From the Departments of Medicine, Physiology and § Pathology, School of Medicine, University of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104
Received for publication, September 5, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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Epithelial sodium channels (ENaC) have a
crucial role in the regulation of extracellular fluid volume and blood
pressure. To study the structure of the pore region of ENaC, the
susceptibility of introduced cysteine residues to sulfhydryl-reactive
methanethiosulfonate derivatives
((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and
[(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET))
and to Cd2+ was determined. Selected mutants within
the amino-terminal portion ( Epithelial sodium channels
(ENaCs)1 are composed of
three homologous subunits, termed We previously reported that selected cysteine substitutions within the
carboxyl-terminal domain of the pore region of mouse Reagents--
All chemicals were from Sigma unless stated otherwise.
Cysteine-scanning Mutagenesis--
Site-directed mutagenesis was
performed on mouse Functional Expression of the Mutant mENaCs in Xenopus
Oocytes--
Complementary RNAs (cRNAs) for wild type and mutant Two-electrode Voltage Clamp--
Two-electrode voltage clamp was
performed 20-72 h after injection at room temperature (22-25 °C)
as described previously (14). Data acquisition and analyses were
performed using pClamp 6.03 software (Axon Instruments) on a Pentium
PC. Oocytes were maintained in a recording chamber with 1 ml of bath
solution containing (in mM) 100 sodium gluconate, 2 KCl,
1.8 CaCl2, 5 BaCl2, 10 HEPES, pH 7.2, and
continuously perfused at the flow rate of 4-5 ml/min. Pipettes filled
with 3 M KCl had resistances of 0.5-5 M
The susceptibility of mutant channel with engineered sulfhydryl groups
to sulfhydryl reagents was examined with the sulfhydryl reagents
(2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA), [(2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET), sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES), and
Cd2+. MTSEA (2.5 mM), MTSET (1 mM),
and MTSES (5 mM) were prepared in bath solution immediately
prior to use. Base-line Na+ currents and Na+
currents at 1-3 min following perfusion of the oocytes with a reagent
were measured. Oocytes were then washed with bath solution for 3 min,
and the currents were monitored for observing the reversibility of the
response to the reagent. Bath solution containing 100 µM amiloride was then applied to the oocyte to determine the
amiloride-insensitive current. Responses are expressed as ratios
of amiloride-sensitive Na+ currents after and before
addition of the reagent (I/I0).
Single Channel Recordings--
Patch clamp was performed in
cell-attached mode as described previously (14). Bath and pipette
solutions were identical and contained (in mM) 110 LiCl or
NaCl, 2 CaCl2, 10 HEPES, pH 7.4, adjusted with LiOH or
NaOH, respectively. Na+ or Li+ currents were
recorded at Statistical Analyses--
Values are expressed as mean ± S.E. Student's t test was used for significance analysis
between wild type and mutant channel using MS Excel 97 (Microsoft,
Inc.).
Responses of Mutant mENaCs to External MTS Derivatives--
ENaC
pore regions are highly conserved among members of the ENaC/degenerin
family (Fig. 1A). Secondary
structure predictions of the pore region suggest that the
amino-terminal portion may exist as either
Wild type
We observed distinct effects of MTS reagents on ENaCs with cysteine
substitutions within the amino-terminal
(
In contrast, channels with cysteine substitutions at multiple sites
within the amino-terminal portion of the pore region of
These increases in whole cell currents occurred rapidly (within a
minute) after application of MTS reagents and were not reversible after
MTSEA or MTSET were removed from the bath solution (Fig. 3C), indicating that the introduced cysteine residues were
irreversibly modified. The inward Na+ currents following
stimulation or inhibition by MTSEA or MTSET were completely inhibited
by 100 µM amiloride (Fig. 3C). These data
suggest that MTS modification of the
MTSEA and MTSET introduce positively charged groups to targeted
cysteine residues. We tested whether the charges carried by MTS
reagents were related to the stimulatory or inhibitory effects on
mutant channels by examining the effects of a negatively charged reagent MTSES (5 mM) on Responses of Mutant mENaCs to External Cd2+--
Group
IIB divalent cations such as Cd2+ and Zn2+ are
able to bind free sulfhydryls with high affinity and therefore have
been used as biophysical probes to study the pore structure of ion channels (19-22). Cd2+ was used in this study as its
crystal radius (0.92 Å) (21) is nearly same as that of Na+
(0.95 Å) (23). We examined whether extracellular Cd2+ (5 mM) altered whole cell Na+ currents in oocytes
expressing either WT or mutant mENaCs. A modest increase in
amiloride-sensitive whole cell Na+ current was observed in
response to Cd2+ in oocytes expressing WT Role of Pore Region in ENaC Gating--
The introduction of
cysteine residues at
We performed single channel analyses of
Similar single channel analyses were performed with Li+ as
the conducting ion in the pipette, as well as in the bath solution. We
observed that MTSET treatment of oocytes expressing The substituted cysteine accessibility method has been used to
probe pore structure of various ion channels (27). In this study, we
used this approach to examine the pore structure of At sites amino-terminal to ENaCs with cysteine substitutions at sites carboxyl-terminal to
Structure of the ENaC Pore Region--
The effects of MTS reagents
and Cd2+ were most prominent when cysteine residues were
placed at sites amino-terminal to
Schild et al. (10) previously demonstrated that ENaCs with
acidic residues at position
The outer pore of the KcsA K+ channel is formed by an
Fig. 7A illustrates a model of the ENaC
The introduction of cysteine residues carboxyl-terminal to the GYG
tract of K+ channels conferred sensitivity to MTS reagents
(Fig. 7, A and C). If ENaC and K+
channels share a similar pore structure, the introduction of cysteine
residues carboxyl-terminal to Role of Pore Region in ENaC Gating--
Our observation that
mutant mENaCs with cysteine substitutions within the amino-terminal
portion of the pore region (preceding
Aside from increasing open probability, external application of MTSET
reduced both Na+ and Li+ unitary currents of
Waldmann et al. (13) reported that
Our model predicts a connection between ENaC gating and permeation and
favors a dynamic selectivity filter. Gating and permeation have been
generally considered as independent processes of ionic channels since
1952 (39). However, recent studies have challenged this concept (40,
41). The notion that pore regions function as ion channel gates has
been proposed for cyclic nucleotide-gated channel (42). Recent studies
support a dynamic selectivity filter challenging another long term
notion of a rigid selectivity filter (43).
Alternatively, if the ENaC pore is similar to that proposed by
Kellenberger et al. (11) and Snyder et al. (15)
(Fig. 7B), gating might also involve rotational movement of
the
We observed that several Val569-
Trp582) of the pore region
responded to MTSEA, MTSET, or Cd2+ with stimulation or
inhibition of whole cell Na+ current. The reactive residues
were not contiguous but were separated by 2-3 residues where
substituted cysteine residues did not respond to the reagents and line
one face of an
-helix. The activation of
S580C
mENaC by
MTSET was associated with a large increase in channel open probability.
Within the carboxyl-terminal portion (
Ser583-
Ser592) of the pore region, only
one mutation (
S583C) conferred a rapid, nearly complete block by
MTSEA, MTSET, and Cd2+, whereas several other mutant
channels were partially blocked by MTSEA or Cd2+ but not by
MTSET. Our data suggest that the outer pore of ENaC is formed by an
-helix, followed by an extended region that forms a selectivity
filter. Furthermore, our data suggest that the pore region participates
in ENaC gating.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-, and
ENaC (1, 2). These subunits assemble to form a hetero-oligomeric,
Na+-selective ion channel with a subunit stoichiometry of
2
:1
:1
(3, 4), although an alternative subunit stoichiometry
has been proposed (5, 6). All three Na+ channel subunits
have cytoplasmic amino and carboxyl termini, two transmembrane domains
(termed M1 and M2), and a large ectodomain (7-9). Previous studies
have shown that selected point mutations within the pore region
preceding M2 of each subunit altered functional properties of the
channel, including cation selectivity, single channel conductance, and
sensitivity to the blocker amiloride (4, 10-14). Specific mutations of
residues in a conserved three-residue tract, (G/S)XS
(where X is Ser, Gly, or Cys), within the pore region of the three ENaC subunits, rendered channels
K+-permeable. Snyder et al. (15) examined the
accessibility of a sulfhydryl-reactive methane thiosulfonate (MTS)
derivative to substituted cysteine residues within the pore region of
human
ENaC, and they proposed a structural model of the channel pore similar to that proposed by Kellenberger et al. (11) but
distinct from the resolved structure of the KcsA K+ channel
pore (16).
ENaC
(
Ser580-
Ser592) altered the
cation selectivity and amiloride sensitivity of the channel and
proposed that this region forms the selectivity filter of the channel
(14). In the current study, we systematically examined accessibility of
sulfhydryl reagents to
mENaCs with engineered cysteine within
the 24-residue pore region of the
-subunit. Channels with selected
cysteine mutations within the carboxyl-terminal portion of the pore
region responded to the external application of MTS derivatives with an
inhibition of amiloride-sensitive Na+ currents. In
contrast, we observed a significant increase in amiloride-sensitive
Na+ currents following the external application of MTS
derivatives or Cd2+ when cysteine residues were introduced
at selected sites within the amino-terminal portion of the pore region
of
mENaC. The pattern of distribution of cysteine mutations that led
to MTS-induced activation of Na+ currents suggests that
this region has an
-helical structure. In addition, the activation
of
S580C
by an MTS reagent was associated with a dramatic
increase in channel open probability. We propose that the ENaC pore
region forms part of the outer pore vestibule with an
-helix
followed by an extended region. ENaC may have limited structural
similarities with the KcsA K+ channel (16, 17). In
addition, our results suggest that the pore region has a role in ENaC gating.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ENaC (18) with a sequential polymerase chain
reaction method using Pfu DNA polymerase (Stratagene, La
Jolla, CA). Amino acids
Val569-
Ser592 of
mENaC were replaced individually with a cysteine residue, and target
mutations were conformed by automated DNA sequencing, as described
previously (14).
-,
wild type
-, and
mENaC were synthesized with T3 RNA
polymerase (Ambion Inc., Austin, TX). Stage V-VI Xenopus
oocyte was injected with 2-4 ng of cRNA for each subunit in 50 nl of
H2O. Injected oocytes were maintained at 18 °C in
modified Barth's saline (MBS, 88 mM NaCl, 1 mM
KCl, 2.4 mM NaHCO3, 15 mM HEPES,
0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM
MgSO4, 10 µg/ml sodium penicillin, 10 µg/ml streptomycin sulfate, 100 µg/ml gentamicin sulfate, pH 7.2).
. Typically, oocytes were clamped to a series of voltage steps from
140 to +40 mV
in 20-mV increments for 450 ms every 2 s, and the whole cell
currents were measured at 400 ms. Amiloride-sensitive Na+
currents were defined as the difference of Na+ currents in
the absence and presence of 100 µM amiloride in the bath solution.
100 mV (membrane potential). To test the effects of MTS
on single channel properties of
S580C
mENaC, 10 mM
MTSET prepared in Na+ bath solution was added into the bath
solution giving a 1 mM final concentration of MTSET, and
currents were recorded within 1 h. Event files were generated from
long current traces (>5 min) using Fetchan 6.05 (Axon Instruments),
and open probability was estimated with Histogen 6.05 (Axon Instruments).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix or
-sheet,
whereas the carboxyl-terminal portion appears to be more irregular in
structure. The center portion is predicted to be a turn region (Fig.
1B). To probe the pore region structure, all residues within
the
mENaC pore region (
Val569
Ser592)
were systematically mutated to cysteine and coexpressed with WT
-
and
mENaC subunits in Xenopus oocytes. We previously
observed that all mutants with cysteine substitutions within the pore
region of
mENaC retained channel activity, although low levels of
expressed currents (<200 nA) were observed with two mutants
(
G587C
and
S589C
) (14).
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Fig. 1.
Sequence alignments and secondary structure
prediction of ENaC pore region.
A, pore region amino acid sequence alignments of
,
,
and
mENaC, deg-1 (degenerin from C. elegans),
mec-4 (mechanosensitive protein from C. elegans),
hBNC1 (human brain sodium channel), and ASIC1 (acid-sensing ion channel
1). Identical amino acids are shaded, and similar residues
are boxed. *, analogous site within the C. elegans proteins deg-1 and mec-4 where
mutations result in neurodegeneration;
, proposed amiloride-binding
site in
-,
-, and
ENaC;
, key residues that limit
K+ permeation. B, secondary structure prediction
of the
mENaC pore region was performed with DNASis 2.6 for Windows
95 (Hitachi Software Engineering Co., Ltd., South San Francisco, CA)
using Chou-Fasman algorithm. Uppercase indicates
probability, and lowercase indicates a possibility that the
residue occurs in the indicated conformation.
mENaC responded to 2.5 mM MTSEA with a
partial inhibition of whole cell Na+ currents
(I/I0 = 0.75 ± 0.05, n = 17; Fig.
2A). This reduction in current is similar to that reported
by other investigators (5, 15). The partial inhibition of wild type
ENaC by MTSEA is likely due to covalent modification of
Cys547 in
mENaC that aligns to Ser588 in
mENaC, as proposed by Snyder et al. (15). Mutation of
Cys547 to serine largely eliminated the MTSEA-induced
partial inhibition of Na+ currents, whereas mutation of an
adjacent cysteine (
C551S) had no effect on the partial inhibition of
Na+ currents by MTSEA (data not show). Amiloride-sensitive
whole cell Na+ currents in oocytes expressing wild type
mENaC were not significantly altered following addition of 1 mM MTSET or 5 mM MTSES to the bath solution
(Figs. 2A and
4A).
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Fig. 2.
A, accessibility to external 2.5 mM MTSEA, 1 mM MTSET, and 5 mM
Cd2+ of wild type and cysteine-substituted mENaCs.
Bars (I/I0) indicate ratios of the remaining
amiloride-sensitive Na+ current at 2 min after external
application of the reagent to the amiloride-sensitive Na+
current before delivery of the reagent into the bath. Data are
presented as mean ± S.E., from 4 to 8 oocytes except for wild
type response to MTSEA (17 oocytes). Filled bars indicate
statistical significance (p < 0.05, mutant
versus WT). ND indicates not determined due to
low level of expressed currents with G587C
and
S589C
.
B, helical wheel analysis of
mENaC pore residues
Thr570-Ser583. Residues where substitution
with cysteine resulted in significant changes in amiloride-sensitive
Na+ currents following external application of the
sulfhydryl reagents are marked with *. Residue Gly579 is
not marked based on the small inhibition by Cd2+.
Val569-
Trp582) and carboxyl-terminal
(
Ser583-
Ser592) domains of the pore
region of the mouse
-subunit (Fig. 2A). Within the
carboxyl-terminal portion of the pore region of
mENaC, only 1 of 8 mutants examined (
S583C
) responded to both MTSEA (I/I0 = 0.04 ± 0.01, n = 4) and MTSET
(I/I0 = 0.31 ± 0.09, n = 4) with a
large inhibition of the amiloride-sensitive inward Na+
current (Fig. 2A and Fig. 3, B and C).
Several mutants (
S588C,
V590C, and
L591C) responded to MTSEA
with an inhibition of the amiloride-sensitive inward Na+
current that was significantly greater than WT. These residues are
located in close proximity to and on either side of a three-residue tract (
Gly587-
Ser589) that has a
critical role in restricting K+ permeation through the
channel (11, 12, 14, 15). One mutant (
S588C) that responded to MTSEA
with a partial inhibition of whole cell Na+ current is
located within this three-residue tract. The MTSEA- and MTSET-induced
inhibition of whole cell Na+ currents remained after these
reagents were removed from the bath solution (Fig.
3C).
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Fig. 3.
Representative two-electrode voltage clamp
recordings showing the responses of mutant mENaCs to external
sulfhydryl reagents. A, effect of external 1 mM MTSET on S580C
is shown. Whole cell
Na+ current traces were obtained before
(MTSET
) and after (MTSET+) application of the
reagent, after washout of the reagent, and after perfusion with 0.1 mM amiloride. Current-voltage curves (IV) were
obtained by plotting amiloride-sensitive Na+ currents
against clamp voltages in the range of
100 to 60 mV. Filled
circles represent basal currents, and open circles
indicate the currents recorded after MTSET treatment. B,
responses of
S583C
to MTSEA (left) and MTSET
(center) are displayed as IV curves before (filled
circles) and after (open circles) external application
of the reagents. Currents are amiloride-sensitive Na+
currents. The right panel shows representative voltage ramp
recordings from an oocyte expressing
S583C
. The oocyte was
clamped at 6-s intervals with a linear ramp from
100 mV to 60 mV over
1 s. Top traces are the currents recorded before (0 s)
and after (6 and 12 s) perfusion of a bath solution containing 5 mM Cd2+. Current trace in the presence of
amiloride is close to zero current level. The zero current level is
indicated by a dashed line. C, time courses of
MTSET-induced changes in amiloride-sensitive Na+ currents
from oocytes expressing WT mENaC (
),
S576C
(
),
S580C
(
), or
S583C
(×). Amiloride-sensitive
Na+ currents were measured at 5-s intervals at
100 mV and
normalized to the current level immediately prior to delivery of a
reagent (at time = 1 min). Normalized currents (I/I0)
are shown. Solid bar indicates the period (1-4 min) when
the oocyte was bathed with 1 mM MTSET. MTSET was washed out
over a 3-min period (4-7 min) with bath solution. Dashed
bar indicates the period (7-8 min) when 100 µM
amiloride was present in the bath solution.
mENaC
responded to MTSEA (i.e.
V572C,
S576C,
S580C,
Q581C, and
W582C) or MTSET (i.e.
V572C,
S576C,
N577C, and
S580C) with a significant increase in
amiloride-sensitive inward Na+ currents (Fig.
2A). Only one mutant (
S573C
) responded to MTSET with an inhibition of whole cell Na+ current
(I/I0 = 0.64 ± 0.03, n = 5). The
residues within the amino-terminal portion of the pore region where
cysteine substitutions responded to MTS reagents with a large change in
whole cell Na+ current line one face of an
-helix, with
the exception of
W582C (Fig. 2B), suggesting that this
region is
-helical in structure.
mENaC pore residues did not
interfere with the blocking effect of 100 µM amiloride, although it has been proposed that amiloride blocked ENaC by binding to
a site formed by
Ser583,
Gly525, and
Gly542 (10). Fig. 3A shows representative
recordings of the response of
S580C
to 1 mM
MTSET.
S576C
,
S580C
, and
S583C
.
S576C
responded to MTSES with a modest
increase in whole cell Na+ current (I/I0 = 1.21 ± 0.04, n = 4), and
S580C
responded
to MTSES with a large increase in whole cell Na+ current
(I/I0 = 3.3 ± 0.1, n = 6, Fig.
4A). In contrast, whole cell currents in oocytes expressing
S583C
were unchanged in response to MTSES. When oocytes
expressing
S583C
were pretreated with MTSES, subsequent
treatment with MTSEA still greatly inhibited whole cell Na+
currents (I/I0 = 0.25 ± 0.04, n = 4),
suggesting that
S583C was still accessible to MTSEA. When either
S576C
or
S580C
was pretreated with MTSES, the
subsequent increase in whole cell Na+ current in response
to MTSEA was significantly less than that observed without MTSES
pretreatment (Fig. 4B). These
data suggest that MTSES reacted efficiently with both
S576C
and
S580C
but that
S583C was largely unmodified by MTSES.
Snyder et al. (15) also observed that human ENaC with a
cysteine substitution at the site analogous to
Ser583
(i.e.
G536C) was modified by MTSET but not by MTSES.
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Fig. 4.
Effects of external MTSES (5 mM)
on wild-type and mutant mENaCs. A, external application
of the negatively charged MTSES increased Na+ currents of
S576C
and
S580C
mENaC without affecting the currents
of wild type and
S583C
mENaCs. Bars represent
amiloride-sensitive Na+ currents (mean ± S.E., from 4 to 8 oocytes) in the presence of 5 mM MTSES normalized to
the current level in the absence of MTSES (I/I0).
Solid bars indicates values are significantly different from
wild type (p < 0.01). B, pretreatment with
5 mM MTSES significantly reduced MTSEA-induced increases in
whole cell Na+ currents in oocytes expressing
S576C
or
S580C
, and the MTSEA induced current
inhibition of
S583C
. Open bars indicate ratios of
currents after and before application of 2.5 mM MTSEA
(I/I0). Solid bars represent the ratios of
currents after and before application of 2.5 mM MTSEA
(I/I0) measured in oocytes pretreated with 5 mM
MTSES. * indicates a statistically significant difference
(p < 0.001, mutant versus WT). # indicates a statistically significant difference (p < 0.05, MTSES-pretreated oocyte versus none-pretreated
oocyte).
mENaC. A
similar response to extracellular Cd2+ was observed with 16 of the 22 mENaC mutants examined. Several mutations within the
mENaC
pore region responded to Cd2+ with a significant increase
(
N577C,
S580C, and
W582C) or a modest decrease (
G579C and
L584C) in whole cell Na+ current. Similar to MTSEA,
Cd2+ abolished whole cell amiloride-sensitive
Na+ currents in oocytes expressing
S583C
(I/I0 = 0.03 ± 0.01, n = 4, Fig.
2A). The blocking effect of Cd2+ on
S583C
was both fast and voltage-dependent, as
evidenced by the minimal block of outward currents, compared with the
large inhibition of inward currents (Fig. 3B, right panel).
This is consistent with the observation of
voltage-dependent block of rat
S583C
ENaC
by external Zn2+ (10).
Val572,
Ser576,
Asn577,
Ser580,
Gln581, or
Trp582 led to channels that responded to MTSEA, MTSET,
MTSES, or Cd2+ with an increase in whole cell
Na+ current. These increases in whole cell currents
occurred rapidly (within a minute) after external application of the
MTS reagent or Cd2+ (Fig. 3C), suggesting that
changes in single channel Na+ conductance or open
probability occurred. Previous studies demonstrated that the
introduction of residues with large side chains at the site analogous
to
Ser576 of mENaC led to a significant increase of
currents in oocytes expressing ASIC2 (or BNC1), an ENaC-related
H+-gated ion channel (24). Similar mutations of
deg-1 or mec-4 (mechanosensitive proteins in
Caenorhabditis elegans) led to neuronal degeneration (25,
26). We examined whether the introduction of a residue with a large
side chain at or in proximity to
Ser576 increased whole
cell Na+ currents. Whole cell Na+ currents in
oocytes expressing either
V572F
or
S576C
were significantly greater than that observed in oocytes expressing WT ENaC,
although Na+ currents measured in oocytes expressing
V572C
or
S576F
were similar in magnitude to WT. In
contrast, whole cell currents measured in oocytes expressing
S580F
or
S580C
were significantly less than that
observed in oocytes expressing WT ENaC (Fig.
5).
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Fig. 5.
Expressed amiloride-sensitive Na+
currents (mean ± S.E., n = 6-11 oocytes) of
wild type and selected mENaC mutants were obtained from paired
measurements. Filled bars indicate statistically
significant differences in whole cell amiloride-sensitive
Na+ currents (p < 0.05, mutant
versus WT). Each oocyte was injected with 1 ng of cRNA for
each subunit and clamped in an alternate order 20-30 h after
injection.
S580C
before and after
treatment with MTSET, to test whether the modification of the
introduced cysteine residue altered channel gating (Fig.
6). The single channel slope conductance
for Na+ of
S580C
was 3.7 ± 0.3 pS
(n = 11), slightly less than the conductance of wild
type mENaC (4.3 pS) (14). This is consistent with our previous
observation that the single channel slope conductance for
Li+ of
S580C
was nearly identical to WT ENaC and
that the Li+/Na+ current ratio for
S580C
was 1.36-fold greater than that of WT (14). The open
probability of
S580C
was 0.07 ± 0.02 (n = 5), determined at potentials between
100 and
60 mV. The reduced single channel Na+ conductance and open probability were
consistent with the reduced whole cell Na+ currents
observed in oocytes expressing
S580C
, when compared with
oocytes expressing WT ENaC (Fig. 5). Following treatment with MTSET,
S580C
exhibited a dramatic change in gating characteristics. When patches were made shortly (within minutes) following MTSET treatment, channels were primarily open but exhibited frequent transitions to the closed state (Fig. 6B). However, when
patches were made minutes later following MTSET treatment, channels
remained open, and very few brief closures were observed (Fig. 6,
C and D). The open probability was not determined
due to too few transitions between open and closed states despite long
(>10 min) recordings; however, open probability was clearly >0.9
(Fig. 6, C and D). The single channel conductance
of MTSET-modified
S580C
was 2.3 ± 0.1 pS
(n = 4), lower than that of unmodified channels
(equaling a 38% decrease). In many recordings we only observed noise,
comparable to open channel noise, with no clear transitions suggesting
that the channel remained open over a recording period of 5-10 min (data not shown). These data indicate that MTSET converted
S580C
to a lower conductance channel, but one that was nearly
continuously open. These MTSET-induced changes in conductance and open
probability are the likely mechanisms of the MTSET-induced increase in
whole cell Na+ current observed with this mutant channel
(Figs. 2A and 3A).
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Fig. 6.
External MTSET markedly increased the open
probability of
S580C
mENaC. Single channel recordings were performed in the
cell-attached configuration at
80 or
100 mV (negative value of
pipette potential) with 110 mM NaCl solution
(A-D) or 110 mM LiCl solution (E and
F) in both the pipette and bath. Scales are displayed next
to the recordings on the right and closed states are marked
with short bars. Traces in A and E are
single channel recording traces of the mutant channel prior to MTSET
treatment. The channel is characterized with short open time and long
close time that result in a low open probability. Trace in B
shows a representative recording obtained shortly (within minutes)
after MTSET treatment. It displays increased open probability due to
increased open time and reduced close time as well as decreased single
channel conductance. The estimated Na+ slope conductance
was 2.3 ± 0.1 pS (n = 4) from MTSET-treated cells
and 3.7 ± 0.3 pS (n = 11) from untreated cells).
Recordings in C and D were obtained more than 5 min after MTSET treatment. The channel stays open most of the time with
very short close states. F shows a Li+ current
trace following MTSET treatment. The channel was primarily open but
exhibited a few transitions to the closed state that can be observed
when the trace is displayed in expanded time scale (low
trace). The unitary Li+ current was decreased (0.3 pA)
when compared with untreated channels (0.7 pA) at
80 mV.
S580C
reduced the unitary Li+ current from 0.7 to 0.3 pA at
80
mV and locked the channel in an open state as only brief closures were
observed (Fig. 6F). The Li+ current reduction in
response to MTSET was larger than Na+ current reduction
following MTSET treatment. These data obtained from single channel
analyses with Li+ as the conducting ion were consistent
with the effect of MTSET on amiloride-sensitive whole cell
Li+ currents measured in oocytes expressing
S580C
.
Unlike the effects of MTSET on whole cell
S580C
Na+ currents, Li+ currents were not
significantly increased by MTSET (I/I0 = 0.93 ± 0.06, n = 4). It is likely that the MTSET-induced reduction in Li+ unitary current balances the MTSET-induced increase
in
S580C
open probability.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ENaC. Several
distinct effects of MTS reagents and Cd2+ on
mENaCs were observed with cysteine substitutions within the
pore region of the
-subunit. MTSEA, MTSET, and Cd2+
inhibited whole cell Na+ currents in oocytes expressing
S583C
, as previously reported (4, 10). Surprisingly, this is
the only mutant that displayed significant block by these three
reagents. The lack of an inhibitory effect of MTS reagents on
engineered cysteines near
Ser583 indicates that this
residue is located within a restricted site. Interestingly, another MTS
reagent (MTSES) with a negative charge did not inhibit
S583C
.
Furthermore, MTSES pretreatment of oocytes expressing this mutant
channel failed to prevent the subsequent inhibition by MTSEA (Fig.
4B), indicating that MTSES did not efficiently modify
S583C
. Schild et al. (10) proposed that
Ser583 is located in the electrical field of the ENaC
pore, as Zn2+-induced inhibition of
S583C
was voltage-dependent. We also observed that
Cd2+-induced block of
S583C
mENaC was
voltage-dependent (Fig. 3B). These results
suggest that a negative potential within the vicinity of
Ser583 hinders the access of MTSES. This notion is
reminiscent of the proposal that the pore helix of KcsA K+
channels generates a negative potential at its carboxyl terminus that
contributes to the stabilization of cations in the pore cavity (28).
Ser583, only
S573C
and
G579C
responded to MTSET or Cd2+ with a
partial inhibition of the whole cell Na+ current. Several
mutant channels with cysteine mutations within the amino-terminal
portion of the pore region (i.e. amino-terminal to
Ser583) responded to sulfhydryl reagents with
significant increases in whole cell current. These residues
(
Val572,
Ser576,
Asn577,
Ser580,
Gln581, and
Trp582) line one face of an
-helix, with the
exception of
W582C (Fig. 2B), consistent with an
-helical structure as suggested by secondary structure predictions
(Fig. 1B). These stimulatory effects do not appear to rely
on the positive charges carried by these reagents, as the negatively
charged MTSES also stimulated
S576C
and
S580C
(Fig.
4A). Snyder et al. (15) also observed
MTSET-induced activation of Na+ currents with cysteine
mutations in this region of human
ENaC, although only three mutants
responded with a gain of function in response to MTSET, and the
location of these residues was not suggestive of an
-helical
structure. These differences in responses of
- and
-subunit
mutants to MTS reagents may reflect, in part, the presence of two
-subunits and only one
-subunit within each channel protein.
Another possibility is that the three ENaC subunits may not be arranged
symmetrically along pore axis. This latter notion is supported by the
observations that mutations at homologous sites in different subunits
led to different changes in channel selectivity, amiloride sensitivity,
and divalent cation sensitivity (10, 12, 15). Although KcsA core pore
is formed by four identical subunits in a symmetrical manner (16),
studies on voltage-gated Na+ channels suggested that the
four-pore segments from Domains I-IV are arranged
asymmetrically (19, 20).
Ser583 either did not responded to MTS reagents and
Cd2+ or, alternatively, were partially inhibited
(i.e.
L584C
,
S588C
,
V590C
, and
L591C
). MTSET did not inhibit channels with introduced cysteines carboxyl-terminal to
Ser583, a region
encompassing the proposed selectivity filter. Given several reports
indicating
Gly587 and
Ser589 have an
important role in conferring cation selectivity and restricting K+ permeation (11, 12, 14), residues
Ser587-
Ser589 and adjacent residues are
likely facing the conducting pore. A simple explanation for a lack of
an inhibitory effect of MTSET on channels with cysteine substitutions
in this region is that these sites are not accessible to the reagent.
Ser583. Only modest
changes in whole cell currents were observed in response to MTSEA or
Cd2+ when cysteine residues were placed carboxyl-terminal
to
Ser583. As these accessible residues (defined by a
large response to MTS reagents or Cd2+) preceded
Leu584, our results support a model of the pore region
of the channel that has been proposed by both Kellenberger et
al. (11) and Snyder et al. (15). These groups suggested
that pore is formed by residues that enter the membrane spanning region
from an extracellular site and transitions to an
-helical second
membrane spanning domain. As the pore enters the membrane, it gradually
narrows to form a site that restricts K+, analogous to a
funnel that narrows to its spout. This model is based, in part, on the
observation that the substitution of cysteine residues in the
- or
-subunits at a position analogous to
Ser583 (
G525C
and
G542C) resulted in a large increase in the Ki values of amiloride. Schild and co-workers (10, 11) proposed that
amiloride interacts directly with these residues and that
Ser583 must be external to
Ser589. A
model of the outer vestibule of the pore, incorporating an
-helical
structure that transitions to a narrow selectivity filter, is
illustrated in Fig. 7B. This
model is consistent with that proposed by Palmer (29) 10 years ago.
View larger version (49K):
[in a new window]
Fig. 7.
Structural models of the
ENaC pore region. A, structure of
KcsA pore region (right) and a model for
mENaC pore
region (left) are presented in stick model with ribbon
rendering (9 shin green lines) using the modeling software HyperChem
5.1 (Hypercube Inc., Gainesville, FL). For KcsA K+ channel,
structure of residues P41-Y60 was generated from coordinates obtained
from the Protein Data Bank (code: 1BL8) and the numbering of the
residues is shown in C of this figure. The structural model
for ENaC pore region was generated by mutating KcsA pore region
residues to Val572-Ser592 of
mENaC
according to the alignments shown in C. Residue
Phe586 was omitted and energy minimization was not
performed. Element colors are as followings: cyan for
carbon, blue for nitrogen, and red for oxygen.
Residues corresponding to Val572, Ser573,
Ser576, Asp577, Ser580,
Gln581, Trp582, and Ser583 of
mENaC are displayed in violet color. Substituted cysteines at these
sites were accessible to sulfhydryl reagents from extracellular side
judged by significant changes in amiloride-sensitive Na+
currents in response to these reagents. KcsA residues homologous to the
residues in voltage-gated and inward rectifier K+ channels
that are accessible to external sulfhydryl reagents are also shown in
violet color. B, an alternative model for the
mENaC pore region was generated by rotating residues
Ser12-Ser20 (corresponding to
Ser583-Ser592 of
mENaC) from the model A
(left panel) by 180° along the X axis.
Externally accessible residues in
mENaC are also highlighted in
violet color as in A. This model includes all 24 residues
(Val569-Ser592) of
mENaC pore region. The
key residues retaining K+/Na+ selectivity
(Gly19 and Ser21, corresponding to
Gly587 and Ser589 of
mENaC) are located in
the narrowest region of the pore. C, sequence alignments and
comparison of accessibility to external sulfhydryl reagents between
K+ channels and ENaC. The alignments of the pore region
residues of KcsA, Shaker K+ channel, inward rectifier
K+ channel Kir 2.1 and
mENaC were performed by aligning
the Gly587-Ser588-Ser589 of
mENaC to the K+ channel GYG track. A gap was introduced
in K+ channels to enhance overall alignments. The plus sign
(+) indicates sites where cysteine substitutions within the pore region
result in channels that were inhibited (or activated) by MTS reagents,
Ag+, or Cd2+ and the minus sign (
) shows
residues not accessible to the reagents. The plus/minus sign (±)
indicates sites where cysteine substitutions result in channels that
were modestly inhibited by MTSEA or Cd2+. The X
indicates the sites not tested for sulfhydryl reagent accessibility.
Ser580 (or at the analogous
positions
Gly522 and
Gly534) were
inhibited by extracellular Ca2+ in a
voltage-dependent manner, suggesting that these residues (i.e. amino-terminal to
Ser583,
Gly525, and
Gly537, respectively) were
accessible to extracellular Ca2+. These data support the
proposed pore structure of Kellenberger et al. (11) and
Snyder et al. (15), whereby the interaction of
Ca2+ with
S580D would block the pore of the channel (see
Fig. 7B). However, we observed that oocytes expressing
S580C
mENaC responded to MTSEA, MTSET, and Cd2+
with a large increase in amiloride-sensitive currents. If the
S580C
side chain extends into the pore lumen, its modification by MTS
reagents or Cd2+ would be expected to inhibit the channel
and not activate it. Furthermore, channels with substituted cysteine
residues at selected sites near
Ser580 (i.e.
Val572,
Ser576,
Asn577,
Gln581, or
Trp582) also responded to
MTSEA, MTSET, or Cd2+ with an activation of
amiloride-sensitive currents. If this proposed pore structure is
correct, it is reasonable to predict that channels with cysteine
residues substituted at multiple sites amino-terminal to the
selectivity filter would respond to MTSEA, MTSET, or Cd2+
with a large inhibition of whole cell currents. However, of the mutants
we have examined, only
S583C
responded to these reagents with
a large inhibition of Na+ current. Our results suggest that
periodic residues within the amino-terminal portion of the
mENaC
pore region are accessible to sulfhydryl reagents externally applied
but do not directly face the conducting pore.
-helix that enters the membrane followed by an extended region
directed toward the extracellular space (16) (Fig. 7A). Is
it necessary to propose a pore structure for ENaC that basically
differs from other highly selective cation channels? Previous studies
have suggested that the pore regions of the voltage-gated
Na+ and Ca2+ channels are similar in structure
to voltage-gated K+ channels, although they clearly differ
in mechanisms for achieving cation selectivity (30). Our data suggest
that pore region residues amino-terminal to
Gln581 form
an
-helix, and our previous results (14) suggested that residues
extending from
Ser580-
Ser589 form an
extended selectivity filter. This secondary structure is similar to
that of the KcsA K+ channel. Furthermore, within the pore
regions of K+ channels (Shaker and inward rectifier Kir2.1)
and
mENaC, the pattern of accessibility to substituted cysteine
residues to cysteine-reactive reagents is strikingly similar (Fig.
7C) (31-34). Our previous observation that mutation of the
GSS tract within the selectivity filter of the
-subunit of mENaC
(
Gly587-
Ser589) to the K+
channel selectivity filter signature sequence GYG rendered the mutant
channel K+-selective (14) is also consistent with the
notion that K+ channels and ENaC may have similar pore structures.
-subunit pore
region using the structure of the KscA K+ channel, aligning
the GYG tract within KscA with the GSS tract within
ENaC. Residues
Val572-
Ser580 form an
-helix;
Leu584-
Ser589 form an extended
selectivity filter; and
Gln581-
Ser583
are located at the turn region where the
-helix transitions into the
selectivity filter. Residues where substituted cysteines responded to
MTS reagents or Cd2+ line one face of the helix, with the
exception of
Trp582 (Fig. 2B). Our model
places
Ser592 at a location external to the GSS track,
consistent with the previous observation that the mutant
S592I
rat ENaC was blocked by external Ca2+ in a
voltage-dependent manner (13). This model (Fig.
7A) is not consistent with amiloride interacting directly
with
G525C or
G542C, residues proposed to interact directly with
amiloride (10). These residues would be within the internal portion of the selectivity filter. However, these mutations (i.e.
G525C or
G542C) might change the structure of the pore and
indirectly alter the Ki values of amiloride, as
suggested by Schild et al. (10).
Ser589 would be expected
to confer sensitivity to MTS reagents. However, we only observed a
modest block of Na+ currents in response to MTSEA when
cysteine residues were present at
Val590 or
Leu591, and no changes in whole cell Na+
currents were observed in response to MTSET or Cd2+. These
results suggest that K+ channels and ENaC differ in their
pore structure, although the nature of these differences has yet to be
defined. Based on our data and previous studies, we propose that the
amino-terminal portion of the
ENaC pore region forms an
-helix,
and the carboxyl-terminal portion forms a selectivity filter, like that
of K+ channels. Furthermore, we propose that the transition
from selectivity filter to
-helical second membrane spanning domain
within ENaC has a structure that differs from K+ channels.
Ser583) responded
to MTSEA, MTSET, or Cd2+ with an increase in whole cell
Na+ current led us to examine whether this region has a
role in ENaC gating. In agreement with our observation that MTS
reagents activated whole cell Na+ currents in oocytes
expressing
S580C
, MTSET induced a large increase in open
probability of
S580C
, indicating that this residue is within a
domain that controls ENaC gating. Our data suggest
Ser576 and
Ser580 are two crucial
residues in this gating domain, as all three MTS reagents dramatically
stimulated whole cell Na+ currents in oocytes expressing
these mutant channels. This proposed role of
Ser576 in
channel gating is in agreement with previous observations suggesting a
similar role of the residue at the analogous position in degenerins and
in H+-gated Na+ channels. The introduction of
bulky residues at a site analogous to
S576C within the C. elegans proteins deg-1 and mec-4 lead to
neurodegeneration that is proposed to occur as a result of an
unregulated activation of a putative mechanosensitive cation channel
(35, 36). Similarly, selected mutations at an analogous site within
this pore region of related H+-gated Na+
channels, termed BNC (or ASIC2), resulted in sustained channel activation that was independent of acidification (24). Based on these
observations, we propose that the ENaC pore region participates in
channel gating.
S580C
. The observed changes in whole cell amiloride-sensitive
Na+ currents of
S580C
in response to MTSET
reflects opposing effects on open probability (increased) and unitary
current (decreased). The mechanism of MTSET-induced reduction in
unitary current is unknown. This may reflect changes in the
conformation of the open channel due to covalent binding of MTSET to
S580C; alternatively, the positively charged MTSET may partially
block the pore.
S589I and
S589F
(analogous to
Ser588 of mENaC) reduced rat ENaC open
probability from 0.89 to 0.09 and 0.04, respectively, and increased
Na+ unitary conductance from 4.6 to 10 pS without changing
Li+ conductance. This study provided evidence that
Ser588, located within the selectivity filter of ENaC,
has a role in ENaC gating. We have also observed that
S588C
mENaC has very low open probability resulting from short open and long
close times (data not show). Fyfe et al. (37) studied the
functional properties of ENaCs with chimeric
-
subunits. Their
results also suggested that this region regulates ENaC gating. These
observations that mutations within both amino-terminal and
carboxyl-terminal portions of the pore region resulted in changes in
channel gating suggest a collaborative role of these two regions in
ENaC gating through intradomain interactions. We propose a working
model for ENaC gating. In this model, the pore helix (amino-terminal
portion of pore region) undergoes rotational movement. This movement
could be in response to conformational changes at other sites within ENaC, such as the ectodomain, the M2 domains, or cytoplasmic domains (i.e. the amino terminus (38)). The rotation of the pore
helix leads to changes in the diameter of the selectivity filter, which in turn allows ion translocation through the pore. A recent study of
KscA gating observed movement of reporter cysteines introduced at the
carboxyl-terminal end of the pore helix in association with the channel
gating (45). If the ENaC pore structure shares the fundamental design
of the KcsA K+ channel pore,
Ser576 and
Ser580 are expected to be close to the carboxyl-terminal
end of the pore helix of ENaC. Substitution of large side chains at
these two sites might hinder rotational movement of the pore helix, essentially locking the channel in an open state. Our model suggests that there are extensive interactions between the amino-terminal and
carboxyl-terminal portions of the pore region, and we propose that this
occurs, in part, through hydrogen bonding. ENaC pore regions have a
large number of serine residues (7 in
mENaC, 2 in
mENaC, and 4 in
mENaC). Given the proposed subunit stoichiometry of 2
, 1
, and
1
, a total of 20 serine residues are present within the pore regions
of the channel. These serine residues, as well as other polar residues
and backbone carbonyl oxygens, are capable of forming a network of
hydrogen bonds. A sliding model of gating is also plausible. A relative
sliding movement between
Ser576-Ser580 and
Ser588-Ser592 could lead to channel
transitions between open and closed states.
-helical region preceding the selectivity filter. This gating
mechanism was proposed by Adams et al. (24) in their studies
of activation of the H+-gated Na+ channel BNC
(or ASIC2).
ENaC mutants in the pore helix, including
V572F
and
S576C
, expressed whole cell currents in oocytes that were significantly greater than that observed with WT
ENaC, consistent with the notion that mutations within this region
affect channel gating. We anticipate that selected mutations within
this domain of human ENaC are likely to be found in the clinical
setting of salt-sensitive hypertension due to enhanced ENaC-mediated
Na+ transport in the distal nephron. In this context,
Melander et al. (44) reported a mutation within human ENaC
(
N530K), a position analogous to mouse
Asn577, in a
patient with hypertension and diabetes, although a causal relationship
was not established. In summary, our data suggest that the
amino-terminal pore region of
ENaC is
-helical in structure and
that this region is involved in channel gating.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK54354 and DK50268 and by the Department of Veterans Affairs.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.
Recipients of postdoctoral fellowship awards from the Cystic
Fibrosis Foundation.
¶ Present address and to whom correspondence should be addressed: Renal Division, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213. Tel.: 412-647-3121; Fax: 412-647-6222; E-mail: kleyman@pitt.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M008117200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ENaC, epithelial sodium channel; WT, wild type; MTS, methane thiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSET, [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; KcsA, potassium channel from Streptomyces lividans; ASIC, acid-sensing ion channel; BNC, brain sodium channel; pS, picosiemen..
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Canessa, C. M., Horisberger, J. D., and Rossier, B. C. (1993) Nature 361, 467-470[CrossRef][Medline] [Order article via Infotrieve] |
2. | 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] |
3. |
Firsov, D.,
Gautschi, I.,
Merillat, A. M.,
Rossier, B. C.,
and Schild, L.
(1998)
EMBO J.
17,
344-352 |
4. |
Kosari, F.,
Sheng, S.,
Li, J.,
Mak, D. O.,
Foskett, J. K.,
and Kleyman, T. R.
(1998)
J. Biol. Chem.
273,
13469-13474 |
5. |
Snyder, P. M.,
Cheng, C.,
Prince, L. S.,
Rogers, J. C.,
and Welsh, M. J.
(1998)
J. Biol. Chem.
273,
681-684 |
6. |
Eskandari, S.,
Snyder, P. M.,
Kreman, M.,
Zampighi, G. A.,
Welsh, M. J.,
and Wright, E. M.
(1999)
J. Biol. Chem.
274,
27281-27286 |
7. |
Canessa, C. M.,
Merillat, A. M.,
and Rossier, B. C.
(1994)
Am. J. Physiol.
267,
C1682-C1690 |
8. |
Snyder, P. M.,
McDonald, F. J.,
Stokes, J. B.,
and Welsh, M. J.
(1994)
J. Biol. Chem.
269,
24379-24383 |
9. |
Renard, S.,
Lingueglia, E.,
Voilley, N.,
Lazdunski, M.,
and Barbry, P.
(1994)
J. Biol. Chem.
269,
12981-12986 |
10. |
Schild, L.,
Schneeberger, E.,
Gautschi, I.,
and Firsov, D.
(1997)
J. Gen. Physiol.
109,
15-26 |
11. |
Kellenberger, S.,
Gautschi, I.,
and Schild, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4170-4175 |
12. |
Kellenberger, S.,
Hoffmann-Pochon, N.,
Gautschi, I.,
Schneeberger, E.,
and Schild, L.
(1999)
J. Gen. Physiol.
114,
13-30 |
13. |
Waldmann, R.,
Champigny, G.,
and Lazdunski, M.
(1995)
J. Biol. Chem.
270,
11735-11737 |
14. |
Sheng, S.,
Li, J.,
McNulty, K. A.,
Avery, D.,
and Kleyman, T. R.
(2000)
J. Biol. Chem.
275,
8572-8581 |
15. |
Snyder, P. M.,
Olson, D. R.,
and Bucher, D. B.
(1999)
J. Biol. Chem.
274,
28484-28490 |
16. |
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 |
17. |
MacKinnon, R.,
Cohen, S. L.,
Kuo, A.,
Lee, A.,
and Chait, B. T.
(1998)
Science
280,
106-109 |
18. |
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 |
19. | 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] |
20. | Yamagishi, T., Janecki, M., Marban, E., and Tomaselli, G. F. (1997) Biophys. J. 73, 195-204[Abstract] |
21. |
Tsushima, R. G.,
Li, R. A.,
and Backx, P. H.
(1997)
J. Gen. Physiol.
110,
59-72 |
22. | Becchetti, A., and Roncaglia, P. (2000) Pfluegers Arch. 440, 556-565[CrossRef][Medline] [Order article via Infotrieve] |
23. | Hille, B. (1992) Ionic Channels of Excitable Membranes , 2nd Ed. , p. 276, Sinauer Associates, Inc., Sunderland, MA |
24. |
Adams, C. M.,
Snyder, P. M.,
Price, M. P.,
and Welsh, M. J.
(1998)
J. Biol. Chem.
273,
30204-30207 |
25. | Garcia-Anoveros, J., Ma, C., and Chalfie, M. (1995) Curr. Biol. 5, 441-448[Medline] [Order article via Infotrieve] |
26. | Hong, K., and Driscoll, M. (1994) Nature 367, 470-473[CrossRef][Medline] [Order article via Infotrieve] |
27. | Karlin, A., and Akabas, M. H. (1998) Methods Enzymol. 293, 123-145[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Roux, B.,
and MacKinnon, R.
(1999)
Science
285,
100-102 |
29. | Palmer, L. G. (1990) Renal Physiol. Biochem. 13, 51-58[Medline] [Order article via Infotrieve] |
30. | Catterall, W. A. (2000) Neuron 26, 13-25[Medline] [Order article via Infotrieve] |
31. | Kubo, Y., Yoshimichi, M., and Heinemann, S. H. (1998) FEBS Lett. 435, 69-73[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Dart, C.,
Leyland, M. L.,
Spencer, P. J.,
Stanfield, P. R.,
and Sutcliffe, M. J.
(1998)
J. Physiol. (Lond.)
511,
25-32 |
33. | Lu, Q., and Miller, C. (1995) Science 268, 304-307[Medline] [Order article via Infotrieve] |
34. | Liu, Y., Jurman, M. E., and Yellen, G. (1996) Neuron 16, 859-867[Medline] [Order article via Infotrieve] |
35. | Driscoll, M., and Chalfie, M. (1991) Nature 349, 588-593[CrossRef][Medline] [Order article via Infotrieve] |
36. | Tavernarakis, N., and Driscoll, M. (1997) Annu. Rev. Physiol. 59, 659-689[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Fyfe, G. K.,
Zhang, P.,
and Canessa, C. M.
(1999)
J. Biol. Chem.
274,
36415-36421 |
38. | Grunder, S., Jaeger, N. F., Gautschi, I., Schild, L., and Rossier, B. C. (1999) Pfluegers Arch. 438, 709-715[CrossRef][Medline] [Order article via Infotrieve] |
39. | Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. (Lond.) 117, 500-544[Medline] [Order article via Infotrieve] |
40. | Tomaselli, G. F., Chiamvimonvat, N., Nuss, H. B., Balser, J. R., Perez-Garcia, M. T., Xu, R. H., Orias, D. W., Backx, P. H., and Marban, E. (1995) Biophys. J. 68, 1814-1827[Abstract] |
41. | Schneggenburger, R., and Ascher, P. (1997) Neuron 18, 167-177[Medline] [Order article via Infotrieve] |
42. | Sun, Z. P., Akabas, M. H., Goulding, E. H., Karlin, A., and Siegelbaum, S. A. (1996) Neuron 16, 141-149[Medline] [Order article via Infotrieve] |
43. | Khakh, B. S., and Lester, H. A. (1999) Neuron 23, 653-658[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Melander, O.,
Orho, M.,
Fagerudd, J.,
Bengtsson, K.,
Groop, P. H.,
Mattiasson, I.,
Groop, L.,
and Hulthen, U. L.
(1998)
Hypertension
31,
1118-1124 |
45. |
Perozo, E.,
Cortes, D. M.,
and Cuello, L. G.
(1999)
Science
285,
73-78 |