From the Department of Physiology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104 and the Departments of
¶ Medicine and of
Cell Biology and Physiology, University
of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received for publication, January 7, 2003, and in revised form, January 27, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epithelial sodium channels (ENaCs) are composed
of three homologous subunits that have regions preceding the second
transmembrane domain (also referred as pre-M2) that form part of the
channel pore. To identify residues within this region of the
The epithelial sodium channel
(ENaC)1 represents the
dominant pathway mediating Na+ reabsorption in the distal
nephron, distal colon, and airway. ENaC has a critical role in
regulating transepithelial Na+ transport and modulating
extracellular fluid volume and blood pressure. ENaCs are composed of
three homologous subunits, termed We observed previously that the systematic introduction of cysteine
mutations within the Materials--
All chemicals were purchased from Sigma unless
specified otherwise.
Site-directed Mutagenesis--
Mouse ENaC ( Channel Expression in Xenopus Oocytes--
Xenopus oocytes
(stage V-VI) were pretreated with 2 mg/ml collagenase (type IV) in a
Ca2+-free saline solution. ENaC cRNAs (1-3 ng/subunit in
50 nl of H2O) were microinjected into oocytes. The oocytes
were incubated at 18 °C in modified Barth's saline (88 mM NaCl, 1 mM KCl, 2.4 mM
NaHCO3, 0.3 mM Ca(NO3)2
0.41 mM CaCl2, 0.82 mM
MgSO4, 15 mM HEPES-NaOH, pH 7.2, supplemented
with 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, and
100 µg/ml gentamycin sulfate. Whole cell currents were measured
18-96 h after cRNA injections.
Whole Cell Current Measurements--
A two-electrode voltage
clamp technique was used to measure the whole cell inward
amiloride-sensitive currents in oocytes expressing wild type or mutant
Pipettes of borosilicate glass capillaries (Worldwide Precision
Instruments Inc., Sarasota, FL) were utilized to pull electrodes with a
micropipette puller (Sutter Instrument Co., Novato, CA). The electrodes
were filled with 3 M KCl, and the resistances were 0.5-3
megaohms in a 110 mM NaCl bath solution. Oocytes were
bathed in a solution containing 110 mM NaCl, 2 mM CaCl2, 2 mM KCl, 10 mM HEPES-NaOH, pH 7.40. For selectivity experiments,
separate bath solutions containing either Na+,
Li+, or K+ as the predominant cation were used.
All measurements were carried out at room temperature (22-25 °C),
and the bath solution was continuously perfused at 5 ml/min by gravity.
Oocytes were typically incubated in the bath solution for at least 10 min before the current was recorded to allow currents to stabilize.
Membrane potentials were clamped from
The accessibility of mutant channels with cysteine substitutions within
the pore region to sulfhydryl reagents was examined using the
sulfhydryl-reactive reagents MTSEA and [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET) (Toronto Research
Chemicals, Inc., North York, Canada). Na+ currents were
measured before and 3 min following perfusion of oocytes with the
methanethiosulfonate (MTS) reagent. Oocytes were washed with an
MTS-free NaCl bath solution for >10 min to determine the reversibility
of the current response. A bath solution containing 0.1 or 1.0 mM amiloride was then perfused into the oocyte chamber to
determine the amiloride-insensitive component of the whole cell current.
Rates of inhibition of Na+ currents by MTSEA were
determined by clamping the membrane potential every 5 s at Data Analyses--
Data are expressed as mean ± S.E.
Student's t test was used for significance analyses with MS
Excel 97 software.
Expression of Channels with Cysteine Mutations of Mutations within the Pore Region of Mutations within the Pore Region of the
Little or no inward amiloride-sensitive currents carried by
K+ were observed with 12 mutations within the pore region
of Selected Mutations Alter the Sensitivity of mENaC to MTS
Reagents--
We examined the accessibility of extracellular MTSEA
and MTSET to substituted cysteine residues within the pore region of
Residues within the pre-M2 region comprise the external portion of the
ENaC pore, and several investigators have proposed that the external
pore gradually narrows as it enters the membrane-spanning region to
form the selectivity filter that restricts K+ (9, 11, 12).
If this model is correct, the relative accessibility of
As MTSEA inhibited wild type channels, it is possible that the
introduction of cysteine residues at
MTSET substitutes a large cationic group (trimethylaminoethyl) onto the
sulfhydryl group of cysteine residues, compared with the smaller
aminoethyl modification with MTSEA. We examined the accessibility of
selected substituted cysteine residues within the pore region of the
Recent studies suggest that a limited region preceding the M2
domain of each subunit forms part of the ENaC pore. Selected mutations
within these regions alter ion selectivity, single channel conductance,
amiloride sensitivity, and channel gating (8-14, 18, 19). Changes in
IK/INa, indicating that
channels exhibited a measurable inward K+ current, were
only observed with mutations within a 3-residue tract
(Gly587-Ser588-Ser589) of the
The introduction of cysteine residues at specific sites within the pore
region of the Previous studies examining the effects of amino acid substitutions or
deletions on the efficacy of channel inhibition by amiloride indicated
that selected mutations of residues at a homologous site within the
We examined the accessibility of substituted cysteine residues within
the pore region of the The response of these mutant channels to the larger MTSET was modest at
best. Furthermore, MTSET did not alter currents in oocytes expressing
The specific sites within the 3-residue tract where mutations
allowed for K+ permeation are not identical among the three
ENaC subunits. Selected mutations of the first residue (mouse
We propose a model of this region where the -subunit that line the pore, we systematically mutated residues
Gln523-Ile536 to cysteine. Wild type and
mutant mouse ENaCs were expressed in Xenopus oocytes, and a
two-electrode voltage clamp was used to examine the properties of
mutant channels. Cysteine substitutions of 9 of 13 residues
significantly altered Li+ to Na+ current
ratios, whereas only cysteine replacement of
Gly529
resulted in K+-permeable channels. Besides
G525C, large
increases in the inhibitory constant of amiloride were observed with
mutations at
Gly529 and
Ser531 within the
previously identified 3-residue tract that restricts K+
permeation. Cysteine substitution preceding (
Phe524 and
Gly525), within (
Gly530) or following
(
Leu533) this 3-residue tract, resulted in enhanced
current inhibition by external MTSEA. External MTSET partially blocked
channels with cysteine substitutions at
Gln523,
Phe524, and
Trp527. MTSET did not inhibit
G525C
, although previous studies showed that channels with
cysteine substitutions at the corresponding sites within the
- and
-subunits were blocked by MTSET. Our results, placed in context with
previous observations, suggest that pore regions from the three ENaC
subunits have an asymmetric organization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
(1), with a subunit
stoichiometry of 2
:1
:1
(2, 3), although an alternative
3
:3
:3
subunit stoichiometry has been proposed (4). These
subunits have a similar topology, consisting of two transmembrane
domains (termed M1 and M2) separated by a large extracellular domain
(5-7). A limited region preceding the M2 domain of each subunit may
form part of the channel pore. Selected mutations within these pore
regions alter ion selectivity, or single channel conductance, as well
as amiloride sensitivity (8-13). A critical 3-residue
((G/S)XS) tract within each subunit appears to primarily
govern ENaC selectivity (8, 9, 11, 14).
-subunit pore region led to changes in the
channel selectivity for cations at 6 residues (8). In contrast, Snyder
et al. (9) observed that cysteine substitutions at two
residues within the pore region of the
-subunit (human
Ser541 and
Ser543) led to changes in
cation selectivity, suggesting that the channel selectivity filter is
restricted to a limited site within the pore region of the
-subunit.
To address the role of residues within the
-subunit in conferring
cation selectivity and amiloride sensitivity, we systematically mutated
residues within the pore region
(
Gln523-
Ile536) to cysteine (Fig. 1).
Mutant
mENaCs were co-expressed with wild type
mENaC and wild
type (or C-terminal truncated)
mENaC in Xenopus oocytes,
and functional characteristics were examined. As with the
-subunit
(8), changes in cation selectivity were observed with most mutants
within this region. Large (>9-fold) increases in the inhibitory
constant of amiloride were observed with mutations at
Gly525,
Gly529, and
Ser531. Responses of mutant channels to
sulfhydryl-reactive reagents indicated that the apparent accessibility
to (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA) differed
depending on whether a residue preceded or followed the selectivity
filter. Furthermore, a putative amiloride-binding site,
previously identified on the basis of site-directed mutations that
dramatically increase the Ki for amiloride (12), was
inaccessible to a large sulfhydryl-reactive reagent. Our observations, placed in context with previously published results suggest that ENaC
pore regions have an asymmetric organization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
)
cDNAs were previously cloned into pBluescript SK(
) (Stratagene,
La Jolla, CA) (15). Amino acids in the region preceding and
transitioning into the M2 region of
mENaC,
Gln523-
Ile536 (with the exception of
Cys534), were individually mutated to cysteine using a
polymerase chain reaction method as previously described (8). A
truncation of the C terminus of
mENaC (R583X) was generated by
insertion of a stop codon. This mutation increases function
Na+ channel expression in Xenopus oocytes (16).
All mutations were confirmed by automated DNA sequencing at sequencing
facilities at the University of Pennsylvania or the University of
Pittsburgh. T3 RNA polymerase (Ambion Inc., Austin, TX) was used to
synthesize cRNAs for mutant and wild type
mENaCs, wild type
mENaC
and
mENaC from linearized DNAs. cRNAs were stored at
80 °C and
diluted in diethylpyrocarbonate-treated water prior to injection.
mENaCs with a DigiData 1200 interface (Axon Instruments,
Foster City, CA) and a TEV 200A Voltage Clamp amplifier (Dagan Corp.,
Minneapolis, MN). Data acquisition and analyses were performed using
pClamp 7.0 software with a Pentium II-based PC (Gateway, 2000 Inc., N. Sioux City, SD). Amiloride-sensitive currents were defined as the
difference of the current in the absence and the presence of 0.1 mM amiloride. Higher concentrations of amiloride (1 or 2 mM) were used to define amiloride-sensitive currents in
oocytes expressing the
G525C,
G529C, and
S531C mutants.
140 to +60 mV in 20-mV
increments with the duration of 900 ms. Currents were measured at 600 ms after initiation of the clamp potential. Intersweep potential was
held at the reverse potential. Macroscopic currents were recorded with
each bath solution when stable membrane potentials were observed. Amiloride inhibitory constants (Ki) were determined
by nonlinear regression analysis with Origin program 5.0 (Microcal Inc., Northampton, MA), using the equation,
where Io and I represent inward
current carried by Na+ at
(Eq. 1)
100 mV clamping potential in
the absence and presence of amiloride (B), respectively, and
n' is the Hill coefficient. To determine cation selectivity
of wild type and mutant channels, oocytes were sequentially perfused
with bath solutions containing K+, Na+, or
Li+ as the predominant cation. Oocytes were subsequently
bathed in the K+, Na+, or Li+
solutions supplemented with 0.1 or 2 mM amiloride.
Amiloride-sensitive currents measured at
100 mV were used to
calculate the ratios of the K+ current
(IK) and Li+ current
(ILi) relative to the Na+ current
(INa).
100
and +60 mV for 900 ms following the arrival of the reagent to the
recording chamber. Currents were measured at 600 ms after the
initiation of the
100 mV clamp potential. Intersweep potential was
held at the reverse potential. The time course of MTSEA inhibition of
the currents is shown as relative currents plotted against time. The
time constants were obtained by fitting the data with single
exponential decay. The rates of inhibition were determined by the
equation,
where R, T, and C represent the
rate (M
(Eq. 2)
1 s
1), time constant
(s), and MTSEA concentration (M), respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mENaC in
Xenopus Oocytes--
Several algorithms predicted that the second
membrane-spanning domain of
mENaC begins between residues
Val590 and
Ser592 (8). The corresponding
residues within
mENaC are
Val532 and
Cys534 (see Fig. 1). In
order to examine the role of residues within the pore region of the
-subunit in conferring cation selectivity, 13 of the 14 residues in
the region preceding and transitioning into the second transmembrane
domain of
mENaC, from
Gln523 through
Ile536, were individually mutated to cysteine.
Cys534 is a naturally occurring cysteine. The functional
characteristics of these
mENaC mutations were determined by their
co-expression with wild type
- and
mENaC in Xenopus
oocytes (Table I). Average whole cell amiloride-sensitive Na+ currents ranged from
1.6 to
12.5 µA for both wild type and mutant mENaCs, with the
exception of
G529C
and
S531C
, whose whole cell
amiloride-sensitive currents were less than
400 nA at
100 mV (data
not shown). We previously observed that channels with cysteine residues
at the analogous sites within
mENaC expressed low levels of
amiloride-sensitive Na+ currents (8, 17). In order to
enhance levels of current expression with these mutants,
G529C and
S531C were co-expressed with wild type
-subunit and a
-subunit
with a truncation of the C-terminal cytoplasmic domain (
R583X; see
Table I). Na+ currents measured in oocytes expressing
R583X were 1.9-fold greater than in oocytes expressing wild
type
ENaC (see Table I), although no change in channel cation
selectivity and only a modest (less than 2-fold) change in the
Ki for amiloride were observed.
View larger version (13K):
[in a new window]
Fig. 1.
Alignments of residues within the pore
regions of -,
-,
and
ENaC are illustrated. Residues within
mENaC (Gln581-Val594),
mENaC
(Gln523-Ile536), and
mENaC
(Gln540-Ile553) (GenBankTM
accession numbers AF112185, AF112186, and AF112187) are shown.
Identical residues are shaded.
Expressed amiloride-sensitive Na+ currents,
Li+/Na+ and
K+/Na+ current ratios, and inhibitory
constant for amiloride (Ki) (mean ± S.E. (n))
,
,
R583X.
G529C and
S531C were co-expressed with wild type
-subunit and
R583X.
mENaC Alter the
Ki for Amiloride--
Previous studies have shown that the
introduction of a cysteine at
Gly525 dramatically
increases the Ki for amiloride (2, 12). We also
observed that
G525C
exhibited a 1400-fold increase in the
amiloride Ki, when compared with wild type ENaC (Table I and Fig. 2). Amiloride is a pore
blocker, and we examined whether the substitution of cysteine residues
at other sites altered the Ki for amiloride. Large
increases in the Ki for amiloride inhibition were
observed when cysteine residues were placed at two sites (
G529C and
S531C; Table I and Fig. 2). These sites are within the critical
three-residue tract within ENaC subunits (8-10, 13) that restricts
K+ permeation through the channel. Modest increases in the
Ki for amiloride inhibition were observed when
cysteine residues were placed at other sites, including
Phe526,
Val532, and
Leu533. The Hill coefficients for wild type ENaC and all
mutants examined, except for
G529C
R583X, were between 0.74 and
0.99. The Hill coefficient for
G529C
R583X was 0.36, suggesting
that this mutation led to negative cooperative interactions among sites
within the channel that participate in amiloride binding.
View larger version (19K):
[in a new window]
Fig. 2.
Amiloride dose-response curves for
selected -subunit mutants. Wild type
channels exhibited a Ki for amiloride of
70 ± 10 nM (n = 7; mean ± S.E.;
). The amiloride Ki for
G525C
was
97 ± 10 µM (n = 5;
).
G529C
and
S531C were co-expressed with wild type
and
R583X.
Ki values for amiloride were increased to 5.1 ± 0.7 and 0.6 ± 0.1 µM, respectively, for
G529C
R583X (n = 7;
) and for
S531C
R583X (n = 4;
). Oocytes expressing
wild type or mutant ENaCs were bathed in Na+-containing
solution, and whole cell currents were recorded at a holding potential
of
100 mV during continuous perfusion with increasing concentrations
of amiloride. The currents measured in the presence of increasing
concentrations of amiloride (IAmil) were
normalized to the amiloride-sensitive (1 or 2 mM) component
of the whole cell current. Lines were from data fitting
with Equation 1.
-Subunit Alter Cation
Selectivity Properties of ENaC--
The cation selectivities of wild
type and
-subunit mutant mENaCs were determined by measuring the
ratio of inward Li+ or K+ whole cell
amiloride-sensitive currents (ILi and
IK) relative to the inward amiloride-sensitive
Na+ current (INa)
at a holding potential of
100 mV. Data
are summarized in Table I and Figs. 3 and
4. Oocytes expressing wild type
mENaC exhibited an
ILi/INa of 1.88 ± 0.14 and no detectable amiloride-sensitive inward current when bathed
in a solution with K+ as the predominant cation, in
agreement with previous studies (8-11). Cysteine substitutions at
multiple sites within the
mENaC pore region altered the
ILi/INa of the channel,
similar to that observed following cysteine substitutions within this
region of the
-subunit (8). The introduction of a cysteine residue
at 9 of 13 sites with this region of
-subunit pore altered the
ILi/INa of the channel,
although the changes in
ILi/INa of
I536C
were modest. Increases in the Li+ to Na+
current ratio of the channel were frequently observed and varied from
2.25 ± 0.14 to 5.56 ± 1.16. Two mutants exhibited a
significant reduction in channel
ILi/INa. The mutants
F526C
and
G529C
R583X exhibited
ILi/INa of 1.20 ± 0.03 (p < 0.01, n = 6) and 0.30 ± 0.09 (p < 10
8, n = 5), respectively. The results suggest that multiple residues in the
vicinity of the 3-residue tract of
ENaC
(
Gly529-
Ser531) that primarily governs
ENaC selectivity affect, either directly or indirectly, cation
selectivity.
View larger version (19K):
[in a new window]
Fig. 3.
Cation selectivity of wild type
(WT) and mutant channels. Oocytes were injected
with wild type or mutant ENaCs. Current ratios
(ILi/INa) were determined
from amiloride-sensitive currents in the presence of a Li+
or Na+ bath solution at a holding potential of 100 mV
(A). Nine of 13
-subunit mutants exhibited an
ILi/INa that differed
significantly from wild type ENaC. Current ratios
(IK/INa) were
determined from amiloride-sensitive currents in the presence of a
K+ or Na+ bath solution at a holding potential
of
100 mV (B). Only
G529C
R583X expressed a
K+-permeable channel (n = 5). Data from
4-8 oocytes are presented as mean ± S.E. The p values
from Student's t tests (wild type versus mutant
channels) were as follows: *, <0.05; **, <0.01.
View larger version (22K):
[in a new window]
Fig. 4.
Selected mutations within the 3-residue GGS
tract of the selectivity filter alter cation selectivity.
Representative whole cell currents recorded in oocytes expressing
either wild type ENaC, G529C
R583X, or
S531C
R583X are illustrated in A. Whole cell
currents were determined in oocytes bathed in a solution containing
Na+, Li+, or K+ at test potentials
between
140 mV to +60 mV (20-mV increments). Whole cell currents
recorded in oocytes expressing wild type ENaC in the presence of 100 µM amiloride are also shown. Dashed
lines indicate zero current. Amiloride-sensitive
current-voltage relationships of wild type,
G529C
R583X, and
S531C
R583X were obtained in the presence of K+
(
), Na+ (
), or Li+ (
) bath solutions
(B). Data are presented as mean ± S.E. from 5-8
oocytes.
mENaC as well as with wild type
mENaC and with
R583X (Table I and Fig. 3). As previously observed (11), the
introduction of a cysteine residue at
Gly529 resulted in
channels that were permeable to K+ (Table I; Figs. 3 and
4). The IK/INa for
G529C (8.9 ± 3.6, n = 5) was remarkably
different (p < 0.001) from wild type. This channel
(
G529C
R583X) displayed outward rectification and its reverse
potential in a K+ bath shifted markedly to the right
compared with wild type ENaC (Fig. 4). Kellenberger et al.
(11) previously observed that placement of an alanine residue at
Gly529 resulted in channels that restricted
K+ permeation but reversed the channel
ILi/INa. In agreement
with these data, we observed that
G529A
R583X channels had
an IK/INa 0.013 ± 0.013 (n = 7, p > 0.90 versus wild type) and an
ILi/INa of 0.60 ± 0.01 (n = 7, p < 0.0001 versus wild type). These results indicate that
G529 is
the only residue in the region of this study crucial for
K+ exclusion, supporting the dominant role of the
3-residue (G/S)XS tract in the determination of ENaC
cation selectivity.
mENaC by measuring the effect of these MTS reagents on
amiloride-sensitive whole cell Na+ currents. The addition
of 0.5 mM MTSEA to the bath solution
resulted in an irreversible decrease of
the amiloride-sensitive Na+ current by 28 ± 9%
(n = 8; Figs. 5 and 6),
in agreement with previous observations and reflects, in part,
modification of Cys547 in the pore region of
mENaC by
MTSEA (9, 17). Amiloride (0.1 mM) blocked the residual
current. The response of
R583X to MTSEA was similar to wild
type ENaC (data not shown). The effects of external MTSEA on whole cell
Na+ currents in oocytes expressing
-subunit mutants were
examined in order to identify residues within pore region accessible to MTSEA. Fractional amiloride-sensitive currents remaining following external MTSEA are shown in Fig. 5A. Cysteine substitutions
at only 4 of 13 sites within the pore region of
mENaC resulted in significantly greater MTSEA-mediated inhibition of amiloride-sensitive Na+ currents than wild type mENaC. These residues were not
contiguous but were either flanked by (
F524C,
G525C, and
L533C) or were within (
G530C) the 3-residue tract
(
Gly529-
Ser531) that has a critical role
in excluding K+ from the channel pore. Both
G525C
and
L533C
responded to MTSEA with >80% inhibition of
amiloride-sensitive INa.
View larger version (31K):
[in a new window]
Fig. 5.
Cysteine substitutions of selected residues
within the pore region of ENaC conferred
sensitivity to MTS reagents. Oocytes were injected with wild type
or mutant ENaCs. Amiloride-sensitive current in presence of 0.5 mM MTSEA, relative to the amiloride-sensitive current in
the absence of MTSEA at a test potential of
100 mV is illustrated in
A. Oocytes expressing wild type
mENaC responded to
0.5 mM MTSEA with a 28 ± 9% (n = 8)
inhibition of amiloride-sensitive whole cell Na+ currents.
Four (
F524C,
G525C,
G530C, and
L533C) of 13 mutants
exhibited MTSEA-mediated inhibition of amiloride-sensitive whole cell
Na+ currents that were significantly greater than the
inhibition observed with wild type channels. Amiloride-sensitive
currents in presence of 0.5 or 2 mM (
G529C,
S531C,
L535C, and
I536C) MTSET, relative to amiloride-sensitive currents
in the absence of MTSET at a test potential of
100 mV are illustrated
in B. Only 3 of 11 mutant channels responded to MTSET with a
modest (less than 50%) reduction in amiloride-sensitive whole cell
Na+ currents (see below). The
IMTSET/I0 values for
G529C
R583X and
S531C
R583X were not significant
different from the
IMTSET/I0 value for
R583X (1.36 ± 0.10, n = 3, 2 mM MTSET). The p values from Student's
t tests (wild type versus mutant channels) were
<0.05 (*) and <0.01 (**). Data are presented as mean ± S.E.
from 4-8 oocytes (A) or 4-7 oocytes (B).
View larger version (28K):
[in a new window]
Fig. 6.
Representative whole cell recordings
(A) and I/V relationships
(B) were obtained in the absence or presence of MTSEA
in oocytes expressing wild type ENaC,
G525C
,
or
L533C
.
Whole cell currents were determined in a Na+ bath solution
at test potentials between
140 mV and +60 mV (20-mV increments) prior
to and following the addition of 0.5 mM MTSEA to the bath.
Dashed lines indicate zero current.
Amiloride-sensitive currents are illustrated in B. Data in
B are presented as mean ± S.E. from 4-8
oocytes.
Gly525 and
Leu533 to MTSEA should differ,
and this difference should be reflected in the rates of channel
inhibition in response to MTSEA. Fig. 7
illustrates the rate of current loss over the initial 180 s following the addition of MTSEA to the bath solution. The rate of
current reduction in response to MTSEA for
G525C
channels (36.7 ± 1.1 M
1 s
1,
n = 6) was approximately twice the rate observed for
GL533C
channels (17.4 ± 1.4 M
1
s
1, n = 5, p < 0.001)
(Fig. 7 and Table II). These results
suggest that
G525C is more accessible to external MTSEA than
L533C, supporting a gradually narrowing pore geometry.
View larger version (24K):
[in a new window]
Fig. 7.
Time course for MTSEA inhibition of ENaC
currents. Relative currents (mean ± S.E.) measured at 100
mV from oocytes (five or six oocytes for each group) expressing
G525C
(open square),
G525C
C547S
(solid square),
L533C
(open
triangle), and
L533C
C547S (solid
triangle) following MTSEA application were plotted against
time. Lines are from curve fitting of the data to
Equation 2.
Rate of MTSEA-mediated inhibition of mutant mENaCs (mean ± S.E.)
Gly525 or
Leu533 enhanced the accessibility of
Cys547 to MTSEA (see above) (9, 17). We examined the
effect of MTSEA on
G525C
C547S and
L533C
C547S
channels. MTSEA inhibited both
G525C
C547S and
G525C
to a similar extent, and the rates of inhibition of these channels by
MTSEA were similar (see Fig. 7 and Table II), suggesting that current
inhibition of
G525C
by MTSEA is due to covalent modification
of the introduced sulfhydryl group at
Gly525. In
contrast, the extent of inhibition of
L533C
by MTSEA was considerably greater than that of
L533C
C547S (Fig. 7).
Furthermore, the rate of inhibition of
L533C
by MTSEA
(17.4 ± 1.4 M
1 s
1,
n = 5) was significantly greater than the rate of
inhibition of
L533C
C547S (11.3 ± 1.4 M
1 s
1, n = 5, p < 0.05), suggesting that the
L533C mutation
enhanced the accessibility of MTSEA to
Cys547. Since
MTSEA-mediated inhibition of
L533C
C547S was greater than both
C547S and wild type mENaC, it is likely that the inhibition of
L533C
mENaC by MTSEA was due to both a direct modification of
L533C and the enhanced modification of
Cys547.
-subunit to MTSET. The amiloride-sensitive Na+ currents
carried by wild type ENaC were not affected by MTSET, as previously
observed (9, 17). Surprisingly, only modest reductions in
amiloride-sensitive INa were observed with 3 of 11 mutants studied (Figs. 5 and 8).
Residues with substituted cysteines that responded to MTSET preceded
the 3-residue tract (Gly529-Ser531) that has a
critical role in excluding K+. Furthermore, the mutant
G525C
did not respond to MTSET (n = 6). This
result was unexpected, since Snyder et al. (9)
observed that the equivalent mutation in human ENaC (
G527C
)
was inhibited by MTSET (67% reduction in current). These differences
may reflect the use of channels derived from different species (mouse
versus human). In contrast, the introduction of a cysteine
residue at the analogous site within the
-subunit of mouse and human
ENaC (
S583C
) or the
-subunit of human ENaC (
G536C)
responded to external MTSET with a large (
70%) reduction in
amiloride-sensitive INa (9, 17).
View larger version (23K):
[in a new window]
Fig. 8.
Representative whole cell recordings
(A) and I/V relationships
(B) were obtained in the absence or presence of
MTSET in oocytes expressing wild type (WT)
ENaC,
Q523C
,
and
W527C
.
Whole cell currents were determined in a Na+ bath solution
at test potentials between
140 and +60 mV (20-mV increments) prior to
and following the addition of 0.5 mM MTSET to the bath.
Dashed lines indicate zero current.
Amiloride-sensitive currents are illustrated in B. Data in
B are presented as mean ± S.E. from 4-7
oocytes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit. Selected mutations of the first (Gly587) or
third (Ser589) residues resulted in channels that were
K+-permeable. Snyder et al. (9) observed that
cysteine substitutions of the third residue within a homologous
3-residue tract
(Ser541-Cys542-Ser543) within the
pore region of the human
-subunit led to changes in cation
selectivity; only mutations in
Ser543 resulted in
K+-permeable channels. A 3-residue tract within the
-subunit (Gly529-Gly530-Ser531)
is present in a position homologous to the GSS and SCS tracts within
the
- and
-subunits, respectively, that restricts K+
permeation through the channel (see Fig. 1). Only substitution of
cysteine (not alanine) in the first residue (
Gly529) of
this tract resulted in expression of K+-permeable channels,
in agreement with previous observations (11). Our results, together
with previous studies that have examined the effects of systematic
amino acid substitutions within the pore regions of the
- and
-subunit, suggest that the selectivity filter of the channel resides
primarily within a conserved 3-residue tract.
- and
-subunit, other than within the conserved
3-residue tract, led to changes in
ILi/INa (8) (Fig.
3A), suggesting that residues adjacent to the conserved 3-residue tract that restricts K+ permeation influence
cation selectivity. It remains unclear at this time whether ENaC has an
extended selectivity filter structure or whether amino acids adjacent
to the conserved 3-residue tract modify
ILi/INa by indirectly
altering the structure of the selectivity filter. Although Snyder
et al. did not observe changes in Li+ to
Na+ permeability ratios following cysteine substitutions
within the pore region of the human
-subunit, other than within the
conserved 3-residue tract (9), we cannot exclude the possibility that cysteine substitutions within this region of
mENaC alter
ILi/INa.
- and
-subunits of ENaC (
Gly525 and
Gly542 in mENaC) led to dramatic increases (100 to
>1000-fold) in the Ki for amiloride (2, 12). In
contrast, cysteine substitutions throughout the pore regions of the
-subunit resulted in modest increases in amiloride
Ki at 9 residues, including the residue
(
Ser583) that is a site homologous to
Gly525 and
Gly542 (8, 12). Mutations at
position
Ser588 within the 3-residue tract of the
-subunit that restricts K+ permeation exhibited
amiloride Ki values that were 20-50-fold higher
that wild type (11, 20). We also observed large (~9-73-fold) increases in the Ki for amiloride following cysteine substitutions of selected residues (
S531C
and
G529C
,
respectively) within the 3-residue tract of the
-subunit that
restricts K+ permeation through the channel. Kellenberger
et al. observed that mutations at position
Gly529 resulted in increases in the
Ki for amiloride of 40-130-fold (11). Whereas
Schild et al. (12) suggested that amiloride binds to
Ser583,
Gly525, and
Gly542
(in mENaC), it is quite possible that amiloride also interacts with
residues within the narrow selectivity filter.
-subunit to methanethiosulfate analogs.
Channels with cysteine substitutions at sites (
F524C,
G525C,
L533C) on either side of the 3-residue tract that restricts K+ permeation through the channel were blocked by MTSEA
(Fig. 5), indicating that these residues were accessible to MTSEA. Our
results suggest that
G525C was more readily accessible to
modification by MTSEA than
L533C (see Fig. 7 and Table II). MTSEA is
a membrane-permeant reagent (21), and it is possible that MTSEA gained
access to
L533C via the internal aspect of the channel pore.
Furthermore, the inhibition of
L533C
by MTSEA was due, in
part, to modification of
Cys547.
G525C
. This was a surprising result, since several groups have
proposed that amiloride interacts directly with this residue and with
residues at the homologous sites within the
- and
-subunits
(i.e.
Ser583 and
Gly542) and
that
Gly525 must be external to
Gly529
(9-11). These investigators proposed a "funnel-like" structure for
the ENaC pore region that gradually narrows as it enters the membrane-spanning region to form the selectivity filter that restricts K+ and then transitions to an
-helical second
membrane-spanning domain (see Refs. 9-11). This proposed structure
differs from the structure of the pore region of the KcsA
K+ channel, whose outer pore is formed by an
-helix that
enters the membrane followed by an extended region containing the
selectivity filter that is directed toward the extracellular space
(22). Our previous study examining of the pore (pre-M2) region of
ENaC predicted that this region is formed by an
-helical domain
that transitions into an extended selectivity filter in a manner
analogous to the K+ channel pore (17). The residues that
form the putative amiloride-binding site (
Ser583,
Gly525, and
Gly542) are at the site where
the pore
-helix transitions to an extended selectivity filter (17).
Our earlier work indicated that the second membrane-spanning domains of
ENaC have a secondary selectivity site, and we suggested that M2 may
have an "inverted teepee" structure similar to that of KcsA (18).
The apparent lack of accessibility of
G525C
to MTSET is
consistent with a pore region structure similar to that of the KcsA
K+ channel. However, both Zn2+ (12) and MTSEA
(Fig. 5A) were able to block
G525C
, indicating the
introduced sulfhydryl group was accessible to smaller reagents. In
contrast, our observation that
G525C
is more accessible than
L533C
to MTSEA was consistent with a "funnel-like"
structure of the ENaC pore region. If the ENaC pore has a
"funnel-like" structure, the apparent lack of accessibility of
G525C to MTSET suggests that this residue resides deeper within the
pore region than the analogous residues in the
- and
-subunits.
Alternative structural approaches are needed to resolve the pore region structure.
Gly529) in this tract within the
-subunit (Fig. 3)
(11), the third residue in this tract (equivalent to mouse
Ser548) within the
-subunit (9), and primarily at the
third residue (
Ser589) (8, 10, 11) but also the first
residue (
Gly587) (8) within the
-subunit resulted in
K+-permeable channels. Furthermore, whereas mutations in
Gly525 and
Gly542 led to large changes in
the Ki for amiloride (2, 12) (Fig. 2), mutations in
Ser583 led to only a modest increase in the
Ki for amiloride (8). These results suggest that the
ENaC pore region has an asymmetric organization.
ENaC pore region is
shifted down toward the cytoplasmic face of the plasma membrane, relative to these regions within the
- and
-subunits (see Fig. 9). Several lines of evidence support an
asymmetric structure of ENaC pore region. Although
G525C was not
modified by MTSET (see Fig. 5), channels with cysteine substitutions at
the corresponding sites in the
- and
-subunits were blocked by
MTSET (8, 9). Furthermore, only mutations in the first residue within
the
-subunit 3-residue selectivity filter resulted in
K+-permeable channels, whereas mutations in the third
residue of the selectivity filter within the
- and
-subunit
allowed for K+ permeation. Sequence comparisons revealed
that the ENaC pore region residues are highly conserved, but are not
identical (Fig. 1). At several sites, glycine residues are present in
both the
- and
-subunits, including
Gly522/
Gly539 and
Gly525/
Gly542, whereas a serine residue
is at the corresponding site in the
-subunit. Pore (pre M2) regions
of
-,
-, and
-subunits have 2, 5, and 3 glycine residues,
respectively. Given the unique structural role of glycine residues in
proteins, it is possible that the number of glycine residues in the
pore regions of different subunits contributes to their secondary
structure and degree of flexibility. Differences have been reported in
the single channel properties of
,
, and
ENaCs
(23) and may reflect different pore structures that are contributed by
different subunits. The accessibility pattern of introduced cysteine
residues within the pore regions of ENaC subunits also supports an
asymmetric arrangement of the pore region residues. For example, MTSET
significantly increased whole cell currents in oocytes expressing
channels with a cysteine substitution at
Ser576 or at
the corresponding site in the
-subunit, by presumably locking the
channel in open state (17, 19). In contrast, MTSET did not alter whole
cell currents in channels with a cysteine substitution at the
corresponding site in the
-subunit (9). Comparisons of the effects
of MTSET on mutant ENaCs with cysteine substitutions in the pore
regions of the
- and
-subunits revealed significant differences
in the responses at multiple sites (9, 17). Asymmetries in the pore
regions of voltage-gated Na+ and Ca2+ channels
have also been observed (24-28), in contrast to the reported symmetric
organization of the pore region of K+ channels (22).
View larger version (14K):
[in a new window]
Fig. 9.
Model of the pore regions of
-,
-, and
mENaC illustrating an asymmetric organization.
The N-terminal portions of the
,
, and
mENaC pore regions are
shown as cylinders representing possible helical structures,
and the C-terminal portions are shown as thick
curves, indicating nonhelical structures. Side chains of
selected residues are shown as broken or
solid wavy lines. The three
dashed horizontal lines indicate
relative levels corresponding to sites involved in channel gating
(Gating), amiloride-binding (Amiloride), and
selectivity filter (Filter) from previous and current
studies. The accessibility of introduced cysteines near these sites to
external MTS reagents is shown to the right of each level
line. yes indicates that an MTS reagent significantly
altered channel activity (current); no indicates that an MTS
reagent failed to produce significant change in channel activity
(current). nd, not determined.
In summary, our results confirm the crucial role of the previously
identified 3-residue
(Gly529-
Gly530-
Ser531)
tract of
ENaC in cation selectivity and suggest that residues surrounding this tract also contribute to
Li+/Na+ selectivity. Based on the observation
that
G529C and
S531C dramatically decreased amiloride
sensitivity, we speculate that the ENaC selectivity filter, in addition
to the previously described amiloride binding site
(
Ser583-
Gly525-
Gly542),
may interact with amiloride. Differences in the accessibility of
introduced cysteine residues within the pore regions of the
-,
-,
and
-subunits to external sulfhydryl reagents were observed (see
above and Refs. 9 and 17). We propose that ENaC pore region residues
are arranged asymmetrically, similar to voltage-gated Na+
and Ca2+ channels but distinct from K+ channels.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Kathleen McNulty for generating the
R583X construct.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK54354.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.
§ Recipient of a postdoctoral fellowship award from the Cystic Fibrosis Foundation.
** To whom all correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. E-mail: kleyman@pitt.edu.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M300149200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ENaC, epithelial sodium channel; mENaC, mouse ENaC; M2, second transmembrane domain; cRNA, complementary RNA; MTS, methanethiosulfonate; MTSEA, (2-aminoethyl) methanethiosulfonate hydrobromide; MTSET, [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide; KcsA, K+ channel from Streptomyces lividans.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Canessa, C. M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J. D., and Rossier, B. C. (1994) Nature 367, 463-467[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Kosari, F.,
Sheng, S.,
Li, J.,
Mak, D. O.,
Foskett, J. K.,
and Kleyman, T. R.
(1998)
J. Biol. Chem.
273,
13469-13474 |
3. |
Firsov, D.,
Gautschi, I.,
Merillat, A. M.,
Rossier, B. C.,
and Schild, L.
(1998)
EMBO J.
17,
344-352 |
4. |
Snyder, P. M.,
Cheng, C.,
Prince, L. S.,
Rogers, J. C.,
and Welsh, M. J.
(1998)
J. Biol. Chem.
273,
681-684 |
5. | Canessa, C. M., Merillat, A. M., and Rossier, B. C. (1994) Am. J. Physiol. 267, C1682-C1690[Medline] [Order article via Infotrieve] |
6. |
Renard, S.,
Lingueglia, E.,
Voilley, N.,
Lazdunski, M.,
and Barbry, P.
(1994)
J. Biol. Chem.
269,
12981-12986 |
7. |
Snyder, P. M.,
McDonald, F. J.,
Stokes, J. B.,
and Welsh, M. J.
(1994)
J. Biol. Chem.
269,
24379-24383 |
8. |
Sheng, S.,
Li, J.,
McNulty, K. A.,
Avery, D.,
and Kleyman, T. R.
(2000)
J. Biol. Chem.
275,
8572-8581 |
9. |
Snyder, P. M.,
Olson, D. R.,
and Bucher, D. B.
(1999)
J. Biol. Chem.
274,
28484-28490 |
10. |
Kellenberger, S.,
Gautschi, I.,
and Schild, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4170-4175 |
11. |
Kellenberger, S.,
Hoffmann-Pochon, N.,
Gautschi, I.,
Schneeberger, E.,
and Schild, L.
(1999)
J. Gen. Physiol.
114,
13-30 |
12. |
Schild, L.,
Schneeberger, E.,
Gautschi, I.,
and Firsov, D.
(1997)
J. Gen. Physiol.
109,
15-26 |
13. |
Kellenberger, S.,
and Schild, L.
(2002)
Physiol. Rev.
82,
735-767 |
14. |
Kellenberger, S.,
Auberson, M.,
Gautschi, I.,
Schneeberger, E.,
and Schild, L.
(2001)
J. Gen. Physiol.
118,
679-692 |
15. | Ahn, Y. J., Brooker, D. R., Kosari, F., Harte, B. J., Li, J., Mackler, S. A., and Kleyman, T. R. (1999) Am. J. Physiol. 277, F121-F129[Medline] [Order article via Infotrieve] |
16. | Schild, L., Canessa, C. M., Shimkets, R. A., Gautschi, I., Lifton, R. P., and Rossier, B. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5699-5703[Abstract] |
17. |
Sheng, S.,
Li, J.,
McNulty, K. A.,
Kieber-Emmons, T.,
and Kleyman, T. R.
(2001)
J. Biol. Chem.
276,
1326-1334 |
18. |
Sheng, S.,
McNulty, K. A.,
Harvey, J. M.,
and Kleyman, T. R.
(2001)
J. Biol. Chem.
276,
44091-44098 |
19. |
Snyder, P. M.,
Bucher, D. B.,
and Olson, D. R.
(2000)
J. Gen. Physiol.
116,
781-790 |
20. |
Waldmann, R.,
Champigny, G.,
and Lazdunski, M.
(1995)
J. Biol. Chem.
270,
11735-11737 |
21. | Holmgren, M., Liu, Y., Xu, Y., and Yellen, G. (1996) Neuropharmacology 35, 797-804[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Doyle, D. A.,
Cabral, J. M.,
Pfuetzner, R. A.,
Kuo, A.,
Gulbis, J. M.,
Cohen, S. L.,
Chait, B. T.,
and MacKinnon, R.
(1998)
Science
280,
69-77 |
23. |
Fyfe, G. K.,
and Canessa, C. M.
(1998)
J. Gen. Physiol.
112,
423-432 |
24. |
Perez-Garcia, M. T.,
Chiamvimonvat, N.,
Marban, E.,
and Tomaselli, G. F.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
300-304 |
25. |
Benitah, J. P.,
Tomaselli, G. F.,
and Marban, E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7392-7396 |
26. |
Wu, X. S.,
Edwards, H. D.,
and Sather, W. A.
(2000)
J. Biol. Chem.
275,
31778-31785 |
27. | Chiamvimonvat, N., Perez-Garcia, M. T., Ranjan, R., Marban, E., and Tomaselli, G. F. (1996) Neuron 16, 1037-1047[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Koch, S. E.,
Bodi, I.,
Schwartz, A.,
and Varadi, G.
(2000)
J. Biol. Chem.
275,
34493-34500 |