From the Howard Hughes Medical Institute and Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extracellular amiloride inhibits all known
DEG/ENaC ion channels, including BNC1, a proton-activated human
neuronal cation channel. Earlier studies showed that protons cause a
conformational change that activates BNC1 and exposes residue 430 to
the extracellular solution. Here we demonstrate that, in addition to
blocking BNC1, amiloride also exposes residue 430. This result
suggested that, like protons, amiloride might be capable of activating
the channel. To test this hypothesis, we introduced a mutation in the
BNC1 pore that reduces amiloride block, and found that amiloride
stimulated these channels. Amiloride inhibition was
voltage-dependent, suggesting block within the pore,
whereas stimulation was not, suggesting binding to an extracellular
site. These data show that amiloride can have two distinct effects on
BNC1, and they suggest two different interaction sites. The results
suggest that extracellular amiloride binding may have a stimulatory
effect similar to that of protons in BNC1 or extracellular ligands in
other DEG/ENaC channels.
A characteristic shared by DEG/ENaC cation channels is that
extracellular amiloride inhibits their current. The ability of amiloride to inhibit the human epithelial Na+ channel
(ENaC) makes it clinically useful as a diuretic (1). Amiloride-sensitivity is also a valuable marker of currents generated by other DEG/ENaC channels from vertebrates, insects, mollusks, and
nematodes (2, 3). However, the mechanism of amiloride's action is not
understood. For example, some data suggest that amiloride blocks
current by occluding the ion-conducting pore (4), whereas other
evidence suggests that amiloride binds a predicted extracellular domain
of ENaC (5, 6).
The relationship between amiloride block and channel activity is
interesting. For example, the Drosophila Na+
channel Ripped Pocket (RPK) generates a minimal Na+ current
that is blocked by relatively high concentrations of amiloride (7).
However, when RPK contains a "Deg" mutation that increases
Na+ current 50-fold, current is blocked by 20 times lower
concentrations of amiloride. In this study we further examined the
relationship between amiloride and channel activity. The DEG/ENaC
channel that we studied was BNC1 (also called MDEG and BNaC1), a
proton-gated channel from human neurons that may be involved in
synaptic transmission and the sensation of pain and sour taste (8-12).
Under basal conditions at neutral extracellular pH, BNC1 generates only
a very small whole cell Na+ current (8). However, lowering
extracellular pH activates a large transient Na+ current
(11).
Like RPK, BNC1 is activated by a Deg mutation, which alters a critical
glycine residue at position 430 (9). Residue 430 is a site where
analogous mutations in some Caenorhabditis elegans family
members cause neurodegeneration (13). In BNC1, mutation of
Gly430 to valine or larger amino acids locks the channel in
an activated state, even at neutral pH (9). In contrast, mutation of
Gly430 to cysteine (BNC1-G430C), generates only small
constitutive currents (14), and confers a unique sensitivity to
sulfhydryl-reactive methanethiosulfonate
(MTS)1 compounds (15). The
ability of MTS compounds to modify Cys430 is
state-dependent. Under basal conditions at extracellular pH 7.4 with the channel closed, Cys430 is largely protected
from extracellular MTS compounds. However when the channel is activated
by protons, Cys430 becomes accessible to extracellular MTS
compounds, which covalently modify Cys430 to make its side
chain larger. As a result, the channel is irreversibly activated
because it is unable to return to the inactive state.
In the course of investigating the role of residue 430 in channel
activation and in inhibition by amiloride, we made the surprising discovery that under certain conditions amiloride could activate current. Here we describe our investigation of the responsible mechanisms.
cDNA Constructs--
BNC1 mutants were constructed by
single-stranded mutagenesis of BNC1 (8) in pBluescript. The validity of
constructs was confirmed by DNA sequencing. Constructs were cloned into
pMT3 (16) for expression.
Expression and Electrophysiological Analysis in Xenopus
Oocytes--
cDNA constructs were expressed in defolliculated
albino Xenopus laevis oocytes (Nasco, Fort Atkinson, WI) by
nuclear injection of plasmid DNA. cDNA encoding BNC1-G430C was
injected at 5 ng/µl. cDNA encoding BNC1-G430V/G437C was injected
at 40 ng/µl. For coexpression of the double mutant BNC1-G430V/G437C
with BNC1-G430V, we injected 10 and 2 ng/µl, respectively. For
coexpression of BNC1-G430V/G437C with wild-type BNC1, we injected 25 and 5 ng/µl, respectively. Following injection, oocytes were
incubated at 18 °C in modified Barth's solution. Generally, cells
were studied 12-24 h after injection; however, cells expressing
BNC1-G430V/G437C (with or without wild-type BNC1) were studied 36-48 h
after injection. Whole cell currents were measured using a
two-electrode voltage clamp. During recording, oocytes were bathed in
frog Ringer's solution (116 mM NaCl, 0.4 mM
CaCl2, 1 mM MgCl2, 5 mM
HEPES, pH 7.4). MTS compounds were from Toronto Research Biochemicals
(Toronto, Ontario) and included cationic reagents, 2-aminoethyl
methanethiosulfonate bromide (MTSEA) and 2-(trimethylammonium)ethyl
methanethiosulfonate bromide (MTSET), and an anionic reagent, sodium
2-sulfonatoethyl methanethiosulfonate (MTSES).
Zn2+ Reversibly Activates BNC1-G430C--
In earlier
work (15) we showed that when Gly430 was mutated to
cysteine, covalent modification of residue 430 by MTS reagents irreversibly locked BNC1-G430C in an activated state. Therefore we
hypothesized that a noncovalent interaction between Zn2+
and Cys430 might also activate the channel, but in a
reversible way. Fig. 1A shows
that extracellular Zn2+ had no effect on current in oocytes
expressing wild-type BNC1. However, at neutral extracellular pH,
Zn2+ reversibly activated current in oocytes expressing
BNC1-G430C (Fig. 1B). The EC50 for activation of
BNC1-G430C by Zn2+ was approximately 300 µM
(Fig. 1C).
Previous studies (15) showed that pH-dependent activation
of BNC1-G430C allowed MTS compounds to modify Cys430. Fig.
1D shows that activation by Zn2+ also allowed
the MTS compound, MTSES, to modify Cys430 and irreversibly
activate the channel. How might both Zn2+ and MTSES
interact with Cys430? Because the BNC1 channel is a
homomultimer composed of multiple BNC1 subunits (17), it is likely that
within one channel there are multiple cysteines with which these agents
may interact. Taken together, these data indicate that Zn2+
binds to Cys430 to reversibly activate BNC1-G430C.
Amiloride Increases the Accessibility of Cys430 to
Zn2+ and MTS Reagents--
Fig.
2A shows that 100 µM Zn2+ activated BNC1-G430C and that current
was blocked by 1 mM amiloride. However, as shown in Fig. 2,
B and C, Zn-activated currents were not smaller
but larger if a submaximal concentration of amiloride (100 µM) was present. In the absence of Zn2+,
amiloride either had no effect (Fig. 2B) or blocked a small amount of current (Fig. 2C), depending on the amount of
basal current generated by BNC1-G430C. The combined effects of
amiloride and Zn2+ concentrations are shown in Fig.
2D; at low concentrations, amiloride enhanced
Zn2+ stimulation of current, and amiloride blocked at high
concentrations. Enhanced stimulation in the presence of intermediate
amiloride concentrations may also explain the transient current
stimulation observed on washing out high concentrations of
amiloride and Zn2+ (Fig. 2A).
These data show that amiloride alone does not stimulate current, but
when both amiloride (intermediate concentrations) and Zn2+
are present, current is greater than with Zn2+ alone. One
interpretation of these results is that amiloride binds to the channel,
and in so doing makes Cys430 more accessible for
interaction with Zn2+. In this respect, amiloride might be
similar to protons; a decrease in extracellular pH causes a
conformational change that exposes Cys430 to the
extracellular solution where it can be irreversibly modified by MTS
reagents (15).
Therefore, we hypothesized that amiloride causes a conformational
change that exposes Cys430 to the extracellular solution.
To test this hypothesis, we asked if amiloride would allow MTS reagents
to irreversibly modify and activate the channel at pH 7.4. Fig.
3A shows that in the absence of amiloride, MTSEA had little or no effect at pH 7.4. Current remained
low when amiloride and MTSEA were simultaneously present in the
extracellular solution. However, once amiloride and MTSEA were
removed, it was apparent that a large current had been activated; this
current was blocked by subsequent treatment with amiloride. We obtained
similar results with two other MTS reagents, MTSET and MTSES (Fig. 3,
B and C). These results indicate that in the presence of amiloride, MTS reagents irreversibly modified
Cys430, locking the channel open. Because the activated
channel remained sensitive to amiloride block, current was only
apparent after amiloride was removed from the extracellular
solution.
The G437C Mutation Reduces Sensitivity to Amiloride
Block--
Extracellular amiloride and protons shared the ability to
expose Cys430 to MTS compounds. However, an important
difference between the two interventions was that protons activated
current (15), whereas amiloride alone did not. We hypothesized that
amiloride might have two effects; like protons, amiloride might bind to
one site to stimulate current, but in addition, amiloride might bind to a second site to block the channel. Thus, any stimulatory effect would
be obscured by the ability of amiloride to simultaneously block the
channel. To test this hypothesis, we asked whether the inhibitory
effect of amiloride could be selectively abolished by mutation. We
tested a mutation equivalent to a mutation that reduces amiloride block
of ENaC: substitution of cysteine for Gly437 (Fig.
4A). Residue 437 lies within
the predicted pore-forming second membrane-spanning domain and is
highly conserved among DEG/ENaC channels (9). In ENaC subunits,
mutations equivalent to G437C had several effects; if present in the
To determine whether the G437C mutation reduced amiloride inhibition,
we studied it in the context of BNC1-G430V, which generates large
constitutive currents (9, 15). BNC1-G430V generated large currents
(904 ± 84 nA; n = 9) that were blocked by
amiloride (Fig. 4B). Expression of the double mutant
BNC1-G430V/G437C failed to generate current; this was consistent with
the observation that mutations abolish ENaC function if present in all
subunits of that channel (18). Therefore, we coexpressed
BNC1-G430V/G437C with a lesser amount of BNC1-G430V (5:1 ratio). This
combination generated large constitutive currents (773 ± 63 nA;
n = 10; Fig. 4C) like BNC1-G430V. However in
contrast to BNC1-G430V, 3 mM amiloride blocked only 38 ± 4% of the current (n = 6). MTSEA blocked a larger portion of the current (69 ± 3%; n = 6, Fig.
4C). MTSEA block was likely due to covalent modification of
Cys437, because block was irreversible, and MTSEA did not
block channels that lacked the G437C mutation (wild-type BNC1 or
BNC1-G430V; not shown). These data indicate that when subunits
containing the G437C mutation are included in a BNC1 channel complex,
the sensitivity to block by amiloride is reduced.
A hint of amiloride stimulation came from examining the response to
increasing amiloride concentrations (Fig. 4C, left part of trace). Amiloride produced a biphasic response, with inhibition at
100 µM, an increase in current at 1 mM,
followed by inhibition at 3 mM. However, because the G430V
Deg mutation was present in every subunit, a stimulatory effect of
amiloride would be minimized.
Amiloride Stimulates Current in Cells Expressing BNC1-G430V/G437C
with Wild-type BNC1--
To test whether amiloride might stimulate
BNC1-G430V/G437C, we coexpressed it with a small amount of wild-type
BNC1 (5:1 ratio). This combination generated no current on its own;
basal current (133 ± 13 nA; n = 10) was not
different from that in uninjected cells (not shown). However, addition
of amiloride stimulated current (Fig. 4D). In contrast,
amiloride did not stimulate wild-type BNC1 (Fig. 4E).
Simulation was reversible and evident only at concentrations greater
than 100 µM. 3 mM amiloride increased current by 279 ± 40 nA (n = 10). This result, coupled
with the ability of amiloride to increase access of residue 430, indicated that amiloride, like protons, could stimulate BNC1.
Amiloride May Interact with Two Different Sites in
BNC1--
Because amiloride had two effects (stimulation and
inhibition), we hypothesized it might interact with two separate sites in BNC1. To test this idea, we determined the effect of voltage on
inhibition and stimulation. Because amiloride is positively charged,
effects should be voltage-dependent if the binding site lies within the membrane electrical field. To test inhibition, we
studied BNC1-G430V. Block was steeply voltage-dependent,
suggesting that amiloride blocked within the membrane electrical field
(Fig. 5, A-D).
Fig. 5, B and C, shows that block depended on the
reversal potential of BNC1 current, suggesting that outward flow of
cations prevented amiloride from reaching a blocking site. These
results suggest that when amiloride blocks, it binds within the channel pore.
To test stimulation, we studied oocytes expressing BNC1-G430V/G437C and
wild-type BNC1 (5:1 ratio). Fig. 5, E and F,
shows that stimulation was not appreciably
voltage-dependent. This result suggested that amiloride
stimulated the channel by binding a site outside the membrane
electrical field, different from the site of amiloride block.
Our data show that amiloride can have two distinct effects on the
BNC1 channel and suggest that there may be two different sites of
interaction. One site appears to lie within the channel pore; when
amiloride interacts with this site it blocks the channel. Three pieces
of evidence suggest that amiloride blocks within the channel pore: the
voltage dependence suggested block within the membrane electrical
field; alteration of the reversal potential and the outward flow of
cations reduced block by extracellular amiloride; and block was
attenuated by mutation of residue 437, which other data suggest lies
within the second membrane-spanning helix (19, 20).
The other site appears to be different from the blocking site; when
amiloride interacts there it can cause a conformational change that
activates the channel and exposes residue 430 to extracellular MTS
reagents. Our data make no predictions about the location of the
stimulatory site, except that it is not likely deep within the pore
because the transmembrane electrical field has little effect on
amiloride-dependent stimulation.
We considered the possibility that amiloride-dependent
stimulation might represent the interaction of amiloride at a single site producing open channel block. However, this mechanism does not
explain stimulation because net current increased in the presence of
amiloride, and amiloride stimulated current in channels that were
largely insensitive to amiloride block. Moreover, membrane voltage had
an effect on block, but not on stimulation. We also considered the
possibility that amiloride might interact with a single binding site
that might move during channel gating to give both
voltage-dependent and -independent effects. This also seems
unlikely, because the G437C mutation selectively attenuated block.
Nevertheless, we cannot exclude an interaction at a single site that
somehow both blocks and activates the channel; the multimeric nature of
BNC1 might allow for such an effect.
Our data suggesting that amiloride may interact with two sites may help
explain earlier studies of other DEG/ENaC channels. Studies of ENaC
suggest that amiloride might block the pore. First, amiloride block is
voltage-dependent (4). Second, amiloride is a more potent
blocker in reduced extracellular Na+, suggesting that
amiloride and Na+ may compete for a binding site in the
channel pore (4). Third, mutations that alter ion conduction or
selectivity also alter amiloride block (4, 18, 20, 21). On the other
hand, studies of ENaC identified residues in the predicted
extracellular domain that bind an amiloride anti-idiotypic antibody;
mutation of those sites altered amiloride sensitivity (5). In addition,
nonfunctional ENaC splice variants that lack the pore-forming second
transmembrane domain but retain much of the extracellular domain retain
binding to phenamil (an amiloride derivative) with high specificity
(6). These findings are consistent with an extracellular
amiloride-binding site on ENaC. The suggestion of two interaction sites
in BNC1 raises the question of whether amiloride will have two effects on all DEG/ENaC family members. Alternatively, some family members may
lack one or both sites and may not be inhibited by amiloride.
The large extracellular domain may regulate the function of DEG/ENaC
channels. In the FMRFamide-activated Na+ channel (FaNaCh),
this region may form a ligand-binding site for channel activation (22).
Consistent with this possibility, mutations in the predicted
extracellular domain reduce sensitivity to
FMRFamide.2 In BNC1, ASIC,
and DRASIC, this region may be responsible for proton activation (2).
In C. elegans DEG/ENaC proteins, genetic studies suggest
that this region may interact with extracellular collagen to affect
channel gating (23, 24). Our data suggest that there is also an
amiloride-binding site in BNC1, perhaps located in the extracellular
domain, that can modulate channel activity. Future studies that
identify this site may help our understanding of the function of the
extracellular domain and could possibly identify agents that activate
or inhibit the channels for therapeutic purposes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Zn2+ reversibly activates
BNC1-G430C. A, effect of extracellular Zn2+
on wild-type BNC1; B, BNC1-G430C; C, effect of
Zn2+ concentration on current in BNC1-G430C. Data are
mean ± S.E. of percent maximal activation, n = 7. D, Zn2+ allows MTSES (abbreviated ES)
to irreversibly activate BNC1-G430C. Zn2+ (100 µM), MTSES (5 mM), and amiloride (1 mM) were present during times indicated by bars.
We previously showed that MTSES alone has no effect on BNC1-G430C when
extracellular pH is 7.4 (see Ref. 15 and Fig. 3C). In all
figures, the 0 indicates zero current and membrane voltage
was 60 mV.
View larger version (22K):
[in a new window]
Fig. 2.
Amiloride enhances stimulation by
extracellular Zn2+. A, effect of 1 mM amiloride on current activated by 100 µM
Zn2+; B, effect of 100 µM
amiloride and 100 µM Zn2+; C,
effect of 100 µM amiloride and 30 µM
Zn2+; D, effect of Zn2+
concentration in the presence of indicated concentrations of amiloride.
Data are mean ± S.E. from at least six oocytes. All experiments
were performed with BNC1-G430C.
View larger version (12K):
[in a new window]
Fig. 3.
Amiloride exposes Cys430 to
extracellular MTS compounds in BNC1-G430C. Panels
A-C, effect of amiloride on modification by 100 µM MTSEA (EA), 100 µM MTSET
(ET), or 3 mM MTSES (ES). Amiloride
concentration was 300 µM. Because of amiloride block,
channel activation was not apparent until amiloride was removed from
the extracellular solution. Membrane voltage was 60 mV in
panels A and B and
40 mV in
panel C.
or
subunits of the channel, the mutations reduced amiloride
block and Na+ conductance. Also, the introduced cysteine
could be modified by MTSET to inhibit current (17, 18). However, if
present in all ENaC subunits, the mutations abolished channel activity (18).
View larger version (26K):
[in a new window]
Fig. 4.
Amiloride stimulates current when the
blocking effect of amiloride is attenuated. A, sequence
pile-up comparing BNC1 (8) with that of the ,
, and
subunits
of human ENaC (25, 26) showing the locations of Gly430 and
Gly437 in BNC1; B, inhibition of BNC1-G430V by
amiloride. Amiloride concentrations are indicated. C, effect
of amiloride on oocytes expressing BNC1-G430V/G437C plus BNC1-G430V in
a 5:1 ratio. Amiloride and MTSEA (1 mM) were applied as
indicated. D, effect of amiloride on current in cells
expressing BNC1-G430V/G437C plus wild-type BNC1 in a 5:1 ratio;
E, effect of amiloride on wild-type BNC1. Membrane voltage
was
60 mV.
View larger version (30K):
[in a new window]
Fig. 5.
Voltage dependence of amiloride block and
amiloride stimulation. Panels
A-D, amiloride block of BNC1-G430V.
Panel A, families of BNC1-G430V currents at
voltages ranging from 120 to +60 mV. Holding voltage (Vh)
was
20 mV. Amiloride concentration is indicated. Panel
B, current-voltage relationship from an oocyte expressing
large BNC1-G430V currents. For several minutes before current
measurements were made, holding voltage was clamped to 0,
40, or
80
mV, as indicated. When holding voltage was
40 or
80 mV,
intracellular Na+ accumulated and the reversal potential
became more negative (27). In oocytes expressing ENaC, a similar
increase in intracellular [Na+] occurs (26). Once the
reversal potential no longer drifted, current was measured during brief
1 s voltage steps in the absence (open symbols) or
presence (closed symbols) of 30 µM amiloride.
Panel C, current values from panel B
normalized to the reversal potential in each condition. Panel
D, current-voltage relationship of amiloride-inhibited current.
Amiloride concentration was 1 µM (squares), 10 µM (inverted triangles), 100 µM
(circles), or 1 mM (triangles). Data
are average ± S.E. from six oocytes where Vh was
20 mV.
Panels E and F, amiloride stimulation of current
in oocytes expressing BNC1-G430V/G437C with wild-type BNC1. Panel
E, current-voltage relationship of amiloride-stimulated current.
Amiloride concentration was 1 mM (
) or 3 mM
(
). Data are average ± S.E. from three oocytes. Panel
F, families of currents at voltages ranging from
120 mV to +40
mV. Amiloride concentration is indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dan Bucher, Dawn Melsson, Ellen Tarr, and Theresa Mayhew for excellent assistance. We especially appreciate the discussions and help of Drs. Margaret Price, Joseph Cotten, and John Rogers. We thank the University of Iowa DNA Core Facility for assistance with sequencing and oligonucleotide synthesis.
![]() |
FOOTNOTES |
---|
* This work was supported by the Howard Hughes Medical Institute.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.
Predoctoral trainee supported by National Institute on Aging,
National Institutes of Health Grant T32 AG 00214.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail: mjwelsh{at}blue.weeg.uiowa.edu.
2 J. Rogers and M. J. Welsh, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MTS, methanethiosulfonate; MTSEA, 2-aminoethyl methanethiosulfonate bromide; MTSET, 2-(trimethylammonium)ethyl methanethiosulfonate bromide; MTSES, sodium 2-sulfonatoethyl methanethiosulfonate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|