From the Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the DEG/ENaC protein family form ion
channels with diverse functions. DEG/ENaC subunits associate as hetero-
and homomultimers to generate channels; however the stoichiometry of
these complexes is unknown. To determine the subunit stoichiometry of
the human epithelial Na+ channel (hENaC), we
expressed the three wild-type hENaC subunits (,
, and
) with
subunits containing mutations that alter channel inhibition by
methanethiosulfonates. The data indicate that hENaC contains three
,
three
, and three
subunits. Sucrose gradient sedimentation of
hENaC translated in vitro, as well as
-,
-, and
hENaC coexpressed in cells, was consistent with complexes containing
nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced
complexes of similar mass. Our results suggest a novel nine-subunit
stoichiometry for the DEG/ENaC family of ion channels.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The DEG/ENaC protein family includes channels with diverse physiologic and pathophysiologic functions. Epithelial Na+ channels (ENaC) absorb Na+ in kidney, lung, and intestine (1, 2), and mutations in human ENaC (hENaC) cause disease (3-6). Several family members from Caenorhabditis elegans, including MEC-4, MEC-10, and DEG-1, play a role in mechanotransduction, and some gain-of-function mutations cause neurodegeneration (7). In Helix aspersa, the FMRFamide-gated channel (FaNaCh) functions as a neurotransmitter receptor (8). Three family members have recently been identified in the mammalian nervous system, BNC1 (MDEG, BNaC1) (9-11), BNaC2 (ASIC) (11, 12), and DRASIC (13).
All members of the DEG/ENaC family appear to function as multimers.
ENaC contains three homologous subunits, ,
, and
(14-18). Functional studies show that simultaneous expression of all three subunits is required to generate maximal Na+ current,
although expression of
ENaC alone can produce small currents. In
addition, biochemical data show that the three human ENaC (hENaC)
subunits associate (19). Genetic evidence suggests that MEC-4, MEC-10,
and DEG-1 also function as heteromultimers (7, 20). However, the
subunit stoichiometry is not known for any DEG/ENaC channel.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNAs and mutations were generated as described previously
(9, 17, 18). FaNaCh was amplified by polymerase chain reaction following reverse transcription of RNA from H. aspersa. We
tagged the C terminus of hENaC with the sequence DYKDDDDK
(
Flag) for immunoprecipitation by anti-Flag M2
monoclonal antibody. This did not alter the function of the
subunit
in Xenopus oocytes or epithelia or its ability to associate
with other subunits (19).
Wild-type or mutant -,
-, and
hENaC (0.2 ng each) were
expressed in Xenopus oocytes by nuclear injection of
cDNA (18). When a mixture of wild-type and mutant cDNAs for a
subunit was coinjected, the total amount of cDNA for the subunit
remained constant. 16-24 h after injection, whole-cell Na+
current was measured by two-electrode voltage clamp at
60 mV (bathing
solution, 116 mM NaCl, 2 mM KCl, 0.4 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4). To increase the affinity for amiloride
of channels containing
G536C, we decreased the
Na+ concentration for experiments in Fig. 1 (25 mM NaCl, 93 mM KCl). Under these conditions, 3 mM amiloride completely inhibits hENaC current. The
fraction of Na+ current inhibited by
MTS1 reagents,
Inh, was determined by measuring current blocked by a
maximal concentration of amiloride before and after addition of MTSET
(Toronto Research Chemicals) or MTSEA to the bathing solution for
100 s. Following coexpression of wild-type and mutant hENaC
subunits, we determined the number of
,
, or
subunits in the channel complex, n, as described previously by
MacKinnon (21) using the equation
where Inhwt is the fraction of current
inhibited in channels containing only wild-type subunits,
Inhmut is the fraction of current
inhibited in channels containing n mutant subunits, and
f is the fraction of subunits expressed that are wild-type
or mutant, as indicated.
cDNAs were transcribed and translated in vitro in the presence of canine pancreatic microsomal membranes, as described previously (22). COS-7 cells were electroporated as described previously (19), pulse-labeled with 100 µCi/ml [35S]methionine (NEN Life Science Products) at 37 °C for 30 min, and then chased for 0-7 h at 15 °C. We have found that hENaC subunits assemble in the ER (19), then become insoluble to detergents prior to transport to the golgi.2 To minimize insolubility, we incubated cells at 15 °C to inhibit transport out of the ER. Proteins were solubilized in cold Tris-buffered saline (50 mM Tris, 150 mM NaCl) containing 1% digitonin, 0.4 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotonin, 20 µg/ml leupeptin, and 10 µg/ml pepstatin A and sedimented on sucrose gradients as described in figure legends.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stoichiometry of hENaC Using the G536C Mutation--
To
investigate the stoichiometry of hENaC, we first asked how many
subunits contribute to the channel complex. Our strategy, similar to
that described by MacKinnon (21), was to coexpress mixtures of
wild-type and mutant
subunits in Xenopus oocytes and
then determine the sensitivity to an inhibitor. We used
MTSET,3 an agent that
covalently modifies cysteines. Wild-type hENaC is relatively
insensitive to MTSET (Fig.
1A). Previous studies indicate
that Gly536 in the
subunit lines the channel pore
(23).3 When we replaced Gly536 with cysteine
(
G536C), MTSET irreversibly decreased current 87% (Fig.
1B) by covalently modifying the introduced cysteine. Wild-type
(
wt) and
G536C produced
equal Na+ currents (Fig. 1E). Interestingly,
when we coexpressed a 0.5:0.5 mixture of
wt and
G536C (with wild-type
and
hENaC), most of the
Na+ current (77%) was inhibited by MTSET (Fig.
1C). When we increased the contribution of
wt
(0.8:0.2
wt:
G536C), MTSET decreased Na+ current 48% (Fig. 1D). The finding that
MTSET decreased Na+ current out of proportion to the
fraction of mutant subunits suggested that hENaC contains more than one
subunit and the
G536C phenotype is dominant.
|
Stoichiometry of -,
-, and
hENaC Using the "Deg"
Mutation--
In contrast to MTSET, MTSEA inhibited current in cells
expressing wild-type subunits by 54% (Fig.
2A). Our unpublished data suggest that MTSEA inhibits by covalently modifying one or more pore-lining cysteines in the
subunit. As a second independent test
of the number of
subunits and to determine the number of
and
subunits, we took advantage of a mutation in the Deg residue that
prevents inhibition. In MEC-4, mutation of Ala442 to bulky amino acids causes neurodegeneration (20), and mutation of the equivalent residue in BNC1 activates the channel (10). In hENaC subunits, the Deg residues are serines. We found that mutation of a Deg
serine to cysteine in any of the subunits (
S549C,
S520C, and
S529C) abolished inhibition by
MTSEA. Instead, MTSEA and MTSET (Fig. 2A) stimulated a
small increase in Na+ current. In this regard, modification
of this residue in hENaC may be similar to the activation that occurs
with a bulky residue at the Deg position in MEC-4 and BNC1. Perhaps
after the cysteine is modified, it provides a steric barrier that
prevents MTSEA from entering the pore where it could alter the
-cysteine(s) and inhibit current.
|
Sucrose Gradient Analysis of Channel Mass--
To further test
stoichiometry, we determined the molecular mass of hENaC by sucrose
gradient sedimentation. Because hENaC can form a homomeric channel,
we first determined the mass of channels containing only the
subunit. We translated
hENaC in vitro with microsomal
membranes. This assay allows subunit multimerization in the ER, while
minimizing the possibility that other cellular proteins will associate
with the hENaC channel complex and alter its molecular mass. Fig.
3A shows that unglycosylated
subunits sedimented mainly in fractions 4 and 5, similar to a
standard of 240 kDa. This migration suggested that most of the
unglycosylated subunits were in the form of dimers or trimers,
consistent with our previous finding that subunit interactions begin
prior to glycosylation (19). In contrast, glycosylated subunits
sedimented in the same fractions (8-10) as a 950-kDa standard (Fig.
3A), consistent with a channel complex containing at least
nine subunits. Because most of the
subunits in fractions 8-10 were
glycosylated, the data suggest that subunits oligomerize during
processing in the ER. Further, multimerization is probably very
efficient, since little glycosylated
was found in fractions 4 and
5. Because most multimeric proteins multimerize in the ER (24), this
assay probably provides an accurate representation of the size of the complex at the plasma membrane.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our functional and our biochemical data support a model of hENaC
containing nine subunits; three , three
, and three
. These
results suggest a novel stoichiometry for an ion channel and contrast
with the stoichiometry of voltage-gated K+ channels (four
subunits) (21) and Na+/Ca2+ channels (four
repeats), nicotinic acetylcholine receptors (five subunits), and gap
junction hemichannels (six subunits) (25).
The finding that BNC1 and FaNaCh migrated on sucrose gradients similar to hENaC suggests that those channels may also be constructed from nine subunits. Thus, a multimeric complex composed of nine subunits may be a conserved feature of the DEG/ENaC family. In addition, genetic evidence suggests that some C. elegans family members contain more than one copy of each subunit. For example, MEC-4, MEC-10, and probably one unidentified subunit may form a functional complex. A loss-of-function mutation on one mec-4 allele suppressed a dominant neurodegeneration-associated mutation on the other mec-4 allele (26). Similar interactions appear to occur with MEC-10 subunits (27). These results suggest that the functional complex contains two or more MEC-4 and two or more MEC-10 subunits (7).
The approach we used has several advantages. First, the
electrophysiological assays allowed us to selectively determine the stoichiometry of functional channels. If different combinations of
subunits produced nonfunctional channels or channels not delivered to
the cell surface, they would not be detected. Second, our approach allowed us to determine the absolute number of ,
, and
subunits in the channel complex, rather than a ratio of one subunit
relative to another. Third, use of two independent electrophysiological assays for the
subunit strengthen our conclusions. Fourth, we were
able to avoid the use of concatameric constructs, which could disrupt
the normal association of subunits, as previously reported for Shaker
K+ channels (28). Finally, we used both functional and
biochemical approaches. Both supported the same conclusion.
Our approach also has limitations. First, our electrophysiological assays would not detect inactive channels. Second, our sucrose gradient assay detected channels in the ER, rather than channels at the plasma membrane. However, earlier work suggested that the hENaC subunits assemble in the ER (19). Finally, our electrophysiological approach relies on two assumptions; that wild-type and mutant subunits express equally and associate randomly and that a single mutant subunit has a dominant effect on MTS sensitivity. As discussed above, these assumptions are likely valid. However, if the assumption that a single mutant subunit is dominant is incorrect, our calculations will underestimate the number of subunits. In the case of all three limitations, the concordance between biochemical and functional data support the validity of our approach.
It is interesting to speculate how nine subunits might assemble to form
a highly selective channel. Based on theoretical considerations, Guy
and Durell (29) independently proposed a model for ENaC that
contains three , three
, and three
subunits. In their model,
the pore is formed by
barrels derived from the residues immediately preceding M1 and M2, and they predicted that all nine subunits contribute to the channel pore. Our experimental data are
consistent with this model.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank H. Robert Guy for discussions about his model of ENaC, Theresa Mayhew, Ellen Tarr, and Dan Bucher for technical assistance, and John B. Stokes, Christopher Adams, Margaret Price, Joseph Cotten, and our other laboratory colleagues for helpful discussions. We thank the University of Iowa DNA Core for assistance.
![]() |
FOOTNOTES |
---|
* 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.
Supported by a Fellowship from the Roy J. Carver Charitable
Trust and by the NHLBI and NIDDK, National Institutes of Health. To
whom correspondence should be addressed: Dept. of Internal Medicine,
University of Iowa College of Medicine, 200K EMRB, Iowa City, IA 52242. Tel.: 319-356-7481.
§ Supported by a National Research Service Award from the NHLBI, National Institutes of Health.
¶ Supported by the Howard Hughes Medical Institute.
1 The abbreviations used are: MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis.
2 L. S. Prince and M. J. Welsh, unpublished observations.
3 P. M. Snyder and M. J. Welsh, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|