(Received for publication, June 28, 1996, and in revised form, June 4, 1997)
From the Department of Physiology and Biophysics,
University of Alabama at Birmingham, Birmingham, Alabama 35294, the
Departments of ¶ Medicine, § Pathology, and
§§ Physiology and
Institute for
Neurological Sciences, University of Pennsylvania and Veterans
Administration Medical Center, Philadelphia, Pennsylvania 19104, and
the ** Department of Physiology, Emory University,
Atlanta, Georgia 30322
Limited information is available regarding
domains within the epithelial Na+ channel (ENaC)
which participate in amiloride binding. We previously utilized the
anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to
delineate amino acid residues that contact amiloride, and identified a
putative amiloride binding domain WYRFHY (residues 278-283) within the
extracellular domain of rENaC. Mutations were generated to examine
the role of this sequence in amiloride binding. Functional analyses of
wild type (wt) and mutant
rENaCs were performed by cRNA expression
in Xenopus oocytes and by reconstitution into planar lipid
bilayers. Wild type
rENaC was inhibited by amiloride with a
Ki of 169 nM. Deletion of the entire WYRFHY tract (
rENaC
278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride
(Ki = 26.5 µM). Similar results were
obtained when either
rENaC or
rENaC
278-283 were co-expressed
with wt
- and
rENaC (Ki values of 155 nM and 22.8 µM, respectively). Moreover,
rENaC H282D was insensitive to submicromolar concentrations of
amiloride (Ki = 6.52 µM), whereas
rENaC H282R was inhibited by amiloride with a Ki
of 29 nM. These mutations do not alter ENaC
Na+:K+ selectivity nor single-channel
conductance. These data suggest that residues within the tract WYRFHY
participate in amiloride binding. Our results, in conjunction with
recent studies demonstrating that mutations within the
membrane-spanning domains of
rENaC and mutations preceding the
second membrane-spanning domains of
-,
-, and
rENaC alters
amiloride's Ki, suggest that selected regions of
the extracellular loop of
rENaC may be in close proximity to
residues within the channel pore.
The diuretic amiloride is a prototypic inhibitor of epithelial
Na+ channels
(ENaCs)1 (1), although
amiloride and its various derivatives inhibit many
Na+-selective transport proteins. Several laboratories have
recently identified domains within the epithelial Na+
channel and the Na+/H+ exchanger that appear to
participate in amiloride binding. Residues within the second
membrane-spanning domain of rENaC may interact with amiloride, as
mutations of a serine residue at position 589 result in a large
decrease of the apparent Ki for amiloride and the
amiloride analog benzamil, as well as alter cation selectivity (2).
Selected mutations of residues within a hydrophobic region, termed H2
(3), immediately preceding the second membrane-spanning domains of the
-,
-, and
-subunits of rENaC (i.e. Trp-
582, Ser-
583, Gly-
525, Gly-
537) and the
-subunit of bovine ENaC (Lys-504, Lys-515) affect the Ki for amiloride, and
several of these mutations affect single-channel conductance (4, 5). Snyder and co-workers have identified splice variants of
rENaC in
which the C-terminal 199 or 216 amino acid residues, including the
second membrane-spanning domain, are truncated (6). These splice
variants are not functional when expressed in Xenopus
oocytes, but retain amiloride and phenamil binding activity, suggesting that at least a portion of the amiloride and phenamil binding domain is
proximal to the C-terminal 216 residues of
rENaC.
Pouyssegur and co-workers generated a mutant NHE1 that had an apparent 30-fold decrease in its affinity for methylpropylamiloride. Sequence analysis identified a single point mutation within the putative fourth transmembrane domain, changing a leucine in position 167 to a phenylalanine. Further analysis of this region by site-directed mutagenesis identified phenylalanine residues at positions 165 and 168 that may participate in amiloride binding (7). Analysis of this leucine residue within the putative fourth transmembrane domain of NHE2 yielded similar results (8).
We have previously raised both polyclonal and monoclonal antibodies to
amiloride (9-11). Amiloride was conjugated to carrier protein with
linking groups located at different positions on the amiloride molecule
to allow distinct sites of the amiloride molecule to be exposed
following immunization (10). One amiloride derivative was coupled to
bovine serum albumin through a hydrocarbon spacer arm on a terminal
nitrogen of its guanidinium moiety (9). This strategy was based on
previous observations that several amiloride analogs with hydrophobic
substituents at this site are potent inhibitors of epithelial
Na+ channels (1). The binding of anti-amiloride antibodies
to amiloride was examined by solid phase immunoassay using
amiloride-bovine serum albumin conjugates adsorbed onto a solid
support. Polyclonal and several monoclonal anti-amiloride antibodies
recognized both benzamil and amiloride, but did not bind ethyl
isopropylamiloride (9, 10), consistent with the rank order of potency
of inhibition of high amiloride affinity epithelial Na+
channels (i.e. benzamil > amiloride ethyl
isopropylamiloride (1)). We utilized one monoclonal anti-amiloride
antibody (BA7.1) as a surrogate amiloride receptor (12) to identify the
amino acid residue types that may form an amiloride binding site, as well as their topologic orientation. Analysis of structural features of
this anti-amiloride antibody led to identification of a structurally related 6-residue tract within the extracellular loop of
rENaC (13).
We now provide evidence that this 6-amino acid residue tract within the
extracellular loop of
rENaC, identified as a putative amiloride
binding site by its homology with the amiloride binding domain within
the anti-amiloride antibody BA7.1 (12), is required to express an
epithelial Na+ channel that is sensitive to nanomolar
concentrations of amiloride.
Amiloride was a gift from Merck. Lipids were
purchased from Avanti Polar Lipids (Alabaster, AL). Moloney murine
leukemia virus reverse transcriptase and DNA tailing kit were obtained
from Life Technologies, Inc., Taq polymerase from Perkin
Elmer, dNTPs from Pharmacia Biotech Inc., Geneclean kit from Bio101,
Sequenase II DNA sequencing kit from U. S. Biochemical Corp., T4
ligase from Boehringer Mannheim, Escherichia coli strains
from Strategene (La Jolla, CA), restriction enzymes and cap analog from
New England Biolabs (Beverly, MA), and pALTER-1 in vitro
mutagenesis system, TNTTM-coupled reticulocyte lysate system, Ribomax
kit, and plasmid WizardTM mini-prep kits from Promega (Madison, WI).
rENaC,
rENaC, and
rENaC cDNAs in the vector pSPORT were a
gift from Drs. B. C. Rossier and C. Canessa (University of
Lousanne, Lousanne, Switzerland). Monomeric actin was purified from
rabbit skeletal muscle (a kind gift from Dr. S. S. Rosenfeld,
University of Alabama, Birmingham, AL) or purchased from Sigma. All
other reagents were purchased from Sigma.
A mutant of rENaC in
which amino acid residues 278-283 were deleted was generated by
designing PCR primers to amplify the 5
and the 3
regions of
rENaC
flanking the region to be deleted, and introducing a unique
XhoI restriction site 3
to the deletion to allow the two
products corresponding to the 5
and 3
ends of
rENaC to be ligated
following digestion with XhoI. The XhoI restriction site was generated by mutating nucleotide C942 to G, and
C945 to G. These mutations did not alter the amino acid residues in
positions 287 and 288. Primer pairs to amplify the 5
region of
rENaC were 5
-GTACCGGTCCGGAATTCCCGGGTCG-3
and
5
-CAGTCTCGAGAGAATGTTGATCTCCCTCACTGCATCCACCCCAGAGGAG-3
. PCR
was performed by denaturing the reaction mixture at 92 °C for
5 min, followed by 35 cycles (0.5 min at 92 °C, 1 min at 58 °C, and 2 min at 72 °C), and a final extension at 72 °C for 7 min. A
product of the predicted size of ~950 base pairs was isolated, purified (Geneclean), and digested with SmaI and
XhoI at 37 °C for 1 h. Primer pairs to amplify the
3
region of
rENaC were 5
-ACCGCTCGAGACTGTCGGACACCTCG-3
and
5
-TCTAAGGGATGCATAGACTGTGTGTTC-3
. PCR was performed by denaturing the
reaction mixture at 92 °C for 5 min, followed by 35 cycles (92 °C
for 1 min, 55 °C for 2 min, 72 °C for 3 min) and a final
extension at 72 °C for 7 min. A product with the predicted size of
1890 base pairs was isolated, purified (Geneclean), and digested with
XhoI and NsiI for 1 h at 37 °C. The 5
and 3
PCR-amplified regions of
rENaC were ligated into pSPORT,
which had been digested with SmaI and NsiI.
Plasmids were amplified, purified, and subjected to DNA sequencing
through the deletion site by Sanger dideoxynucleotide sequence analysis (14) to confirm that the construct was correct.
rENaC site-directed mutants were generated using the Altered SitesTM
in vitro mutagenesis system according to the manufacturer's instructions. Wild type
rENaC was excised from pSPORT-1 by digesting with SalI and SphI, and then ligated into
pALTER-1 via EcoRI and SphI sites with an
EcoRI-NotI-SalI adaptor.
Single-stranded pALTER-1-
rENaC template was prepared, and
appropriate mutagenic oligonucleotide primers, including an ampicillin
repair primer, were annealed to the template and second strand
synthesis was performed with T4 polymerase and T4 DNA ligase. Following
transformation of E. coli ES1301 mutS, cells
containing mutated pALTER-1-
rENaC were selected on the basis of
ampicillin resistance. Plasmid was then purified from individual
colonies and subjected to DNA sequencing to confirm the mutation of
rENaC. The primer used to generate H282D was
5
-ACCGCTTCGATTACATCAAC-3
; the primer used to generate H282R was
5
-TACCGCTTCCGCTACATCAAC-3
; the primer used to generate R280G was
5
-TACGGCTTCCATTACATCAAC-3
. Plasmids were linearized by overnight
digestion with SphI, and cRNA was synthesized using T7 RNA
polymerase in the presence of the cap analog
m7G(5
)ppp(5
)G using Promega's Ribomax kit according to
the manufacturer's instructions.
Oocytes were injected
with a total of 25 ng of either ,
,
rENaC cRNA or
278-283,
,
rENaC cRNA, and current recordings were performed
2 days later, as described previously (15). Briefly, recordings were
carried out at room temperature (20 °C). Oocytes were impaled with
recording current and voltage electrodes filled with 3 M
KCl (resistances ranging from 0.4 to 3 megohms). Two external
electrodes (1 voltage and 1 current) made from Ag-AgCl and were
connected to the chamber via 3% agar bridges filled with 3 M KCl. The recording and references electrodes were
connected to a four-electrode voltage clamp (TEV-200, Dagan,
Minneapolis, MN). Oocytes were voltage-clamped to 0 mV, and their
membrane voltage was stepped for 450 ms from
100 to +100 mV in 20-mV
increments to measure whole-cell currents. The potential was returned
to 0 mV for 50 ms between each voltage step. Current-voltage
(I/V) curves were constructed as described previously (16).
Increasing concentrations of amiloride were sequentially added to the
bath solution, and current was allowed to stabilize for 5 min between bath additions. Currents measured in the presence of varying
concentrations of amiloride were normalized to the currents obtained in
the absence of amiloride.
Membrane
vesicles from oocytes injected with rENaC,
rENaC
278-283,
rENaC H282D, or
rENaC H282R cRNA, vesicles from oocytes injected
with
,
,
rENaC cRNAs, and vesicles from oocytes injected with
278-283,
,
rENaC cRNAs were made essentially as described earlier (17). Briefly, 30 oocytes in each group were rinsed and
homogenized in high [K+]/sucrose medium containing
multiple protease inhibitors. Membranes were isolated by discontinuous
sucrose gradient centrifugation and resuspended in 300 mM
sucrose, 100 mM KCl, and 5 mM MOPS (pH 6.8).
This material was aliquoted into 50-µl fractions and stored at
80 °C until use.
Planar lipid bilayers were made as described
previously (17) using a phospholipid solution containing a 2:1:2
mixture of diphytanoyl-phosphatidylethanolamine/diphytanoyl-phosphatidylserine/oxidized cholesterol in n-octane (final lipid concentration = 25 mg/ml). Bilayers were bathed either with symmetric 100 mM
NaCl containing 10 mM MOPS-Tris buffer (pH 7.4) or with
this solution in the trans compartment and a buffer
containing 100 mM KCl, 10 mM MOPS-Tris (pH 7.4)
in the cis compartment. In selected experiments, actin was
added to the cis compartment. Actin was diluted prior to use to a final concentration of 4-10 µg/ml with a buffer containing 2 mM Tris, 0.2 mM CaCl2, 0.2 mM MgATP, 0.2 mM -mercaptoethanol (pH 8.0).
The mixture of actin with recombinant plasma gelsolin (dissolved at a
concentration of 10 µg/ml in 100 mM KCl, 10 mM HEPES buffer (pH 7.4), and dialyzed against 0.2 mM EGTA buffer) at 1:1 ratio was added to both sides of the
bilayer as described previously (18). All solutions were made with
Milli-Q water and were filter-sterilized by passing the solution
through 0.22-µm filters (Sterivax-GS filters, Millipore Corporation,
Bedford, MA). Current measurements were performed using a high gain
amplifier circuit, as described previously (17). Here and elsewhere
throughout this report, applied voltage is referred to the
trans chamber, which was connected to the current-to-voltage
converter and therefore was held at virtual ground. Under these
experimental conditions, the channels were oriented with their
amiloride-sensitive (extracellular) surface exposed to the
trans compartment in over 90% of the incorporations. The
probability of successful incorporation of single channels versus multi-channel incorporations depends upon the density
of channels in oocyte membranes. In our experience, these probabilities were highly variable, and we have developed a procedure of empirical dilution of oocyte vesicles yielding predominately single-channel incorporations (17). The experiments when no channels were evident in
the membrane were considered unsuccessful incorporations as described
previously. The rate of successful incorporations from oocyte membrane
was 1 in 50-200 attempts. Amiloride was added to the trans
chamber at concentrations indicated in the figures.
Acquisition and analysis of single-channel recordings were performed using pCLAMP software and hardware (Axon Instruments) as described previously (17). Data were stored digitally, and were filtered at 300 Hz with a 8-pole Bessel filter prior to acquisition at 1 ms/point. All the analyses were performed for membranes containing only single Na+ channels. The single-channel open probability was calculated for at least 3 min of continuous recording using Equation 1.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Limited information is available regarding ENaC domains that
participate in amiloride binding. We previously utilized the anti-amiloride antibody BA7.1 as a surrogate amiloride receptor (12) to
delineate amino acid residues that contact amiloride. We observed that
the sequence YYGHY contained in the CDR3 domain of the heavy chain of
mAb BA7.1 aligned with the sequence tract WYRFHY, corresponding to
residues 278-283 within rENaC (13, 19). It is likely that the
-subunit of the epithelial Na+ channel possesses an
amiloride binding site, as expression of the
-subunit alone is
sufficient to induce expression of a Na+ current in
Xenopus oocytes, which is inhibited by amiloride with apparent inhibitory constants (Ki) nearly identical
to that observed with expression of
,
,
rENaC
heterotrimers (Ki values of 100 and 104 nM, respectively). Similar results have been reported with
expression of
rENaC alone or expression of
,
,
rENaC
heterotrimers in planar lipid bilayers (Ki values of
170 nM for both channels) (3, 15, 17, 19). The putative
amiloride binding tract WYRFHY is within the extracellular domain of
rENaC (20-22). To examine the role of this sequence in amiloride
binding, we generated several mutants of
rENaC. One mutant,
rENaC
278-283, has a deletion of residues 278-283 (i.e.
WYRFHY). Our analysis of the binding of amiloride to BA7.1 suggested
that the histidine within the CDR3 region of the heavy chain stabilized
amiloride binding via electrostatic interactions with the halide (Cl)
on the pyrazine ring of amiloride (12). Therefore, two site-directed
mutants were generated:
rENaC H282D, in which the histidine at
position 282 was mutated to aspartic acid, and
rENaC H282R, in which
the histidine at position 282 was mutated to arginine. An additional
site-directed mutant,
rENaC R280G, was also generated.
A major technical difficulty in examining the functional
properties of rENaC
278-283 in the Xenopus oocyte
expression system is the low level of Na+ current observed
when the
-subunit is expressed alone (i.e. without
co-expression of
- and
rENaC) (3). Therefore, experiments using
this system were performed with the heterotrimeric channel. Fig.
1 shows the results of measurements of
macroscopic currents in Xenopus oocytes expressing wt
,
,
rENaC or
278-283,
,
rENaC. Interestingly, oocytes
expressing
278-283,
,
rENaC displayed a current that was
~40% smaller than that in oocytes expressing wt
,
,
rENaC.
The deletion of residues 278-283 within
rENaC may affect the
association of subunits forming the channel and alter subsequent
transit of the channel to the cell surface, or alter single-channel
properties. The amiloride-sensitive portion of the current (10 µM amiloride added to the bath) was much larger in the
oocytes expressing wt
,
,
rENaC (63 ± 8%) than in oocytes expressing
278-283,
,
rENaC (19 ± 3%). These
observations are consistent with those reported by Busch et
al. (23). A portion of the base-line current in water-injected
oocytes was also found to be amiloride-sensitive (17 ± 4%).
Inhibition of wt
,
,
rENaC by amiloride can be described in
terms of Michaelis-Menten kinetics (Fig.
2) with a Ki of
231 ± 46 nM (n = 4), in reasonable agreement with previous observations (3). The low current induced by
278-283,
,
rENaC and the limited inhibition by 10 µM amiloride precluded the detailed analysis of
amiloride-induced inhibition of
278-283,
,
rENaC expressed
in oocytes. A rough estimation of Michaelis-Menten kinetics, based on
analyses of the normalized current and assuming that 60% of the total
current is mediated by the Na+ channel, gave a
Ki of 35 ± 10 µM
(n = 9) for the mutant channel. In view of the low
levels of macroscopic current observed in Xenopus oocytes
expressing
rENaC alone, further studies examining the properties of
rENaC
278-283rENaC, and of
rENaC with selected mutations
within the WYRFHY tract (residues 278-283) were performed in planar
lipid bilayers.
When reconstituted in planar lipid bilayers, rENaC
278-283,
rENaC R280G,
rENaC H282D, and
rENaC H282R formed
Na+ channels essentially indistinguishable by conductance
or gating from those produced by wt
rENaC (Fig.
3). All mutated
rENaC channels display
a concerted type gating between 13 and 39 pS states consistent with
what was reported previously (17). We have previously observed this
gating pattern for
rENaC alone, and for
,
rENaC or
,
rENaC
heterodimers, which also display a concerted type gating between the 13 pS and 39 pS states and which is quite distinct from the gating pattern
observed with heterotrimeric channels (17). However, similar to the
experiments with heterotrimeric channels containing
rENaC
278-283, the mutant channels exhibited altered sensitivities to
amiloride (Figs. 3 and 4). Wild type
rENaC was inhibited by amiloride with a Ki of
169 ± 15 nM (n = 13), as determined
by fitting the amiloride dose-response data to the first order
Michaelis-Menten equation. Both the
rENaC
278-283 mutant and
rENaC H282D were largely insensitive to submicromolar concentrations
of amiloride, with Ki values of 26.5 ± 3.5 µM (n = 7) and 6.52 ± 0.45 µM (n = 10), respectively (Fig. 4 and
Table I). However, a conservative mutation of His-282 (
rENaC H282R) led to a decrease in the apparent Ki for amiloride by 5.8-fold. The
rENaC mutant
R280G had an apparent Ki for amiloride of 830 ± 70 nM (n = 6), a 4.9-fold increase when
compared with wt
rENaC. Similar apparent Ki
values for amiloride were obtained by fitting the amiloride
dose-response data to either the first order Michaelis-Menten equation
or the Michaelis-Menten equation with the Hill coefficient (Table I).
These data support the hypothesis that residues within the tract WYRFHY
are required for expression of epithelial Na+ channels that
are sensitive to nanomolar concentrations of amiloride, and suggest
that amiloride binds to, or interacts with, residues within this
tract.
|
Single-channel properties of rENaC channels studied in planar lipid
bilayers under these conditions differ from heterotrimeric rENaC
channels expressed in Xenopus oocytes and studied by the patch-clamp technique (3, 17). Therefore, we examined the properties of
wt
,
,
rENaC and of
278-283,
,
rENaC in planar lipid
bilayers. Incorporation of membrane vesicles obtained from oocytes
co-expressing wt
,
,
rENaC or
278-283,
,
rENaC in
planar lipid bilayers generated channels which displayed an essentially identical gating pattern with a predominant residence in a 13-pS state
and occasional openings to 39 pS (Fig. 5,
top traces) and consistent with our previous findings of the
heterotrimeric ENaC currents observed in bilayers (17). However, the
sensitivity of these channels to inhibition by amiloride was
significantly different. The heterotrimeric channel containing
rENaC
278-283 was inhibited by amiloride at concentrations more than 2 orders of magnitude higher than wt
,
,
rENaC (Table I).
Amiloride dose-response curves are illustrated in Fig.
6. The amiloride inhibitory constants (Ki values) for the deletion mutant (i.e.
278-283,
,
rENaC) and wt
,
,
rENaC were 22.8 ± 3.1 µM (n = 8) and 155 ± 14 nM (n = 12), respectively, determined by
fitting the amiloride dose-response data to the first order
Michaelis-Menten equation. Similar apparent Ki
values were obtained by fitting the amiloride dose-response data to the
Michaelis-Menten equation with the Hill coefficient (Ki values of 20.1 ± 2.2 µM and
189 ± 28 nM, respectively; see Table I). These
results are similar to the apparent Ki for amiloride
of wild type
,
,
rENaC and the estimated apparent Ki for amiloride of
278-283,
,
rENaC
expressed in oocytes.
We have previously demonstrated that the addition of actin to the
cis compartment alters properties of wt ,
,
rENaC
reconstituted into planar lipid bilayers, by reducing single-channel
conductance to 6 pS and by increasing mean open and closed times,
characteristics similar to those observed for ENaCs expressed in native
tissues and analyzed by patch-clamp (18, 24). The sensitivities to amiloride of wt
,
,
rENaC and of
278-283,
,
rENaC
were not altered when actin was added to the cis compartment
(Figs. 6 and 7), although single-channel
conductance was reduced to 6 pS.
Additional studies were performed to examine whether deletion of the
WYRFHY tract (residues 278-283) or mutations within this tract affect
selectivity properties of the channel. The
Na+:K+ selectivity of rENaC
278-283,
rENaC R280G,
rENaC H282D, and
rENaC H282R did not differ from
wt
rENaC (Fig. 8), as determined in
symmetrical NaCl and bi-ionic (NaCl trans:KCl
cis) conditions in planar lipid bilayers. In addition,
Na+:K+ selectivity ratio of the heterotrimeric
Na+ channel was not altered by deletion of residues
278-283, as determined by the incorporation of channels in planar
lipid bilayers (Fig. 9), or by expression
of channels in Xenopus oocytes using the two electrode
voltage clamp technique (Fig. 10). The
plots in these graphs represent fit of the data obtained in
Na+ to K+ substitution experiments using the
Goldman-Hodgkin-Katz (GHK) equation,
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
Amiloride is the prototypic inhibitor of epithelial
Na+ channels. Amiloride analogs have been used by a number
of investigators as tools to isolate and characterize
amiloride-sensitive Na+ channels, and may have an important
role as Na+ channel inhibitors in the treatment of selected
forms of hypertension (29, 30). Previous studies demonstrated that it
is the charged, protonated species of amiloride that inhibits
Na+ channels (31-34). This channel block is dependent, in
part, on the apical membrane potential. Analysis of the kinetics of
amiloride binding to the Na+ channel in the presence of a
varying apical plasma membrane potential suggests that amiloride senses
between 10% and 45% of the membrane electric field (31, 34). This
observation, when taken together with studies utilizing voltage-clamped
cells to demonstrate that amiloride binding kinetics are altered by
Na+ or Li+ loading cells to generate an outward
current through the channel (35, 36), supports the idea that amiloride
interacts within the channel pore. This hypothesis is further supported
by recent studies of Waldmann et al. demonstrating that
substitutions of the first or second putative transmembrane region of
rENaC with the corresponding domains within Mec-4 led to a decrease
amiloride sensitivity by 3- and 14-fold, respectively (2). Mec-4 has significant sequence similarity with ENaCs and is a member of the
Caenorhabditis elegans degenerin family that is associated with mechanotransduction (19). Mutation of a serine to
phenylalanine in position 589 of the second membrane-spanning
domain of
rENaC increases the Ki for amiloride
and alters cation selectivity and single-channel conductance,
suggesting that serine 589 participates in amiloride binding and
resides within the channel pore (2). In addition, residues preceding
the second membrane-spanning domains of
-,
-, and
rENaC and of
bovine
ENaC may also form part of the channel pore, and selected
mutations within these regions dramatically affect amiloride
sensitivity (4, 5).
Organic cations other than amiloride and related analogs, such as
2,4,6-triaminopyrimidine, also function as epithelial Na+
channel inhibitors, although with Ki values much
greater than that of amiloride (37), suggesting that it is the
guanidine moiety of amiloride that is interacting with the channel
pore. However, the substituted pyrazine ring of amiloride is required for the drug to inhibit ENaCs with a submicromolar
Ki, and may have a critical role in stabilizing
amiloride bound to the channel (1, 38). The putative amiloride binding
site on the anti-amiloride antibody BA7.1 that we previously
characterized primarily interacts with the substituted pyrazine ring
moiety of amiloride (12). By analogy, the 6-amino acid track within the
extracellular loop of ENaC we identified based on its homology with
the amiloride binding site on BA7.1 likely binds amiloride via
interactions with the substituted pyrazine moiety of amiloride, and is
not necessarily associated with the pore region of the channel. Our
results demonstrating that the single-channel conductances and
Na+:K+ selectivity ratios of
rENaC
278-283,
rENaC R280G,
rENaC H282D, and
rENaC H282D are
indistinguishable from wt
rENaC suggest that residues 278-283 do
not form part of the Na+ channel pore. Li and co-workers
have identified splice variants of
rENaC, in which the C-terminal
199 or 216 amino acid residues are truncated, including the second
membrane-spanning domain (6). These splice variants are not functional
when expressed in Xenopus oocytes, but retain amiloride and
phenamil binding activity, suggesting that part of the amiloride and
phenamil binding site is proximal to the C-terminal 216 residues of
rENaC, again consistent with our findings. The previously published
results suggesting that residues within the second membrane-spanning
domain of
rENaC and within a hydrophobic (putative pore) region
preceding the second membrane-spanning domains of
-,
-, and
rENaC bind amiloride, and our results, indicating that residues
within the extracellular loop of
rENaC (i.e. residues
278-283) bind amiloride, suggest that amiloride contact residues are
derived from different regions of rENaC and that selected regions of
the extracellular loop of
rENaC may be in close proximity to
residues within the Na+ channel pore.
Our previous analysis of the amiloride binding site on the
anti-amiloride antibody BA7.1 suggested that a histidine residue within
the CDR3 region of the heavy chain primarily interacts with the Cl atom
on the pyrazine ring moiety of amiloride through an electrostatic
interaction (12). Therefore, we examined the effects of mutations of
the histidine residue (His-282) present within the 6 amino acid
putative amiloride binding domain on the extracellular loop of ENaC.
Mutagenesis of this histidine to aspartic acid (H282D) resulted in a
change in charge of this residue from cationic to anionic and was
associated with a 39-fold increase in the apparent
Ki for amiloride. Alternatively, mutagenesis of this
histidine to arginine (H282R) with conservation of the cationic charge
was associated with a 6-fold decrease in the apparent Ki for amiloride. H282R is the first mutation of
ENaC described that is associated with a large decrease in the
amiloride Ki. These data suggest that His-282 may
have an important role in stabilizing the binding of amiloride to the
Na+ channel. Although it is possible that His-282 primarily
interacts with the Cl atom on the pyrazine ring moiety of amiloride,
there is no direct evidence to support this hypothesis. H282R and H282D have a difference in their apparent Ki values for
amiloride of 225-fold. The
rENaC mutant R280G was inhibited by
amiloride with a Ki of 830 nM, a
4.9-fold increase in the apparent Ki for amiloride
when compared with wt
rENaC suggesting that residues within the
tract 278-283, other than His-282, may bind amiloride or affect the
topology of this site.
The Hill coefficient of 2.4 that we observed in our studies of
amiloride inhibition of wt ,
,
rENaC and wt
rENaC
reconstituted into planar lipid bilayers is in reasonable agreement
with previous observations (17). ENaC stoichiometry has not been
determined. If amiloride interacts primarily with the
-subunit, a
Hill coefficient of 2.4 indicating cooperative binding suggests that
ENaCs have more than one
-subunit or, alternatively, that there are
multiple sites, or domains, within the
-subunit which participate in
amiloride binding. Interestingly, the Hill coefficient decreased with
rENaC mutations that increased amiloride's Ki,
suggesting that the amiloride binding domain we have identified may
affect subunit-subunit interactions, may affect intramolecular
interactions within the
-subunit, or alternatively may alter
-subunit stoichiometry.
The amiloride binding domain WYRFHY is conserved within ENaC in all
species that have been cloned and sequenced to date, including rat,
human, bovine, Xenopus, and mouse (15, 19, 39-41). A nearly
identical tract, WYHFHY, is present in the recently cloned
subunit
of a Na+ channel that appears to be expressed in both
epithelial and nonepithelial tissues (42). Both
ENaC and
ENaC are
sufficient, by themselves, to induce the expression of
Na+-selective, amiloride-sensitive channels in
Xenopus oocytes. The current levels observed with expression
of
ENaC or
ENaC increase by approximately 100-fold when
co-expressed with
- and
ENaC. An amiloride- and
benzamil-sensitive FMRFamide peptide-gated Na+ channel
(FaNaCh) was recently cloned from marine snail neurons (43).
Interestingly, FaNaCh does not have a WYRFHY tract, although a related
tract WLRFIQKF is present in the putative extracellular domain of
FaNaCh that shares some features with the amiloride binding domain we
have identified within
ENaC, including the presence of planar and
cationic amino acid residues. Further studies are required to examine
whether residues within the tracts WYHFHY in
ENaC and WLRFIQKF in
FaNaCh participate in amiloride binding.
The sensitivity of rENaC to amiloride does not appear to be
dependent upon co-expression with
- and
rENaC, as wt
rENaC and
wt
,
,
rENaC reconstituted into planar lipid bilayers have similar Ki values for amiloride (155 nM
and 169 nM, respectively (Table I)). This was also observed
with
rENaC
278-283, as
rENaC
278-283 and
278-283,
,
rENaC have nearly identical sensitivities to
amiloride (Ki values of 22.8 µM and
26.5 µM, respectively; Table I). These data support the
hypothesis that residues required to form high affinity amiloride
binding domains reside within the
-subunit. However,
- and
ENaC also participate in amiloride binding (4). Interestingly, a
tract WYKLHY (residues 230-235) within the extracellular loop of
rENaC bears striking similarity to the amiloride binding domain we
identified within the extracellular domain of
ENaC (3). Additional
studies are required to determine whether this region within
rENaC
(i.e. residues 230-235) participates in amiloride binding.
It is conceivable that mutations we have generated alter the
stoichiometry of subunit association, which might affect amiloride
binding. Previous studies from our laboratory suggest that
rENaC,
reconstituted alone or as
/
or
/
heterodimers, primarily
exhibits 13-pS and 39-pS conductance states, whereas the
/
/
heterotrimer primarily exhibits a 13-pS conductance state (17). The
conductance states observed with both wt
,
,
rENaC and with
278-283,
,
rENaC (Fig. 3A) indicate that
278-283,
,
rENaC is reconstituted into the lipid bilayer as
a heterotrimer.
In summary, the analysis of an anti-amiloride antibody resulted in the
identification of an amiloride binding domain on ENaC. Although
there are other sites within
ENaC, as well as within other ENaC
subunits that participate in amiloride binding, our data clearly
suggest that residues 278-283 within
rENaC, particularly His-282,
are part of an amiloride binding site. In addition, these studies
support the hypothesis that selected anti-ligand antibodies may serve
as surrogate ligand receptors, and that in selected systems these
antibodies may provide useful tools to develop models of tertiary
structural features of naturally occurring ligand receptors.
We thank Drs. B. C. Rossier and C. Canessa for providing -,
-, and
rENaC cDNAs.