From the Institut de Pharmacologie et de Toxicologie, Université de Lausanne, 1005 Lausanne, Switzerland
The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three
subunits. The structures involved in the ion permeation pathway have only been partially identified, and the
respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel
block by amiloride. Cys substitutions of Gly residues in
and
subunits at position
G525 and
G537 increased
the apparent inhibitory constant (Ki) for amiloride by >1,000-fold and decreased channel unitary current without
affecting ion selectivity. The corresponding mutation S583 to C in the
subunit increased amiloride Ki by 20-fold,
without changing channel conducting properties. Coexpression of these mutated
subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the
S583C mutant showing a Ki for Zn2+of 29 µM. Mutations of residues
W582L or
G522D also increased amiloride Ki, the later mutation generating a Ca2+blocking site located
15% within the membrane electric field. These experiments provide strong evidence that
ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of
subunits may
form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.
The epithelial sodium channel (ENaC) is a highly selective cation channel involved in the reabsorptive movement of Na+ions in epithelia of distal colon, airways,
and distal nephron (Rossier et al., 1994). This electrogenic vectorial transport of Na+is accomplished by a
two-step transport system involving the apical ENaC channel and the basolateral Na+-K+ pump. The functional
characteristics of ENaC have been studied in isolated
renal tubular segments using patch-clamp techniques and can be defined as a small 4-6 pS conductance channel in isotonic NaCl with high selectivity for Na+andLi+over K+ (<20:1) and slow gating kinetics. In addition, ENaC is highly sensitive to the diuretic amiloride
with an apparent inhibitory constant of 10
7 M.
The primary structure of ENaC has been elucidated
by expression cloning and is composed of three homologous subunits (Canessa et al., 1994b
). The ENaC
subunits are members of a new gene superfamily of ion
channels including a peptide-gated Na channel recently cloned in the snail Helix aspera, a voltage-insensitive brain Na channel showing high selectivity for Na+ions and amiloride sensitivity. More distant relatives of
ENaC are gene products identified in the nematode
Caenorhabditis elegans called degenerins (MEC-4, DEG-1),
whose functional properties are not yet known, but are
thought to be involved in mechanosensation (Corey
and García-Añoveros, 1996
).
The predicted membrane topology of the ENaC subunits reveals two hydrophobic domains separated by a
large hydrophilic loop that represents the major portion of the protein. Compelling evidences indicate the
NH2 and COOH termini are facing the cytoplasmic
side leaving the large hydrophilic loop in the extracellular milieu (Canessa et al., 1994a; Renard et al., 1994
;
Snyder et al., 1994
). This membrane topology makes
ENaC resemble other channel types such as the inward-rectifier potassium channel, the ATP-gated cation channel (P2X receptor), or the mechanosensitive channel of
Escherichia coli (North, 1996
).
Expression of the ENaC subunit in Xenopus oocytes
results in a small amiloride-sensitive current which is
greatly potentiated by coexpression with the
and
ENaC subunits (Canessa et al., 1994b
). Expression of
and
subunits alone or together do not result in a detectable amiloride-sensitive current. These experiments suggest that the
subunit is a pore-forming subunit
which contains all the biophysical and pharmacological
properties of ENaC. Recent labeling experiments have
shown that only the heteromultimeric
channel can
be detected at the cell surface indicating that the mature channel is a heteromultimeric channel made of
three
subunits with an unknown stoichiometry
(Firsov et al., 1996
). The functional role of the
and
subunits has not yet been clearly established. From
their structural homology with the
subunit, it is conceivable that the
or
subunits also participate in the
formation of the channel pore. Alternatively, the ion
conduction pathway could be made exclusively of
subunits with the
and
subunits representing accessory subunits that modulate ENaC activity.
To address these questions about the subunit structure of ENaC, we have used a site-directed mutagenesis
approach to identify amino acid residues in the ,
,
and
subunits that play a role in ion permeation or
channel sensitivity to amiloride block. In addition, we
have performed different type of amino acid substitutions such as sulfhydryl or carboxylate residues with the
purpose of engineering divalent cation binding sites for
Zn2+or Ca2+ions that would block the channel. Within a
short segment preceding the second membrane-spanning domain we have identified Gly and Ser residues in
all three
subunits, which when mutated alter the
conducting properties, channel's affinity for amiloride,
or create binding sites for blocking cations. These mutagenesis experiments support the notion that the
amiloride binding site is located within the ion permeation pathway and that all three channel subunits participate in the formation of the conducting pore.
Preparation of the ENaC mutants
Rat ENaC channel cDNA encoding the subunits cloned into
the pSD5 (
and
subunits) or pSport (
subunit) vectors were used for site-directed mutagenesis. Mutations were introduced by
PCR using a three-step protocol as described previously (Schild et al., 1996
). The first PCR step used a 5
mutagenic primer and a
3
inverse hybrid primer carrying an unrelated sequence. The PCR product was elongated during the second step, and finally the cDNA was selectively amplified using a primer that primes synthesis only from DNA containing the unrelated sequence introduced in step 1. The final PCR product containing the desired
mutation flanked by two unique restriction sites allowed its isolation and ligation into the ENaC cDNA. Mutations in the
subunit were introduced in a cDNA cassette flanked by unique Sfu1
and BamH1 restriction sites, mutations in the
subunit in a cassette flanked by Sac1 and Spe sites, and a cassette delineated by
unique BglII and Apa 1 was used for
mutations. cDNA sequencing confirmed the presence of the desired mutation.
In addition, the G525 to C,D and
G537 to C mutations were
respectively introduced in
and
subunit cDNA containing a
FLAG reporter octapeptide DYKDDDDK located ~30 residues
downstream the first transmembrane domain which can be recognized by anti-FLAG M2 IgG1 mouse monoclonal antibodies
(Firsov et al., 1996
). Introduction of the FLAG epitope does not
alter the functional properties of the ENaC subunits and allows
quantification of ENaC channel subunits expressed at the cell
surface using a specific binding assay (Firsov et al., 1996
).
Expression of ENaC Channels in Xenopus Oocytes
Complementary RNAs of each ENaC subunits were synthetized in vitro, and equal amount of subunit cRNA at saturating
concentrations for maximal channel expression (5 ng total
cRNA) were injected into stage V oocytes (Schild et al., 1995
).
Electrophysiological measurements were performed 24 h after
injection.
Macroscopic whole-oocyte Li+currents (ILi) resulting from
ENaC channel expression were measured using the two-electrode voltage-clamp technique and determined as the amiloride-sensitive inward current at 100 mV holding potential generated
by substitution of 20 mM KCl with 20 mM LiCl in the external sucrose buffer solution.
Single channel recordings were obtained using patch-clamp technique in the cell-attached configuration. Data were sampled at 2 kHz and filtered at 100 Hz for analysis. Construction of current-voltage curves (I-V curves) for the channel open states was done from amplitude histograms containing at least 50 current transitions between open and closed states. All electrophysiological measurement were performed at room temperature.
Surface expression of ENaC channels was determined by specific binding of [125I]M2IgG1 (M2Ab) to oocytes expressing FLAG-tagged channel and
subunits as described previously (Firsov
et al., 1996
). The equilibrium dissociation constant of specific
[125I]M2IgG1 (M2Ab) binding was 3 nM. Accordingly, 12 nM
M2Ab were added to a Modified Barth's Saline (MBS) solution
supplemented with 5% calf serum incubating the oocytes. After 1 h
incubation at 4°C, oocytes were washed eight times with 1 ml
MBS solution and transferred individually in a
counter, and
then used to measure amiloride-sensitive Li+ current. M2Ab nonspecific binding was detected with oocytes injected with ENaC
cRNAs encoding nontagged subunits.
Solutions
Oocytes were incubated in a MBS solution containing in mM: NaCl 85; KCl 1; NaHC03 2.4; MgSO4 0.82; CaCl2 0.4; HEPES-NaOH 16, pH 7.2. Measurement of macroscopic currents were performed in sucrose buffer solutions containing in mM: either KCl, 20 or LiCl, 20 or NaCl, 20; sucrose, 150; CaCl2, 0.2; HEPES-KOH, 30, pH 7.2.
The pipette filling solution in patch-clamp experiments contained in mM: NaCl or LiCl, 110 mM; KCl, 2.5; CaCl2, 1.8; HEPES-OH, 10, pH 7.2. The bathing solution in these experiments was identical to the pipette solution except that 100 mM KCl replaced LiCl or NaCl.
Analysis of the Data
To analyze titration curves for inhibition of macroscopic Li+current (ILi), the ratio ILi/ILi0 measured in the presence (ILi) of a particular blocker B to that in the absence of the blocker (ILi0) is described by a Langmuir inhibition isotherm:
![]() |
(1) |
where Ki is the inhibitory constant of the blocker, n is a pseudo-Hill coefficient.
In case of a voltage-dependent block, Ki(V) is the voltage-dependent inhibitory constant, which has been expressed by Woodhull (Woodhull, 1973) as a Boltzmann relationship with respect to the voltage,
![]() |
(2) |
where Ki(0) is the inhibitory constant at 0 mV, z is a slope parameter, and F, V, R, and T have their usual meanings. z
is equal to
the product of the actual valence of the blocking ion z and the
fraction of the membrane potential (or electrical distance)
acting on the ion.
ENaC channel subunits are comprised of two hydrophobic segments M1 and M2 that probably form transmembrane helices (Fig.1). Preceding the second transmembrane domain a short segment (pre-M2 segment)
shows a high degree of homology among all the members of the ENaC gene family identified in epithelia or
in CNS that exhibit sensitivity to amiloride block. It has
been postulated that this segment forms an
barrel
structure extending to the extracellular entrance of the
channel pore (Guy and Durell, 1995
). Sequence alignment of the pre-M2 segment in the
ENaC subunits is shown in Fig. 1. This stretch of amino acid residues is
strictly identical among the different
ENaC genes
from Xenopus laevis, rat, or human. To investigate the
relationship of this amino acid segment with the channel pore, we specifically mutated the Ser residues (shown
in the boxes) at positions S580 and S583 in the
subunits and the Gly residues at corresponding positions
in the
(G522, G525) and
(G534, G537) subunits. The
functional consequences of these site specific mutations
on the ionic permeation properties, and on the blocking
pharmacology of the channel were investigated.
Amino Acid Substitutions in the S583,
G525,
G537
Positions
Among the unique functional
characteristics of ENaC are its higher ionic permeability for Li+than Na+ ions, and its unmeasurably low permeability for K+ ions (Canessa et al., 1994b). We have
taken advantage of this ionic permeability property to
assess functional expression of ENaC at the cell surface
by measuring the inward current after substitution of
20 mM K+ by 20 mM Li+in a K-sucrose buffer solution.
Fig. 2 shows representative recordings of macroscopic Li+currents (ILi) obtained with expression of
wild-type
(wt) 1 ENaC in Xenopus oocytes. Substitution of external
K+ by Li+induced an inward current that was inhibited
by increasing concentrations of external amiloride. Under these experimental conditions >98% of ILiwas inhibited by 5 µM amiloride, indicating that the amiloride-sensitive inward Li+current reflects channel activity of
amiloride-sensitive ENaC expressed at the cell surface.
Single amino acid substitutions of either S583,
G525, or
G537 induced a robust inward Li+current
in the µA range (Table I). With the exception of the
G525D, for which ILiwas <1 µA, the expressed current
for the mutants were not different from the ENaC wild
type. No ILicould be detected with coexpression of the
Cys mutations in the three
subunits (
G583C +
G525C +
G537C). The absence of ILiexpressed by
the triple Cys mutant could result either from impaired
channel expression at the cell surface or from a nonconducting channel.
Table I. |
To address these possibilities, we measured cell-surface expression of the triple Cys mutant (G583C +
G525C +
G537C) using a binding assay with a iodinated antibody (M2Ab) of high specific activity against
a FLAG epitope placed in the extracellular domain of
and
subunits. This epitope placed ~30 amino acids
downstream from the first transmembrane M1 domain
does not affect ENaC channel function (Firsov et al.,
1996
). Fig. 3 compares ILiwith specific binding of M2Ab
in oocytes expressing either FLAG-tagged
ENaC wild type, the FLAG-tagged triple Cys mutant or FLAG-tagged
subunits. For the FLAG-tagged wt the mean
ILicurrent expressed corresponded to ~0.3 fmol of
M2Ab specifically bound per oocyte at the cell surface.
Surface expression of the triple Cys mutants was not
statistically different from wild type as shown by M2Ab binding assay, but no detectable ILicould be measured.
The absence of ILiand specific M2Ab binding in oocytes
injected with only
and
subunits tagged with the FLAG
epitope demonstrate that single
,
subunits or
dimers are not expressed at the cell surface, and that
surface targeting of ENaC requires co-assembly of the
three
subunits. The specific M2Ab binding in oocytes with the triple Cys mutant implicates that the heterotrimeric channel mutant is correctly expressed at
the membrane surface but is nonconducting. As shown
in Table I, surface expression of the
G525D mutant was
comparable to the wild type, and the lower ILifor this
mutant is consistent with a lower ionic permeability.
The consequences of Cys mutations of S583,
G525, and
G537 residues on ion permeation were
further investigated at the single channel level. Fig. 4 illustrates a single channel recording of the
G525C mutant coexpressed with the
and
wt in the presence of
external Na+as conducting ion. This mutant displays
characteristic long opening and closing events as well
as shorter transitions between open and closed states, a
gating behavior similar to that described for the ENaC
channel wt (Canessa et al., 1994b
; Palmer and Frindt, 1996
). Changes in the single channel conductance are
evidenced by the current-voltage relationships comparing wt ENaC, or
S583C mutant with
G525C and
G537C. The
S583 to C mutation did not significantly
affect channel conductance (8.9 pS versus 10 pS for
ENaC wt). The Cys mutations at corresponding positions in the
and
subunits resulted in an ~40% decrease in single channel cord conductance without affecting Na+over Li+permeability ratio. Channel conductance for Li+ions were 5.8 and 6.2 pS for
G525C
and
G537C mutants.
Taken together these results indicate that the Gly to
Cys mutations in the and
subunits affect ion permeation through the channel without changing its Li+
over Na+ selectivity or its apparent gating properties.
The effect of these mutations was additive and resulted
in a nonconducting channel when all three subunits
were mutated.
In early
experiments with expression of G525C mutant together with
and
subunits, it became rapidly evident
that the inward Li+current was less sensitive to amiloride.
Representative recordings of macroscopic ILifor the
G525C mutant and its dose-dependent inhibition by
amiloride is illustrated in Fig. 5 A. ILiexpressed by
G525C mutant could only be partially inhibited by increasing amiloride concentrations up to 100 µM, indicative of a decrease in amiloride sensitivity compared to
wt ENaC (see Fig. 2). Amiloride titration curves of ILiis
shown in Fig. 5 B for wt ENaC and the
,
, or
or Cys
mutants. In the presence of 20 mM external Li+, the inhibitory constant (Ki) for amiloride block of wt ENaC was 42 nM. The
S583 to C mutation coexpressed with
and
wt decreased channel affinity for amiloride
block to 1 µM. A more dramatic decrease in affinity for
amiloride was observed with the Gly to Cys mutations in
the
and
subunits with an ~1,000-fold increase in
amiloride Ki. As shown on Table I similar amiloride resistance were conferred by the
G525 to A mutation,
and the G525D mutant was completely resistant to
amiloride block at concentrations up to 100 µM. Finally double Cys mutations in the
and
subunits were not additives with respect to amiloride sensitivity,
whereas mutations in the
and
subunits further increased the Ki for amiloride. Thus Cys mutations in
subunits affect external channel block by
amiloride, but the effects of mutations in the
and
are quantitatively more important and coincides with
their respective decrease in single channel conductance.
Introduction of Cys residues within the primary sequence of different channel types has been useful to
confer sensitivity to channel block by divalent cations
such as Cd2+ or Zn2+and to identify residues directly
involved in the ionic permeation pathway (Favre et al.,
1995; Chiamvimonvat et al., 1996
). We tested the possibility that the Cys mutation makes ENaC channel sensitive to external block by Zn2+. Fig. 6 compares the ability of external Zn2+ to block the Li+current expressed
by wt ENaC or Cys mutants. The wt ENaC is completely insensitive to external Zn2+ at concentrations up to the
millimole (Kifor Zn2+ > 10 mM) whereas the
S583C
mutants is blocked by µM concentrations of Zn2+ with
a Ki of 29 µM. The
G525C and
G537C displayed a
significant block by Zn2+ compared to the wt, but with a
40- to 70-fold lower affinity for Zn2+ (Ki = 1.1 and 1.9 mM, respectively) than the
S583C mutant. The Zn2+
block characteristics of S583C mutant at the single
channel level is illustrated in Fig. 7. In the absence of
external Zn2+,
S583C mutant shows long openings
and closures; after addition of 0.2 mM Zn2+ the channel
exhibit a flickery block characterized by short opening and closing events lasting <100 ms. Hyperpolarization
of the membrane increased channel block, indicative
of a slight voltage dependence for Zn2+ block. These experiments indicate that Zn2+ has access to the Cys residues at position
S583,
G525,
G537 in the
subunits in order to block the channel, implicating that
these residues are located in the ion permeation pathway. The voltage dependence of Zn2+ block suggests
that
S583 residue is located within the transmembrane electric field.
Assuming that the residue at position 583 in the subunit is a subsite of an external amiloride binding
site, we tested the hypothesis that Zn2+ binding at
Cys583 in the
subunit competitively interacts with amiloride at a common binding site. Dose-response
curves of channel block by amiloride were performed
in the presence of 0.2 mM external Zn2+. As shown in
Table I, the
S583C mutant shows an approximately sixfold decrease in the affinity for amiloride block
(Ki = 5.7 µM) compared to the Ki obtained in the absence of Zn2+ (Ki= 1.04 µM). This shift in amiloride Ki
by Zn2+is strong evidence for a competitive interaction
between Zn2+ and amiloride as predicted by the following equation relating amiloride Kiin the presence
(Kapp) or in absence of Zn2+ (Kiamil):
![]() |
(3) |
where KiZn represents the Ki for Zn2+ block. In the presence of 0.2 mM Zn2+, the theoretical Kapp value for amiloride was 8.1 µM, a value close to that determined experimentally in Table I. Our result suggest that Zn2+ competitively interacts with amiloride for channel block at a common binding site located within the ion conduction pathway.
Contribution of aromatic residues in amiloride sensitivity.Amiloride is also a potent inhibitor of the Na+/H+ exchanger in which a stretch of amino acids including
Phe residues (FFLF sequence) has been implicated in a
putative amiloride binding site (Counillon et al., 1993).
In addition, amiloride contains a guanidinium moiety
and, interestingly, guanidinium toxins such as TTX or
STX which block voltage-gated Na channels with high affinity, interact with aromatic residues at the entrance
of the channel pore (Favre et al., 1995
). We investigated the possible contribution of the aromatic Trp
and Phe residues (
W582 and
F524) in ENaC block
by amiloride. As shown in Table I, the W582L mutation decreased the channel affinity for amiloride block with
a Ki of 1.4 µM in the same range of that obtained with
the
S583 to C mutation. By contrast the
F524A or D
mutations adjacent to
G525 did not change channel
affinity for amiloride.
Amino Acid Substitutions at S580,
G522,
G534
Positions
Asp or Glu substitution of the S
and G residues at position S580,
G522,
G534 did
not significantly change the level of macroscopicILi(data not shown). However changes in single channel conductance were observed in single channel recordings as shown in Fig. 8 A. The
G522D mutant exhibits
both long and short transitions between closed and
open states with a reduced single channel conductance compared to ENaC wild type (upper tracing). This reduction in single channel conductance is particularly
evident from the recording obtained in oocyte coexpressing both wt
and
G522D mutant together with
and
subunits (lower tracings). In this tracing two
channels with different conductances can be observed,
the wt ENaC channel characterized by a larger conductance, and a channel of smaller conductance, the
G522D mutant that spend most of the time in the open
conformation. The I-V relationships in Fig. 8 B (left
panel) shows a reduced single channel conductance for
the
S580D and
G522D mutants in the presence of
Na+or Li+ions compared to
G522S or wt ENaC (see
Fig. 4 B). The I-V relationship for S580D and
G522D
does not conform the predictions of the constant field
equation as observed with the wt ENaC or the
G522S
mutant, but rather suggests a voltage-dependent block
of the channel. As shown in Fig. 8 B (right panel), removing external Ca2+from the pipette solution restored a single channel conductance of 11.5 pS with
Li+and 6.7 pS with Na+for both mutant that was similar to wt ENaC or the G522S mutant. This Ca-dependent
decrease in single channel conductance can be interpreted as a fast channel block which involves coordination of C a2+ ions with the negatively charged Asp
at position
S580 and
G522, whereas the
G522S mutant remains insensitive to Ca2+ block. The accessibility of the
residues
S580 and
G522 by Ca2+ions and the resulting channel block further support the notion that the
segment encompassing
S580 and
S583 residues are
facing the pore lumen.
Equilibrium kinetics parameters of channel block by
external Ca2+ were determined from Ca2+titration of
macroscopic Li+currents shown in Fig. 9 A. The wt
ENaC exhibits a low sensitivity to external Ca2+block,
as it was previously shown for external Zn2+. The
G522D mutant was the most sensitive for Ca2+block
among the mutants tested with a Ki of 0.4 mM. The
S580D and
G534E shows similar affinities for Ca2+block with a Ki around 2 mM. As suggested by the single channel I-V relation, block of
G522D mutant by
external Ca2+ is voltage-dependent, and Fig. 9B shows
the block of Li+current by Ca2+ as a function of the
membrane potential. For different external Ca2+concentrations, hyperpolarization of membrane increased
external Ca2+block as shown by a decrease in the Li+current. In order to obtain empirical parameters for
the voltage dependence of Ca2+block, the data were
fitted according to the Woodhull model (Woodhull, 1973
). A mean slope parameter z
for the voltage dependency of Ca2+block was 0.30 ± 0.03 for the three
different experimental conditions. According to the
Woodhull formalism, this slope parameter equals the
valence of the blocking ion and the fraction of membrane potential acting on the ion at its blocking site.
This fraction of the voltage drop sensed by Ca2+ions or
the electrical distance was ~15% of the transmembrane electric field, suggesting that Asp at position
G522 of the pre-M2 segment is located within the
outer entrance of the channel pore.
Pharmacological properties.
The S580 to D and
G534
to E mutations did not significantly change the channel
affinity for amiloride block as shown in Fig. 10. Mutations at the corresponding position in the
subunit resulted in decrease in amiloride sensitivity with Ki for
amiloride of 3.4 ± 0.4 and 0.63 ± 0.09 µM for the
G522D and
G522S mutants. Since these titration experiments were performed in the presence of external
Ca2+(1.8 mM) the lower affinity of
G522D mutant for
amiloride block compared to the
G522S mutant can
account for a competitive interaction between Ca2+and amiloride at a common binding site. The consequences of these mutations on amiloride sensitivity were
however less important than the mutations described
previously and located three residues further downstream in the amino acid sequence.
Block by Amiloride
This study identifies residues in a short amino acid segment preceding the second transmembrane domain of
subunits that when mutated decrease channel affinity for amiloride block. These amino acid residues
have been identified in all three subunits at corresponding positions in a locus that comprises Ser residues in the
subunits and Gly in
and
subunits. A
more than 1,000-fold decrease in amiloride sensitivity
resulted from substitutions of a Gly residue in the
and
subunits, whereas mutation of the corresponding
Ser residue in the
subunit resulted in a smaller (~20-fold) change in the channel's affinity for amiloride block. These observations indicate that all three
subunits
of the heteromultimeric ENaC channel are involved in
the channel block by amiloride. In addition, the three
subunits likely display specific structural characteristics in their respective molecular binding interactions
with amiloride and/or the modulation of this effect.
The W582 mutation in the
subunit decreases Kifor
amiloride but not the corresponding aromatic residue
F524 in the
subunits. Conversely, mutations of the
G522 in the
subunit changed amiloride Ki but not the
corresponding mutations in the
and
subunits.
The observation that mutations of G525 and
G537
in
and
subunits have the most important effect on
channel sensitivity to amiloride suggests that these residues are critical for the binding interaction between
amiloride and its receptor on the channel. However expression of ENaC
subunit alone in Xenopus oocytes induces an amiloride-sensitive current of low magnitude (~20 nA range) that exhibits a high affinity for amiloride
of ~100 nM, suggesting that this pharmacological
property can be conferred to the channel by the
subunits alone (Canessa et al., 1994b
). It should be pointed
out that there is presently no direct evidence that functional channels in oocytes expressing
subunits alone
are exclusively made of
subunits, and therefore the
contribution of other endogenous subunits in the formation of the channel cannot be ruled out. Thus the
pharmacological profile of a homomultimeric channel
made of
subunits still remains an open question.
Among the recently cloned ENaC subunits, the
subunit shows the highest degree of homology with the
subunits, and within the stretch of residues shown in
Fig. 1, the
subunit differs from the
ENaC by a Leu at
position 581 and Tyr at position 582, both subunits having a conserved Ser residue at position 583 (Waldmann
et al., 1995a
). Similar to the
subunit, the
subunit associates with
and
subunits to form functional heteromultimeric channels at the cell surface. However, the
channel differs from the
channel by a 30 times
higher Ki for amiloride, which is in the same order of
magnitude as those observed after mutations of
S583
and
W582 in the
subunit. The structural basis for this difference in amiloride sensitivity remains to be determined and could possibly involve the Trp to Tyr and
Gln to Leu substitutions, although they represent rather
conservative amino acid substitutions. Thus there is no
doubt that the
subunit is important in determining the pharmacological properties of the channel, but the
almost complete resistance to amiloride obtained with
mutations in the
and
subunits stresses the important contribution of both subunits in the binding interactions between channel and amiloride.
It may be too early to speculate on the molecular basis of the high affinity of ENaC for amiloride. Conserved Gly residues typically tend to play a structural
role in folding of the peptide backbone, so the Gly mutations may change the structure in this region and
consequently alter amiloride block. However the Zn2+
and Ca2+ block clearly show that these residues must be
accessible to solution and the competitive interactions
between amiloride and divalent cations are consistent
with the notion that S583,
G525 and
G534 form an
external binding site for amiloride. These results do
not exclude the possibility that other functional domains of ENaC subunits may be involved in binding interactions with amiloride. A study on structure activity
relationships of amiloride and analogs has proposed
that ENaC block by amiloride involves a two-step process with distinct binding sites on the channel molecule
(Li et al., 1987
). Based on sequence relationships among
amiloride binding proteins, Kieber-Emmons et al. identified a EWYRFHY locus in the extracellular loop of
ENaC as a candidate site for amiloride binding (Kieber-Emmons et al., 1995
).
Block by Divalent Cations
Divalent cations such as Ca2+, Mg2+, or Ba2+ block
ENaC from the external side in a voltage dependent
manner and with very low affinity (Palmer, 1985). In
the toad urinary bladder the estimated Ki for these divalent cations exceeds 200 mM. Introduction of negatively charged residues at position
S580,
G522, or
G534 increased the channel's affinity for external Ca2+block. This block is characterized by a reduction in unitary
current often described as a "very fast" block in which
the flickering between open and blocked state is so fast
that the channel conductance appears lowered (Hille,
1992
). In the
G522D mutant, the voltage dependence
of block by Ca2+predicts a Ca2+ interaction with the channel at a site located 15% within the transmembrane electric field. This voltage-dependence of Ca2+ block is similar
to that reported for ENaC block by amiloride, further supporting the notion that the
G522 and likely the adjacent
G525 residues within pre-M2 segment are involved in
an external amiloride binding site (Palmer, 1985
).
Cys substitution at S583 position renders the channel
sensitive to block by external Zn2+ions with a Ki in the
µM range. The flickering channel behavior induced by
Zn2+is very similar to channel block by amiloride with
rapid opening and closing events lasting <100 ms and a
slight voltage dependence (Palmer and Frindt, 1986
). Coordination of Zn2+with the sulfhydryl group of Cys at position 583 in the
subunit further decreased channel affinity for amiloride, as shown by the competitive interaction
between these two blocking molecules. Thus Zn2+ ions
have similar blocking effects than amiloride when binding a common site within the transmembrane electric field.
All the divalent blocking experiments provide strong
evidence that the Ser580 and Ser583 as well as the corresponding Gly residues in the and
subunits are accessible by external cations and are facing the ion permeation pathway. The location of these residues within the
transmembrane electric field suggest that the pre-M2
segment forms the outer entrance of the channel pore.
ENaC Channel Pore
In a model proposed by Palmer and based on diverse
functional experimental findings, the ENaC channel
pore can be envisioned as a progressively narrowing
funnel that allows only the smallest monovalent cations
such as H+, Li+, or Na+ to permeate the channel
(Palmer, 1990). The outer mouth of the channel accept
blocking cations with a molecular diameter of <5 Å which bind with low affinity at a single site located at
~15% of the transmembrane electric field. Several pieces
of evidence support the notion that amiloride is a channel pore blocker, plugging the conducting pore and
preventing ion permeation. These evidences include a
voltage dependence of amiloride block very similar to that measured for monovalent and divalent cations and
the interactions between amiloride and permeating or
blocking cations (Palmer, 1985
, 1990
). According to its
size, only the guanidinium moiety of amiloride would
be able to reach the outer mouth of the channel.
In support of this model are the observations that residues in subunits involved in amiloride binding
are facing the ion permeation pathway, and the striking
similarity between amiloride and Zn2+ blocking effects
when binding at a commun site. The molecular mechanism underlying channel block by amiloride is not yet
understood. Evidence for competitive interactions between permeating Na+ions and amiloride has been
provided in experiments where lowering external Na+increased the association rate constant for amiloride
(Palmer and Andersen, 1989
). In our study,
G525C
and
G537C mutations decrease channel conductance
and coexpression of these mutations with the corresponding mutation S583C in the
subunit resulted in
a nonconducting channel expressed at the cell surface.
This indicates that these residues are important for
both ion permeation and amiloride binding. The lower
single channel conductance observed for mutations
that have the more dramatic effect on amiloride Ki are
consistent with a binding interaction between amiloride and Na+ ions at an external Na+binding site. Furthermore, these findings point to the role of
and
subunits
in ion permeation through the channel. The presence of
amino acids in
and
subunits that when mutated affect
single channel conductance and may participate in an external Na+binding site raises questions about the ionic
permeation properties of
homomeric channels, properties that have not yet been convincingly established.
A recent study has identified two Ser residues (Ser
589, Ser 593) in the M2 membrane-spanning segment
of rENaC that when mutated change unitary current
and ionic selectivity of the channel (Waldmann et al.,
1995b
). The Ser589 also decreased channel affinity for
amiloride with a Ki of ~2 µM. According to an
helical
structure of the M2 segment these Ser residues could
line the conducting pore, suggesting that the M2 segment extend deeper into the channel pore where the
selectivity filter could be located.
The picture of the channel pore emerging from our
mutagenesis experiments is still crude but shows a pore
formed by the three subunits. The question as to
whether the channel pore is composed of strictly identical subunits has been debated since the identification of the heterotrimeric structure of the channel. The fact
that the functional properties of heteromultimeric
or
channels are indistinguishable from those measured with expression of
or
subunits alone has been
interpreted as evidence that
or
subunits are the
pore-forming subunits and that the
and
subunits
represent accessory subunits modulating channel activity (Waldmann et al., 1995a
). Clearly, mutations in the
and
subunits that change amiloride affinity, single
channel conductances, and generate binding sites for
channel block by divalent cations represent strong evidence that all these subunits participate in the ion permeation pathway. Thus our experiments favor a model in
which the
subunits are arranged pseudosymmetrically around a central conducting pore in a way similar
to the nicotinic acetylcholine receptor. However the subunit stoichiometry of ENaC remains to be determined.
The residues identified in this study, involved in
amiloride block and in ion permeation, are located in a
short segment with a high degree of homology that precedes the second membrane-spanning M2 segment.
Proteolytic digestion of ENaC subunit is compatible with the presence of a short segment preceding M2
that dips into the membrane (Renard et al., 1994
). This
observation is consistent with the localization of
G522
within the transmembrane electric field, and suggests
that the pre-M2 segment forms a pore loop structure
similar to the H5 domain (or P region) of voltage-gated K or Na channel. These channels, as well as inward rectifier K channels and cyclic nucleotide-gated channels
which contain two membrane-spanning segments, are
thought to contain pore loops, short polypeptide segments extending from one side of the membrane to
the aqueous pore of the channel (MacKinnon, 1995
).
In many channels these pore loops comprise the channel selectivity filter, binding sites for permeating ions
and for channel blockers such as diverse neurotoxins.
By analogy with these channels, the pre-M2 segment of
ENaC may also form a loop region that extends to the outer entrance of the channel pore where amiloride
binds at the same site as permeating ions, blocking
their way into the channel pore.
Original version received 11 September 1996 and accepted version received 28 October 1996.
Address correspondence to Dr. L. Schild, Institut de Pharmacologie & Toxicologie de l'Université, rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Fax: 41-21-692-5355; E-mail: Laurent.Schild{at}ipharm.unil.ch
We thank to B.C. Rossier, and J.-D. Horisberger for helpful discussions and comments on the manuscript.
This study was supported by a grant from the Swiss National Foundation for Scientific Research to L. Schild (No. 31-39435.93) and by the E. Muchamp Foundation.
wt, wild-type.