From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of with either wild-type
or
subunits and
with carboxy-terminal truncated
(
T) or
(
T) subunits in Xenopus laevis oocytes.
and
T channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 µM amiloride in the pipette solution could
channel activity be resolved; both channel types had short open and closed times. Mean channel open probability
(Po) for
was 0.54 and for
T was 0.50. In comparison,
and
T channels exhibited different kinetics:
channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high Po (0.78), or short
openings with long closed times, resulting in a low Po (0.16). The mean Po for all
channels was 0.48.
T (6.6 pS
in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time
of 0.4 s, with a mean Po of 0.6, similar to that of
. Inclusion of 0.1 µM amiloride in the pipette solution reduced
the mean open time of
T to 151 ms without significantly altering the closed time. We also examined the kinetics
of amiloride block of
,
T (1 µM amiloride), and
T (0.1 µM amiloride) channels.
and
T had similar
blocking and unblocking rate constants, whereas the unblocking rate constant for
T was 10-fold slower than
T. Our results indicate that subunit composition of ENaC is a main determinant of Po. In addition, channel kinetics and Po are not altered by carboxy-terminal deletion in the
subunit, whereas a similar deletion in the
subunit affects channel kinetics but not Po.
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INTRODUCTION |
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The epithelial sodium channel (ENaC)1 mediates sodium reabsorption from the distal nephron, colon,
lungs, and other epithelia. The channel consists of
three subunits: ,
, and
that share ~35% identity at
the amino acid level (Canessa et al., 1993
, 1994
). Each
subunit has two transmembrane domains linked by a
large extracellular loop, and the amino and carboxy
termini (COOH termini) in the cytoplasm. Biophysical
properties of ENaC at the single channel level consist of
a small conductance (~5 pS), ionic selectivity of Li+ > Na+ >> K+, and high affinity (Ki of 0.1 µM) for the
open-channel blocker amiloride (reviewed extensively
by Garty and Palmer, 1997
).
Injection of the three subunits of ENaC in Xenopus
laevis oocytes induces the expression of channels with
properties similar to the ones exhibited by channels in
native tissues (Canessa et al., 1994).
subunits alone
can induce amiloride-sensitive currents, but the very
low level of expression (1% of
current) has precluded their characterization at the single channel
level. Coexpression of
and
or
and
, but not
and
, subunits induces whole-cell amiloride-sensitive
currents that reach 10-20% of the magnitude obtained
with
,
, and
together.
Previous characterization of whole-cell currents induced by and
channels showed that these channels differ in many properties (McNicholas and Canessa, 1997
). For instance,
channels have 10-fold
higher Ki for amiloride than
channels (1 and 0.1 µM, respectively); and the ion selectivity of
channels is Li+
Na+, whereas for
channels it is Li+ > Na+. These findings, together with the demonstration
of differential subunit expression within epithelial tissues (Farman et al., 1997
), may explain the variability in
single channel properties of amiloride-sensitive ENaCs
in native tissues (e.g., Palmer and Frindt, 1986
;
MacGregor et al., 1994
) and suggest that alteration of
subunit composition may be physiologically important.
In this study, we sought to define the single channel
properties of and
channels. We examined single
channel conductances and amiloride kinetics of channels formed by
and wild-type or truncated
(
T) and
(
T) subunits. Furthermore, we also examined the kinetics and open probability (Po) of
and
channels
and compared them with those of COOH-terminally
truncated channels
T and
T. The importance of
the COOH termini of the
and
subunits on the activity of ENaC was first shown by mutations that produce
deletions of part or almost all the COOH termini of
or
subunits in patients with Liddle's syndrome, a hereditary form of autosomal dominant hypertension
(Shimkets et al., 1994
; Hansson et al., 1995
). Indeed,
expression of COOH-terminally truncated
or
subunits in Xenopus oocytes induces amiloride-sensitive whole-cell currents that are three- to fivefold larger
than those observed with wild-type subunits (Schild et al.,
1995
). At least two mechanisms have been proposed to
account for the increase in current observed with the
truncated subunits. The first mechanism is an increase
in channel number at the plasma membrane, and the second is an increase in channel Po. Using cell-attached
single channel patches from oocytes expressing either
wild-type (
or
) or truncated (
T or
T) subunits, we have examined whether the COOH terminus
alters channel kinetics and Po.
The findings of this study demonstrate that subunit
composition confers distinct kinetics of amiloride block
to and
channels and that amiloride block is not
affected by deleting the COOH termini. We also show
that subunit composition is a major determinant of
channel Po. We found that
channels exhibited a
very high Po that approximated 1, whereas
channels
exhibited a mean Po of 0.5. Expression of
subunit
with a
chimera (CH), which has only the M2 domain and short segment of amino acids preceding M2
from
, and the rest of the sequence from the
subunit, induced channels with very high Po similar to
channels, indicating that this region of the
subunit
confers the very high Po to the channels. The contribution of the COOH terminus of
and
subunits to
channel kinetics was assessed by examining
and
T
channels and
and
T channels. Channel kinetics
and Po of
and
T channels were not affected by the
COOH terminus of the
subunit; the Po of both types
of channels remained close to 1. In contrast, COOH-terminal deletion of the
subunit changed the gating
kinetics without an overall effect on the mean Po. The
main effect of deleting the COOH termini of
or
on
channel activity was an increase in the density of channels at the cell surface that could account for the three-
to fivefold increase in whole-cell currents.
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METHODS |
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Oocyte Isolation and cRNA Injection
Xenopus laevis were anesthetized using 0.17% tricaine (3-aminobenzoic acid, methanesulfonate salt), stage V-VI oocytes were removed by partial ovariectomy and placed in hypotonic Ca2+-free ND96 containing (mM): 96 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4. Subsequent treatment of the oocytes with collagenase type I (Worthington Biochemical Corp., Lakewood, NJ) at 2 mg/ml in Ca2+-free ND96 for 60-90 min essentially removed follicular membranes. After incubation in this solution for the allotted time, oocytes were washed several times in ND96 containing 1.8 mM CaCl2, and then further washed in supplemented ND96 (50 µg/ml Gentamycin [Life Technologies Inc., Grand Island, NY] and 2.5 mM sodium pyruvate was added).
cRNAs were in vitro transcribed from plasmid psD5 with SP6 RNA polymerase using Message Machine (Ambion Inc., Austin, TX) according to the supplier's instructions. Oocytes were injected 24 h after isolation and defolliculation. Equal amounts of subunit cRNA were used at all times (1-3 ng) in a constant injectate volume of 50 nl. After injection, oocytes were kept in the supplemented ND96 medium with 1 µM amiloride was added to prevent sodium loading of oocytes upon expression of ENaC. Recordings were made from oocytes 24-48 h after injection, depending on the subunit composition of the channels.
Electrophysiology
Before patch clamping, the vitelline membrane was removed manually using fine forceps after allowing the cell to shrink for ~5 min in a hypertonic solution containing (mM): 220 N-methyl- D-glucamine, 220 aspartic acid, 2 MgCl2, 10 EGTA, 10 HEPES, pH 7.4.
Two standard solutions were used throughout this study. They were a Li+ pipette solution containing (mM): 150 LiCl, 1 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, and a K+ bath solution containing (mM): 150 KCl, 5 EDTA, 5 HEPES, pH 7.4. Amiloride was included in the pipette solution where indicated. All experiments described herein were performed at ambient room temperature.
Patch pipettes were pulled from fine borosilicate capillary glass
(LG16 glass; Dagan Corp., Minneapolis, MN) in two stages, using
a microelectrode puller (PP-83; Narishige International USA
Inc., East Meadow, NY). They were not fire polished and typically had tip resistances between 3 and 5 M when partially filled with pipette solution.
Channel currents were recorded from oocytes using the cell-
attached configuration of the patch clamp technique (Methfessel et al., 1986). Recordings were made, lasting anywhere between several minutes (for
) to up to 1 h (for
), using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) interfaced, using a DigiData 1200 interface (Axon Instruments), to an IBM-compatible 166 MHz Pentium PC (Gateway 2000 Inc., N. Sioux
City, SD). Data was acquired at 5 kHz, filtered at 250 Hz during
acquisition using an eight-pole low pass Bessel Filter (Frequency
Devices Inc., Haverhill, MA), and stored directly as data files on
the hard drive of a personal computer. The pClamp 6 (Axon Instruments) suite of software was used for data acquisition and its
subsequent analysis.
Data was further filtered digitally at 50 kHz during analysis and display. In all channel current traces, a downward deflection represents channel opening. Open and closed time histograms were generated using Pstat within pClamp. Po, and in some cases nPo for patches with more than one channel, was calculated using a specialized nPo program that was written by Jinliang Sui (Mount Sinai School of Medicine, New York) and is available online (http://www.axonet.com/pub/userware/popen). nPo was converted to mean channel Po in patches where the number of active channels could accurately be determined; in most cases, only single channel patches were used or patches with at most two active channels, based upon the number of current transitions observed using channel recordings lasting from several minutes to 1 h. Statistical analysis, where appropriate, was performed using the Student's paired t test.
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RESULTS |
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Single Channel Currents and Conductance
Oocytes injected with - or
T-expressed large
amiloride-sensitive whole-cell currents measured with
two-electrode voltage clamp (between 1-3 µA for
and
10 µA for
T, not shown), but we could not discern channel activity when these oocytes were patch
clamped. Upon inclusion of 1 µM amiloride in the pipette solution, which is the Ki for
channels (McNicholas and Canessa, 1997
), we detected channel currents.
Fig. 1 shows representative examples of the currents
observed for
and
T at
40 mV; at this potential,
both channel types displayed fast, flickery transitions between an open and a closed level. A common observation from all
and
T single channel records was
the absence of long closed states.
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Fig. 2 shows that channels were found to exist in
two modes. One gating mode had long closed states
with shorter less frequent openings and had a low
channel Po. Conversely, the other gating mode had
long open states with infrequent transitions to shorter closed states and had a high channel Po. No
channels with intermediate Po were found. All
T channels,
on the other hand, existed in only one state and had
shorter and more frequent openings and closings when
compared with
. However, the frequency of the open
states exceeded that of the closed states. When 0.1 µM
amiloride was included in the pipette, fewer open
states were observed and their length was decreased.
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In oocytes injected with either T or
T, the incidence of channel activity was greater than in oocytes injected with
or
. The observation percentage
(number of patches with channel activity/number of
patches tested) was 40.7% (24/59) for
T, 34.7%
(108/309) for
T, 12.8% for
(10/78) with 1 µM
amiloride (0/24 without amiloride), and 17.3% (21/
121) for the combined populations of
channels.
Multiple channel-containing patches (between four
and eight channels per patch) were very frequent with
T - or
T -expressing oocytes, whereas most patches
from oocytes injected with
or
contained usually one or at most two channels. Therefore, the analysis of
Po and kinetics in the following sections primarily came
from patches that contained only one (kinetics) or at
most two electrically active channels (Po).
Single channel conductance (slope conductance)
was estimated by measuring currents at a range of voltages with 150 mM Li+ in the pipette. Fig. 3 shows the
conductance-voltage relationships for the various channel types. There was no difference between and
T
(9.6 vs. 8.7 pS, P > 0.4) or
and
T (6.7 vs. 6.6 pS, P > 0.4), although the channels containing
and
T subunits had slightly larger conductances than those with
the corresponding
subunits.
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Channel Po
Fig. 4 shows the effect of voltage on channel Po for
and
T channels. These measurements were made
with 1.0 µM amiloride in the pipette.
channel Po
was ~0.65 at 0 mV and 0.5 at
80 mV. As can be seen,
the plots of Po against membrane voltage for
and
T were almost identical and were not greatly affected by voltage. The high channel Po in the presence of an
amiloride concentration equal to the Ki, and the absence of channel gating without amiloride, suggests
that when amiloride is absent, channel Po would be
close, if not equal, to 1.
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Table I shows a comparison of the mean Po values obtained for the various channel types at 40 mV. The Po
values of
(0.54 ± 0.03) and
T (0.50 ± 0.03) were
almost identical, suggesting that truncation of the
COOH terminus of the
subunit did not alter channel
Po (P > 0.15). We were unable to obtain similar plots of
Po vs. voltage for the
and
T channels due to the channel kinetics. For example, to obtain an accurate
and meaningful plot for
, we would have had to
record from a cell-attached patch for at least 90 min
(e.g., 0,
40,
80 mV for at least 30 min each), but
found that oocytes became difficult to patch after 1 h in
the bath. Therefore, we chose to measure channel Po
(and single channel kinetics) for
and
T at
40
mV. This voltage was selected to minimize the activity
of a variety of channels endogenous to oocytes (Stühmer and Parekh, 1995
). A mean Po of 0.16 ± 0.04 was
calculated for the low Po and 0.78 ± 0.05 for the high
Po
channels, with the mean Po for the entire
channel types being 0.47. Preliminary experiments indicate that after excision, patches containing high Po
channels adopt a low Po mode instantaneously
(Fyfe, G.K., and C.M. Canessa, unpublished observations). The mean Po of
T was found to be 0.60 ± 0.01, a value that is not significantly different to the
mean value for the
channels (P > 0.19). Inclusion
of 0.1 µM amiloride in the pipette (the Ki value of
amiloride for
T channels determined by two-electrode voltage clamp; McNicholas and Canessa, 1997
) reduced the mean Po of the
T channels to 0.30 ± 0.02.
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We have also measured the Po of channels formed by
the subunit and a
chimera. This CH combined
the amino terminus, the first transmembrane region
(M1), and the extracellular domain of the
subunit
with the second transmembrane (M2) region and COOH terminus of the
subunit. With 1.0 µM
amiloride in the pipette, the mean Po of these
CH
channels was 0.62 ± 0.06 (n = 3).
Fig. 5 shows two plots of Po vs. time. Fig. 5 A shows a
representative example of a single channel high Po
patch that was recorded for 1 h. Note that although the
mean Po for this example was 0.67, the Po of the channel (measured every 30 s) varied greatly from close to 1 to near 0 constantly throughout the entire recording. In Fig. 5 B, a representative example of an
T patch in
the absence and presence of 0.1 µM amiloride is
shown. Although the length of these recordings is
shorter, it appears truncation of the COOH terminus
has given the
T channel a more stable Po; i.e., a variation of 0.5-0.8, as opposed to that of 0-1 for
.
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Single Channel and Amiloride Blocking Kinetics
Fig. 6 shows open- and closed-time histograms for ,
T, and
T in the presence of amiloride. The kinetics
for
and
T were very similar. On the other hand,
T had relatively longer open and closed times.
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Table II shows a summary of the mean open and
closed times at 40 mV for all channel types and, in
the case of
, the high and low Po modes. High Po
channels had a relatively long mean open time of 6.9 s
and a shorter mean closed time (1.9 s). Conversely, low
Po
channels had a relatively short open time lasting
<1 s and a longer closed time lasting almost 5 s.
T
channels exhibited different kinetics compared with
channels. They had a mean open time of 1.2 s and a
relatively short mean closed time of 400 ms. Channel Po
calculated from the mean open and closed times using
the equation
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(1) |
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yields values almost identical to that calculated using the nPo analysis program (Table I).
Having obtained the open and closed times for
and
T at a range of voltages between 0 and
80 mV
in the presence of 1 µM amiloride, we were able to calculate the blocking (KON) and unblocking (KOFF) rate
constants for amiloride at each of the voltages. KON was
calculated from the equation
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(2) |
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(3) |
Fig. 7 shows the plot of KON and KOFF vs. voltage for
and
T. There was very little difference between
the two channel types. The KON and KOFF, dissociation
constant at 0 mV (Kd(0)), and dissociation constant at
40 mV (Kd(
40)) values are shown in Table III.
Amiloride block is very weakly voltage dependent because the Kd(0) and Kd(
40) for both
(2.7 and 1.5 µM) and
T (1.7 and 1 µM) are close to each other.
The difference in the amiloride blocking kinetics when
comparing
T (and
) to
T is in the off rates: the
off rate for
T was calculated to be 39 s
1 and for
T
was 3 s
1, 1/13 of the
T value. At
40 mV, the Kd of
amiloride for
T was calculated to be 0.05 µM, a value
similar to the amiloride Ki (0.1 µM). We also estimated
, the electrical distance sensed by amiloride, for
and
T. Both values were found to be ~0.3.
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DISCUSSION |
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Subunit Composition and Channel Kinetics
The results presented in this work indicate that subunit
composition is a major determinant of channel kinetics. Combination of with
subunits favored a conformation in the open state, giving rise to
channels
with a very high Po. In the presence of 1 µM amiloride
in the pipette (concentration that corresponds to the
amiloride Ki of
channels), the Po was 0.5, indicating that without amiloride the Po of
channels is close to
one. These channels do not exhibit the characteristic
long closed states as seen in oocytes injected with
,
, and in cells from native tissues.
channels seem
to be constitutively open, no discernible channel transitions were observed in seals obtained in the absence of
amiloride, suggesting that
channels do not have a
closed state.
Channels formed by the subunit and a
CH when
patched with 1.0 µM amiloride in the pipette also exhibited a very high Po similar to
channels (amiloride
Ki of
CH channels is 1.0 µM). However, in recordings
from
CH channels, we occasionally detected very brief
closed states that were never seen in
channels. The
CH comprises the first amino-terminal 2/3 of the
subunit and the last COOH-terminal 1/3 of the
subunit that includes the intracellular COOH terminus,
the M2 domain, and a short segment of amino acids
preceding M2 that has been implicated in amiloride binding (Waldmann et al., 1995
; Schild et al., 1997
).
Similarity in channel kinetics observed in
and
CH
indicates that the 1/3 of the
subunit is responsible
for conferring the high Po on these channels. As we discuss later, the intracellular COOH terminus of the
subunit does not affect the Po of
channels; therefore, the high Po is a property of M2 and/or the region
preceding it.
In contrast to channels,
channels exhibited kinetics that were characteristic of wild-type
with
closed states that varied from a few milliseconds to several seconds. Two populations of
channels were
clearly distinguishable: one consistently had a low Po
(0.16 ± 0.04) with long closed states, and the other had
a high Po (0.78 ± 0.05) with long open states. The
mean Po for the whole population of
channels was
0.47. Similar findings have been documented for ENaC
expressed in cortical collecting tubules where channels
exhibited either predominately low or high Po; but,
when all channels were averaged, the mean Po was 0.5 (Palmer and Frindt, 1996
).
Although the low and high Po channels may represent the extremes of continuous open probabilities,
this does not seem to be the case because all the channels we observed fitted well into the low or high Po
modes, no intermediate Po values were seen. In spite of
the long recording times, we did not see conversion of
channels from a low to a high Po mode or vice versa
when cell attached. Furthermore,
channels in the
two gating modes can coexist; occasionally, we obtained patches with two channels: one with high and
the other low Po. Although not shown here, our observation that high Po channels, when excised, adopt a low
Po state instantaneously before running down several
minutes later suggests that some intracellular factor or
process may be required to maintain a high Po. It has
been reported that hyperpolarization of the membrane
potential and low concentration of extracellular Na+
can shift channels to a high Po state (Palmer et al.,
1998
). For reasons explained in the RESULTS, we did
not attempt these maneuvers; all of our experiments
were performed at
40 mV with 150 mM Li+ in the pipette.
The COOH terminus of the subunit may contain
elements that allow channels to adopt a high or low Po
state. Phosphorylation/dephosphorylation of residues
in
may induce conformational changes that stabilize either high or low Po channels. Indeed, we have recently demonstrated that ENaC is phosphorylated in
the
subunit (Shimkets et al., 1998
). Alternatively,
binding of accessory proteins could be another way of
inducing conformational changes.
Effect of COOH Terminal Deletion on Po and on Channel Density at the Plasma Membrane
Previous experiments have shown that oocytes expressing ENaC containing and
subunits truncated at
their COOH termini had larger whole-cell currents
than oocytes expressing wild-type
or
. There are at
least three mechanisms by which an increase in current
could arise in cells expressing truncated subunits: increased single channel conductance, increased channel Po, or increased expression of conducting channels
at the plasma membrane. Single channel conductance
was found to be unaltered (Schild et al., 1995
; Snyder
et al., 1995
), and the number of channels expressed at
the cell surface was found to be increased, but an effect
on channel Po has remained controversial. For example, Firsov et al. (1997)
, using a flag epitope inserted
into the extracellular domain of the
subunit, demonstrated that cells expressing truncated subunits had an
increased number of channels in the plasma membrane when compared with oocytes expressing wild-type
subunits. The calculated number of channels at
the cell surface did not account for the total increase in
amiloride-sensitive current measured in the same cells,
leading to the inference that channel Po had also been
altered to further increase whole-cell currents. Awayda et al. (1997)
, using a similar approach (fluorescent antibodies to detect surface expression of channels), arrived at the same conclusion because they found only a
small rise in the fluorescent signal when comparing oocytes expressing wild-type
and
T subunits.
We have addressed this problem by measuring directly the single channel conductance and Po of ,
T,
, and
T channels using the cell-attached configuration of the patch clamp technique. Oocytes injected with
T or
T channels expressed three- to fivefold larger whole-cell currents than oocytes injected
with
or
. The single channel conductances of
and
were unaltered by COOH-terminal truncations
(Fig. 3). Therefore, with
or
, changes in single
channel conductance cannot explain the increase in
whole-cell currents. A slight difference in the channel
conductances of
and
T vs.
and
T may reflect
differences in the channel pore structure.
Fig. 4 and Table I show that the Po of and
T
channels in the presence of 1 µM amiloride in the pipette were 0.54 and 0.50, respectively. These results indicate that both types of channels have a very high endogenous Po, ~1, and that it is not affected by deletion
of the COOH terminus of the
subunit.
channels existed in two gating modes corresponding to low and high Po (Fig. 2 and Table I); each of
these modes appeared with equal frequency such that
the mean Po of the entire population was 0.47. Truncation of the COOH terminus of the
subunit produced a single population of
T channels with more homogenous kinetics, distinct from
channels. The mean
open and closed times of
T channels were shorter
and less variable when compared with
channels (Table II). Also, the Po of
channels fluctuated less through time (Fig. 5, A and B). Although the mean Po
of
T channels (0.6) was slightly higher than that of
channels (0.48), the difference does not account for
the observed increase in whole-cell current.
We think that our estimate of channel Po is accurate
for the following reasons. Due to the nature of the kinetics of and
T channels (with amiloride in the
pipette), multichannel patches were evident instantly
and overestimation of channel Po was unlikely. Regarding
and
T channels, the kinetics were slower and,
in most cases, multichannel patches were not evident
until 1-2 min after seal formation. Therefore, we recorded patches with
for longer periods to be confident of the number of electrically active channels
present in the patch; only patches with one or two
channels were used to calculate Po. Our results clearly
demonstrate that truncations of the COOH termini of
the
or
subunits do not increase channel Po.
In contrast, the number of active channels at the
plasma membrane was larger in oocytes injected with
T or
T subunits. The number of successful seals that
contained channels and the frequency of patches having more than one channel was greater in oocytes expressing
T or
T, indicating that truncated channels
are expressed at a higher density. These results are consistent with the presence of endocytosis signals in the
COOH terminus of
and
that, when deleted, increase the number of channels in the cell surface
(Shimkets et al., 1997
).
Staub et al. (1997) have proposed another mechanism to explain the increase in number of channels at
the plasma membrane. According to these investigators, the protein Nedd4 binds to the COOH termini of
the
,
, and
subunits and subsequently ubiquitinates several conserved lysines in the amino terminus of
and
, but not
. Only ubiquitinated channels could be
removed from the plasma membrane; whereas ubiquitination of
had a modest effect on the rate of endocytosis, ubiquitination of
appeared to be a prerequisite. Therefore, the hypothesis predicts that oocytes expressing
and
T channels should express a similar number of channels at the cell surface. Instead, we found
that the whole-cell currents and the density of channels
in the plasma membrane were larger in cells expressing
T channels.
Amiloride Block
Amiloride is an open channel blocker with high affinity
for ENaC (Kd of 0.1 µM). The binding site(s) for
amiloride is located in the extracellular side of the
channel protein. Previous work has shown that mutations of residues in M2 of the subunit (Waldmann et al.,
1995
) and the region preceding M2 of
,
, and
subunits (Schild et al., 1997
) change the affinity for
amiloride. The Kd's for amiloride of
and
channels, measured by inhibition of whole-cell currents, are
1 and 0.1 µM, respectively (McNicholas and Canessa,
1997
). Here, we have examined the kinetics of amiloride block of
and
channels. Direct measurements of
KON and KOFF for amiloride on
channels (Table II)
showed that these channels have a high Kd for amiloride,
and that the decrease in affinity is due to a 10-fold
larger KOFF when compared with values reported for
channels (4 s
1; Palmer and Frindt, 1986
) or
T
(2.5 s
1; Schild et al., 1995
). Both the KON and KOFF of
amiloride for
channels showed a weak voltage dependence, with a calculated electrical distance sensed
by amiloride of 30%. As expected, deletion of the COOH
terminus, which is an intracellular domain of the
subunit, did not affect the kinetics of amiloride block as
shown in Figs. 1 and 7. The KON and KOFF values for
and
T were almost identical (Table II).
CH channels resemble
channels in their amiloride affinity
with a Kd of 1 µM measured by inhibition of whole-cell
currents. The kinetics of amiloride block of
CH channels were also indistinguishable from
channels, a result that agrees with the notion that amiloride interacts
with M2 and the amino acids preceding it.
The kinetics of amiloride block of channels were
markedly different from
channels. Although the
KONs were similar for
T and
T when measured with
1 and 0.1 µM amiloride, respectively, in the patch pipette (Table III), the KOFF of amiloride for
T channels was much slower, suggesting that amiloride may either occupy an inhibitory site on the channel (or reside
in the channel pore) for longer times. The observation
that the closed time for
T in the presence of amiloride remained unaltered when compared with the absence of amiloride (365 and 400 ms, respectively, Table II), and that the corresponding histogram was described well by one exponential, suggests that the channel closed time and amiloride block time were similar
or had the same order of magnitude. We are confident
that the value attributed to the mean blocked time for
amiloride is correct because the Kd (0.05 µM) calculated from the KON and KOFF is similar to the values obtained with two independent measurements: inhibition
of whole-cell current (Ki = 0.1 µM) and mean Po (a
50% reduction in Po with 0.1 µM amiloride).
The kinetics of amiloride block of channels
seemed very similar to those described for
. Overall, except for the lower magnitude of whole-cell current due to the lower number of channels expressed at
the cell surface,
channels exhibited similar properties to
channels.
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FOOTNOTES |
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Address correspondence to Dr. C.M. Canessa, Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520. Fax: 203-785-4951; E-mail: cecilia_ca nessa{at}yale.edu
Original version received 7 May 1998 and accepted version received 2 July 1998.
Portions of this work were previously published in abstract form (Fyfe, G.K., and C.M. Canessa. 1997. The Physiologist. 40:268. Fyfe, G.K., and C.M. Canessa. 1998. Biophys. J. 74:A403).The authors thank Drs. Gordon Gregor MacGregor and Carmel McNicholas for critical discussion.
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Abbreviations used in this paper |
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CH, chimera; ENaC, epithelial sodium channel.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Awayda, M.S.,
A. Tousson, and
D.J. Benos.
1997.
Regulation of a
cloned epithelial Na+ channel by its ![]() ![]() |
2. | Canessa, C.M., J.-D. Horisberger, and B.C. Rossier. 1993. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470 [Medline]. |
3. | Canessa, C.M., L. Schild, G. Buell, B. Thorens, I. Gautschi, J.-D. Horisberger, and B.C. Rossier. 1994. Amiloride-sensitive epithelial Na+ channel is made up of three homologous subunits. Nature 367: 463-467 [Medline]. |
4. |
Farman, N.,
C.R. Talbot,
R.C. Boucher,
C.M. Canessa,
B.C. Rossier, and
J.P Bonvalet.
1997.
Non-coordinated expression of ![]() ![]() ![]() |
5. |
Firsov, D.,
L. Schild,
I. Gautschi,
A.-M. Mérillat,
E. Scneeberger, and
B.C. Rossier.
1997.
Cell surface expression of the epithelial
Na channel and a mutant causing Liddle syndrome: a quantitative approach.
Proc. Natl. Acad. Sci. USA.
93:
15370-15373
|
6. |
Garty, H., and
L.G. Palmer.
1997.
Epithelial sodium channels: function, structure and diversity.
Physiol. Rev.
77:
359-396
|
7. | Hansson, J.H., C. Nelson-Williams, H. Suzuki, L. Schild, R.A. Shimkets, Y. Lu, C. Canessa, T. Iwasaki, B.C. Rossier, and R.P. Lifton. 1995. Hypertension caused by a truncated epithelial sodium channel subunit: genetic heterogeneity of Liddle syndrome. Nat. Genet 11: 76-82 [Medline]. |
8. |
MacGregor, G.G.,
R.E. Olver, and
P.J. Kemp.
1994.
Amiloride-sensitive Na+ channels in fetal type II pneumocytes are regulated by G
proteins.
Am. J. Physiol.
267:
L1-L8
|
9. |
McNicholas, C.M., and
C.M. Canessa.
1997.
Diversity of channels
generated by different subunit combinations of epithelial sodium channel subunits.
J. Gen. Physiol.
109:
681-692
|
10. | Methfessel, C., V. Witzemann, T. Takahashi, M. Mishina, S. Numa, and B. Sakmann. 1986. Patch clamp experiments on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflügers Arch 407: 577-588 [Medline]. |
11. | Palmer, L.G., and G. Frindt. 1986. Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc. Natl. Acad. Sci. USA 83: 2767-2770 [Abstract]. |
12. | Palmer, L.G., and G. Frindt. 1996. Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch. J. Gen. Physiol 107: 35-45 [Abstract]. |
13. |
Palmer, L.G.,
H. Sackin, and
G. Frindt.
1998.
Regulation of Na+
channels by luminal Na+ in rat cortical collecting tubule.
J. Physiol. (Camb.)
509:
151-162
|
14. | Schild, L., C.M. Canessa, R.A. Shimkets, I. Gautschi, R.P. Lifton, and B.C. Rossier. 1995. A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc. Natl. Acad. Sci. USA. 92: 5699-5703 [Abstract]. |
15. |
Schild, L.,
E. Schneeberger,
I. Gautschi, and
D. Firsov.
1997.
Identification of amino acid residues in the ![]() ![]() ![]() |
16. |
Shimkets, R.A.,
D.G. Warnock,
C.M. Bostis,
C. Nelson-Williams,
J.H. Hansson,
M. Schambelan,
J.R. Gill,
S. Ulick,
R.V. Milora,
J.W. Findling, et al
.
1994.
Liddle's syndrome: heritable human hypertension caused by mutations in the ![]() |
17. |
Shimkets, R.A.,
R.P. Lifton, and
C.M. Canessa.
1997.
The activity of
the epithelial sodium channel is regulated by clathrin-mediated
endocytosis.
J. Biol. Chem.
272:
25537-25541
|
18. |
Shimkets, R.A.,
R.P. Lifton, and
C.M. Canessa.
1998.
In vivo phosphorylation of the epithelial sodium channel.
Proc. Natl. Acad.
Sci. USA
95:
3301-3305
|
19. | Snyder, P.M., M.P. Price, F.J. McDonald, C.M. Adams, K.A. Volk, B.G. Zeiher, J.B. Stokes, and M.J. Welsh. 1995. Mechanism by which Liddle's Syndrome mutations increase activity or a human epithelial Na+ channel. Cell. 83: 969-978 [Medline]. |
20. |
Staub, O.,
I. Gautschi,
T. Ishikawa,
K. Breitschopf,
A. Ciechanover,
L. Schild, and
D. Rotin.
1997.
Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.
EMBO (Eur. Mol. Biol. Organ.) J.
16:
6325-6336
|
21. | Stühmer, W., and A.B. Parekh. 1995. Electrophysiological recordings from Xenopus oocytes. In Single-Channel Recording. 2nd ed. B. Sakmann and E. Neher, editors. Plenum Publishing Corp., New York. 341-356. |
22. |
Waldmann, R.,
G. Champigny, and
M. Lazdunski.
1995.
Functional
degenerin-containing chimeras identify residues essential for
amiloride-sensitive Na+ channel function.
J. Biol. Chem
270:
11735-11737
|