fold internal symmetry) (27, 29, 31, 32). As mentioned above, the
analysis of subunit stoichiometry rests on the assumption that the
integration of wild type or mutant subunits into Na+
channels during assembly is a random event. The validity of this assumption is supported by analyses of currents expressed by oocytes injected with mixtures of wild type and mutant subunits that
demonstrated that the distribution of these subunits was indeed
binomial. Our analysis also rests on the assumption that a single
drug-sensitive subunit confers blocker sensitivity to the channel.
Again, this is a valid assumption. We demonstrated that channels that
have one wild type and one mutant (i.e.
S583C)
-subunit, or channels that have two mutant
-subunits, are
completely blocked by MTSEA (Figs. 2, B and C,
and 4). As Na+ channels have only one
-subunit and one
-subunit, the presence (or lack) of a mutant
-subunit
(
,
G525C,
) or mutant
-subunit (
,
,
G542C) determines
the sensitivity to amiloride.
Firsov and co-workers (33) have recently reported an ENaC subunit
stoichiometry in agreement with what we observed, using both a
biophysical approach as well as analysis of ENaC subunit concatemers,
although limitations in the use of concatemers to determine
stoichiometry of ion channels have been described (34). Snyder and
co-workers (35) have recently reported an entirely different ENaC
stoichiometry of 3 -, 3
-, and 3
-subunits. This proposed
stoichiometry of 9 subunits would be unique for an ion channel. In
utilizing a biophysical approach to determine subunit stoichiometry, a
major assumption is that other than differential sensitivities to
inhibitors, functional properties of mutant and wild type channels are
similar (27, 28). These authors utilized 2 different mutants to
determine
-subunit stoichiometry, one of which was used in our study
(
G542C-mENaC) (35). mENaCs that have this mutant have a single
channel conductance that is 69% of wild type. If we did not include a
correction for the reduced conductance of the mutant channel, our
results would indicate a
-subunit stoichiometry of between 1 and 2. The effects of the other subunit mutations (
S549C,
S520C,
S529C) on human ENaC functional (single channel) properties were not
reported. These mutations are within largely hydrophobic regions
preceding the predicted second membrane spanning domain of the channel
that appear to participate in the formation of the channel pore. If these mutations introduce changes in human ENaC (hENaC) functional properties in the presence (or absence) of the methanethiosulfonate derivatives used in their study, analyses of the response to inhibitors might lead to an error in determination of subunit stoichiometry. In
addition, a determination of stoichiometry requires that the relative
amounts of Na+ subunits expressed in oocytes are precisely
known. The authors injected hENaC subunit cDNAs into oocytes and
examined subsequent functional expression. In this regard, both
transcription and translation efficiencies among the different subunits
must be similar. The use of cDNAs, rather than cRNAs, introduces an
independent variable that is not present in our expression studies.
Snyder and co-workers (35) also examined the mass of hENaC expressed in
COS-7 cells and synthesized in vitro in the presence of
microsomal membranes by sucrose density sedimentation. A predicted mass
of ~950 kDa was observed, and the authors suggest that this size is
consistent with an ENaC stoichiometry of 3 -, 3
-, and 3
-subunits. However, ENaC is likely associated with cytoskeletal proteins, and these associated proteins may contribute to the apparent
size of the ENaC complex, assessed by sucrose density sedimentation.
For example, we have observed that ankryin,
-spectrin, and the
protein Apx (Apical protein Xenopus) (36)
co-immunoprecipitate with Xenopus
ENaC.3
-Subunits by themselves can form ENaC channels (12, 17). Recent
studies, presented in abstract form, suggest that these
-subunit
channels are composed of four subunits (37), consistent with the
tetrameric structure that we report for
,
,
Na+
channels. These studies used an
-subunit that has a deletion of a
6-residue tract (rat
-ENaC
278-283 (
-rENaC
278-283)) that results in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (38). Oocytes expressing
278-283,
,
-rENaCs have current levels that are markedly
lower than currents observed in oocytes expressing wild type
,
,
-rENaCs (38). This precluded the use of
-rENaC
278-283 in our analysis of
,
,
-ENaC stoichiometry. We
previously observed that
-subunit channels formed by the mutant
-rENaC H282D was insensitive to submicromolar concentrations of
amiloride (38). Interestingly,
H282D,
,
-rENaC is inhibited by
amiloride with an IC50 similar to wild type
,
,
-rENaC (data not shown), again precluding the use of this
mutant in our analysis of
,
,
-ENaC stoichiometry.
,
-, Or
,
-subunits expressed in oocytes form functional Na+
channels (12). McNicholas and Canessa (39) suggested that
,
-rENaC
and
,
-rENaC are composed of equal numbers of
- and
-subunits or of
- and
-subunits. As our results suggest that ENaCs have a tetrameric structure, we propose that these channels are
composed of 2
- and 2
-subunits or of 2
- and 2
-subunits.
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ACKNOWLEDGEMENT |
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We express our sincere thanks to Dr. Martin Pring for assistance with statistical analyses.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK51391, DK50268, and HL07027, the Department of Veterans Affairs, and the Cystic Fibrosis Foundation.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: Medical Research (151), VA Medical Center, University and Woodland Aves., Philadelphia, PA 19104. Tel.: 215-823-5177; Fax: 215-823-5171; E-mail: kleyman{at}mail.med.upenn.edu.
1 The abbreviations used are: ENaC, epithelial Na+ channel; mec, mechanosensation defects; deg, degenerin; PCR, polymerase chain reaction; MTSEA, (2-aminoethyl)methanethiosulfonate.
2 Y. Ahn, F. Kosari, J. Li, and T. R. Kleyman, manuscript in preparation.
3 J. B. Zuckerman, X. Chen, T. R. Kleyman, and P. R. Smith, manuscript in preparation.
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REFERENCES |
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