From the Departments of Physiology II and Otolaryngology, Research Group of Sensory Physiology, University of Tübingen, D-72076 Tübingen, Germany
Received for publication, February 6, 2003 , and in revised form, April 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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According to the concept of Na+ feedback regulation, a rising intracellular Na+ concentration inhibits ENaC activity (5). Molecular mechanisms mediating this negative feedback regulation possibly include retrieval of surface-expressed channels by ubiquitin-mediated endocytosis. This feedback mechanism depends on interaction of a "PY" motif at the carboxyl terminus of ENaC subunits with the ubiquitin ligase Nedd4-2 (6, 7). However, the way the intracellular Na+ concentration is sensed is unknown.
In contrast, the model of Na+ self-inhibition proposes that the extracellular Na+ ion itself can reduce the ENaC activity (5, 810). Na+ self-inhibition has been best described in amphibian tissues such as Rana skin (811) and Necturus urinary bladder (12). In these tissues, an increase in the extracellular Na+ concentration leads to a decline of the Na+ current within <5 s. The speed of self-inhibition with no measurable increase in intracellular Na+ concentration has led to the proposal that the extracellular Na+ ion itself mediates self-inhibition. Self-inhibition leads to efficient reduction of Na+ transport whenever the extracellular Na+ concentration rises. This would protect the cell from Na+ overload and distribute Na+ transport more evenly along an epithelium such as the collecting duct. However, the molecular basis for Na+ self-inhibition is unknown.
ENaC has been best characterized in Xenopus laevis among amphibian
species. In Xenopus, five ENaC subunits are known. Besides the
,
, and
subunits
(13), which assemble into the
classical heteromeric ENaC with an
2
stoichiometry (1), two isoforms
of
xENaC and
xENaC have been identified (
2xENaC
and
2xENaC, respectively)
(14). These additional
isoforms are probably due to polyploidy of Xenopus. Channels
containing the
2 subunit are characterized by a relatively
high affinity for Na+, leading to current saturation at low (20
mM) Na+ concentration
(14). However, Na+
self-inhibition is not easily observed with any of the possible combinations
of subunits. Very recently, using fast solution exchange, Chraibi and
Horisberger (15) observed
self-inhibition with recombinant
xENaC channels. However,
self-inhibition was too fast to be reliably determined.
Here, we report the cloning of a new ENaC subunit from X. laevis
(xENaC).
xENaC-containing channels displayed strong Na+
self-inhibition with a reduction of the current amplitude of
70%.
Na+ self-inhibition of
xENaC-containing channels was
significantly slower than that of
xENaC-containing channels,
facilitating the investigation of this process. In this study, we identify a
region in the extracellular domain of the channel protein that controls the
speed of inactivation.
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EXPERIMENTAL PROCEDURES |
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For expression studies, the entire coding sequence of xENaC was
amplified from kidney cDNA by PCR with ExpandHighFidelity (Roche Applied
Science, Mannheim, Germany). The PCR primers were
xENaC-5'
(5'-GCCGGATCCTTTATTATGGAGTCCACAG-3') and
xENaC-3'
(5'-GCCAGATCTTAGGAAAAGGTATTTATTTCCCC-3'). Using terminal
restriction endonuclease recognition sequences (BamHI and
BglII), the PCR product was ligated in the oocyte expression vector
pRSSP containing the 5'-untranslated region from Xenopus
-globin and a poly(A) tail. Several clones from two independent PCRs
were sequenced to exclude PCR errors. Mutant cDNAs were constructed by
recombinant PCR using standard techniques.
Semiquantitative Reverse Transcription-PCRTotal RNA was
isolated from Xenopus tissues with peqGOLD RNApure (peqLab, Erlangen,
Germany). It was digested with DNase; RNA integrity was checked on an agarose
gel; and 2 µg was reverse-transcribed with oligo(dT) using SuperScript
(Life Technologies). One-thirteenth of the reaction was amplified for 30
cycles at 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 5 s. The
PCR primers were xENaC-RT-u (5'-GTCCGTCTCAACTGCTCTCG-3') and
xENaC-RT-l (5'-TTGCCCTTTGACTGCAAGCC-3'). Amplification
products were transferred to nylon membrane and then probed with a 300-bp
32P-labeled BamHI/PvuII cDNA fragment from
xENaC. The primers for control amplification of
glyceraldehyde-3-phosphate dehydrogenase were xGAPDH-RT-u
(5'-TCACAACCACAGAGAAGGCC-3') and xGAPDH-RT-l
(5'-CCATCTCTCCACAGCTTGCC-3'). Amplification was for 25 cycles at
94 °C for 30 s, 60 °C for 30 s, and 72 °C for 10 s.
Electrophysiology and Data Analysis0.110 ng of cRNA was injected into stage VVI oocytes of X. laevis. Oocytes were kept in a low Na+ oocyte ringer-2 medium (10 mM NaCl, 72.5 mM N-methyl-D-glucamine, 1 mM Na2HPO4, 2.5 mM KCl, 5 mM HEPES, 0.5 g/liter polyvinylpyrrolidone, 1 mM MgCl2, and 1 mM CaCl2) at 19 °C for 15 days. Oocytes were superfused with a normal frog ringer solution containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES (pH 7.3); and amiloride (50 µM)-sensitive current was measured using two-electrode voltage clamp. Ionic selectivity was determined by replacing NaCl with LiCl or KCl. The holding potential was 60 mV. For determination of apparent amiloride affinity, oocytes were superfused with a solution containing 50 µM amiloride. This solution was then replaced with solutions containing amiloride at 010 µM. Current values were subtracted from the current measured in the absence of amiloride to obtain the value of the inhibited current. Data were fitted for each experiment, and current values were normalized to the maximal value obtained from the respective fit. All experiments were performed at room temperature (2226 °C).
For determination of the time constant of self-inhibition, the oocytes were
superfused with the bath solution at a rate of 20 ml/min. Using a single
exponential function, the time constant of self-inhibition of
xENaC-
and
xENaC-containing channels was determined. This time constant was
used to calculate the time at which self-inhibition was complete by >97%.
This time was considered as the quasi-steady state.
Na+ affinity was determined for the initial current after amiloride washout (Imax) and for the current in the "steady state." For these long measurements, we had to use a slower solution flow rate, limiting the time resolution. Therefore, in these measurements, we considered 2 min as the steady state. Solutions containing different Na+ concentrations (140, 90, 35, 10, 3, and 0 mM), in which Na+ was replaced by equimolar amounts of N-methyl-D-glucamine, were used. To account for the channel rundown in these long measurements, current values were normalized to currents that were obtained under identical experimental conditions, but with an identical Na+ concentration (90 mM). Current values were then normalized to the current measured with 140 mM NaCl. Data were fitted for each experiment. The holding potential was 100 mV.
Apparent affinity for amiloride and Na+ was fitted to the following equation: I = a + (Imax a)/(1 + (C50/[B])n), where [B] is the amiloride or Na+ concentration, Imax is the maximal current, a is the residual current, C50 is the concentration at which half-maximal response occurs, and n is the Hill coefficient. C50 values are expressed as mean ± S.D. Error bars on Figs. 3 and 4 represent S.E. Statistical analysis was done with Student's t test.
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RESULTS |
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Functional Characterization of xENaCWe
investigated the electrophysiological characteristics of
xENaC by
functional expression in Xenopus oocytes. When injected alone in
oocytes,
xENaC elicited only small (<10 nA) amiloride-sensitive
Na+ currents. Coexpression with either
xENaC or
xENaC
increased the current amplitude, but only coexpression with both
xENaC
and
xENaC resulted in full expression with amplitudes on the order of
several µA (Fig.
3A). This feature resembles
ENaC and
ENaC
(2,
17) and identified
xENaC
as an
-like subunit, which efficiently co-assembles with
ENaC and
ENaC to form surface-expressed heteromeric channels. Substitution of
extracellular Na+ with Li+ or K+ revealed
that channels containing
xENaC are highly selective for Na+
over K+, with a Li+
Na+ >>
K+ permeability sequence, similar to
xENaC-containing
channels (Fig. 3B).
xENaC-containing channels were blocked by amiloride with an apparent
IC50 of 2.50 ± 0.82 µM (n = 12),
which is 10 times higher than the IC50 for
xENaC-containing
channels (0.22 ± 0.01 µM; p << 0.05)
(Fig. 3C). Together,
xENaC-containing channels show the electrophysiological hallmarks of
ENaCs: high selectivity for Na+ and block by amiloride at a low
µM concentration.
As illustrated in Fig.
4A, the time dependence of the current after washout of a
saturating amiloride concentration (50 µM) was very different
for xENaC- and
xENaC-expressing
oocytes. For both channels, the current amplitude rose within seconds due to
unblocking of the ion pore. However, whereas
xENaC-expressing oocytes showed a constant inward current
with no strong time dependence over a time period of minutes, the inward
current of
xENaC-expressing oocytes strongly declined
within a few seconds to a quasi-steady state
(Fig. 4A). At this
quasi-steady state (25 s after amiloride washout; see "Experimental
Procedures"), the current amplitude was only 28 ± 7% (n
= 10) of the peak amplitude (Table
I). In addition to this fast current decline of
xENaC-expressing oocytes, both
ENaC- and
ENaC-expressing oocytes displayed a linear
"rundown" of channel activity on a time scale of minutes
(Fig. 4A). The time
course of this rundown was much slower than the initial fast decline of
xENaC-expressing oocytes, and both processes can be clearly
distinguished. The initial fast decline was well fitted to an exponential
function with a time constant of 8.2 ± 1.3 s (n = 10).
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We investigated the dependence of both the initial and steady-state
currents of xENaC-expressing oocytes on extracellular
Na+ by equimolar replacement of increasing amounts of
Na+ with N-methyl-D-glucamine. The initial
current after amiloride washout saturated at
100 mM
Na+ with an apparent EC50 of 15.3 ± 5.3
mM (n = 14), similar to
xENaC-containing channels
(20.5 ± 7.2 mM (n = 8); p = 0.03)
(Fig. 4B). However,
after the time-dependent decline in the current amplitude, the steady-state
current saturated with an apparent EC50 of 4.1 ± 3.0
mM (n = 14; p << 0.05)
(Fig. 4B). Thus, the
time-dependent decline was seen only at high (>10 mM)
Na+ concentration (Fig.
4B).
The rapid inhibition with high concentrations of extracellular
Na+ was also observed after switching from an extracellular
solution containing a low (1 mM) Na+ concentration to
one containing a high concentration (115 mM). Initially, the
current amplitude rapidly rose due to the strongly increased driving force for
the inward Na+ current. But then, the current of
xENaC-expressing oocytes relaxed by 70% with a time
constant of 5.9 ± 1.5 s to a quasi-steady-state value (n = 10)
(Fig. 4C), comparable
to the behavior after amiloride washout. However, rapidly changing the
extracellular Na+ concentration uncovered this fast inhibition also
for
xENaC-expressing oocytes. Currents relaxed by
40%
with a time constant of 1.6 ± 0.2 s (n = 10)
(Fig. 4C). Thus,
inhibition of channels containing
ENaC is significantly faster than
that of channels containing
ENaC (p << 0.05). The speed of
inhibition of channels formed by
xENaC probably leads to
an underestimation of the peak current. Therefore, the apparent ratio of
steady-state to peak current may be overestimated for these channels.
Such a fast inhibition with high concentrations of extracellular
Na+ is known as Na+ self-inhibition
(810).
The characteristic feature of self-inhibition is that it is mediated by the
extracellular Na+ ion itself and not by a rise in the intracellular
Na+ concentration. However, due to the large current amplitude on
the order of several µA in our experiments, a rapid increase in the
intracellular Na+ concentration was difficult to prevent. To show
that a rise in the intracellular Na+ concentration is not
responsible for the rapid current decline, we therefore used an experimental
setup that allowed us to use different concentrations of extracellular
Na+ leading to an inward current of comparable size. This was
achieved by using a low extracellular Na+ concentration together
with a large negative holding potential and a high extracellular
Na+ concentration together with a small positive holding potential.
These experiments were performed successively on the same
xENaC-expressing oocyte. As shown in
Fig. 4D, amiloride
washout gave rise to an inward current in both cases. The initial amplitude of
this current was 1.48 ± 0.20-fold larger in the 2 mM
Na+ solution (n = 5; p = 0.05), indicating a
larger Na+ influx under this condition. However, despite the
smaller Na+ influx, a rapid current decline was observed only in
the 115 mM Na+ solution (the current amplitude 30 s
after the peak was 43 ± 3% of the peak amplitude (n = 5);
p < 0.05). In the 2 mM Na+ solution, no
significant current decline was registered (the current amplitude 30 s after
the peak was 101 ± 2% of the peak amplitude (n = 5);
p = 0.5) (Fig.
4D). These results strongly suggest that the rise in the
extracellular (and not intracellular) Na+ concentration was the
basis of the rapid current decline.
Moreover, we observed self-inhibition of a comparable degree for current
amplitudes ranging from 1 to 50 µA. Finally, it is well documented that
feedback inhibition, which is due to a rise in the intracellular
Na+ concentration, has a slower time course on the order of minutes
(6). Therefore, feedback
inhibition probably underlies the slow linear rundown described above, which
follows Na+ self-inhibition. At least one mechanism of feedback
inhibition is internalization of membrane-expressed channels. However, the
fast current decline was partially but rapidly reversible after application of
an amiloride analog (see below), most likely too fast for a substantial
extrusion of the Na+ load. Together, our results suggest that the
extracellular Na+ ion itself was responsible for the fast current
decline seen in xENaC-expressing oocytes.
Na+ self-inhibition can be relieved in Rana skin by the
amiloride analog benzimidazolylguanidine (BIG)
(9,
11,
18,
19). We exposed
xENaC-expressing oocytes to a solution containing 115
mM NaCl. After the current had relaxed to its steady state, we
applied 1 mM BIG. Following an initial decrease in the current
amplitude, the current amplitude increased rapidly
(Fig. 4E). This
behavior revealed that BIG had, in addition to its inhibitory effect, a
dominant stimulatory effect on
ENaC-containing channels
(Isteady-state/Ipeak = 0.29 ±
0.04 without BIG and 0.48 ± 0.03 with BIG (n = 8); p
<< 0.05). After BIG washout, the current amplitude initially increased due
to relief of blockade by BIG, but then decreased again to the value before BIG
application (Fig. 4E).
This behavior can be explained by reversible relief of self-inhibition by BIG.
ENaC-expressing oocytes showed a qualitatively similar
reaction to BIG application (Fig.
4E). However, here the effect of BIG was mainly
inhibitory (Isteady-state/Ipeak = 0.67
± 0.03 without BIG and 0.62 ± 0.04 with BIG (n = 6);
p < 0.05). The relief of the rapid inhibition by BIG lends
additional support to the interpretation that it reflects Na+
self-inhibition.
Identification of Molecular Determinants of Na+
Self-inhibitionSelf-inhibition of xENaC-containing
channels differed in two ways from that of
xENaC-containing channels.
It was slower; however, a larger fraction of the channels apparently passed
into the inhibited state. Assuming a simple two-state model
(Scheme 1), with an active
state (A) and an inhibited state (I) linked by an
inactivation rate constant (ki) and an activation
rate constant (ka), the rate constant
ki would determine the speed of self-inhibition.
The steady-state level that we measured as
Isteady-state/Ipeak depends on both
ki and ka. Because,
in
xENaC-containing channels, the kinetics of self-inhibition changed
inversely to the steady-state level, this would imply that the
xENaC
subunit changes ka as well as
ki. Compared with the
xENaC subunit, the
ki would be reduced, but the
ka would be even more reduced, leading to the
increased but slower inhibition.
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To identify regions in the channel protein responsible for this
differential behavior of xENaC and
xENaC, we constructed a set of
chimeric channels with parts of
xENaC exchanged with the corresponding
parts of
xENaC. Self-inhibition of these channels was then examined by
rapidly switching from solutions of low (1 mM) to high (115
mM) Na+ concentrations
(Fig. 5 and
Table I). As is shown in
Table I, the kinetics of
self-inhibition was significantly different (p < 0.05) between
either the wild-type
xENaC or
xENaC subunit and all of the
chimeric channels. Channels in which only the N terminus was exchanged showed
slow self-inhibition, as seen with wild-type
xENaC subunits (C1,
=
4.07 ± 1.42 s). However, all of the chimeras in which also the first
transmembrane domain was exchanged showed self-inhibition that was
significantly faster than the self-inhibition of even wild-type
xENaC-containing channels (C2C5,
= 0.22 ± 0.14 to
1.16 ± 0.17 s) (Fig. 5
and Table I), suggesting
unspecific effects in these chimeric constructs. Therefore, we constructed
variants that retained both intracellular termini and both transmembrane
domains from
xENaC and in which only parts of the extracellular loop
were exchanged with the corresponding parts of
xENaC (C3exC5ex)
(Fig. 5). A variant in which
approximately the first third of the extracellular loop was exchanged showed
self-inhibition with an intermediate time course (C3ex,
= 2.69 ±
1.05 s), whereas exchange of bigger parts led to self-inhibition that was
similar to that of
xENaC-containing channels (C4ex,
= 1.79
± 0.67 s; and C5ex,
= 1.27 ± 0.49 s). Altogether, these
results suggest that crucial regions that determine the kinetic differences
between
xENaC and
xENaC reside in the extracellular loop.
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In addition to the time course of self-inhibition, we analyzed the ratio of
steady-state to peak current
(Isteady-state/Ipeak) for the chimeric
constructs (Table I).
Interestingly, for the chimeric channel C2, which contained the cytoplasmic N
terminus together with the first transmembrane domain of xENaC, more of
the peak current was inhibited at steady state (90%) than for
xENaC.
However, inhibition of this channel was faster than for
xENaC. This
shows that a low Isteady-state/Ipeak
ratio can also be measured for rapidly inhibited channels, suggesting that the
difference in Isteady-state/Ipeak
between
xENaC- and
xENaC-containing channels is real. For all of
the chimeras that contained part of the extracellular loop of
xENaC,
Isteady-state/Ipeak was high (ranging
from 0.65 ± 0.05 to 0.85 ± 0.08)
(Fig. 5 and
Table I). This result suggests
that the extracellular loop, especially the proximal third, determines not
only the inactivation rate constant ki, but also
the activation rate constant ka (see
Scheme 1 above).
Finally, we addressed the molecular basis for the lower apparent amiloride
affinity of xENaC-containing channels. Amiloride is an open channel
blocker, and amino acids at the beginning of the second transmembrane domain
are involved in amiloride binding
(20). Sequence alignment of
all known
-like subunits (Fig.
6, upper) shows that there is only one amino acid at the
beginning of the second transmembrane domain that is different in
xENaC
compared with all other
-like subunits: Trp513. Replacement
of Trp513 with the amino acid present in
xENaC confirmed
that Trp513 is responsible for the low apparent amiloride affinity
of
xENaC-containing channels. Channels containing the single amino acid
exchange W513L showed a significantly increased apparent affinity for
amiloride (IC50 = 0.38 ± 0.33 µM (n =
11); p << 0.05). In addition, these channels showed a slow current
rising phase and no apparent self-inhibition after amiloride washout
(Fig. 6, lower, left
trace). However, rapidly changing from a low to high Na+
concentration also uncovered self-inhibition for this mutant channel
(right trace). Self-inhibition was as slow as in wild-type
xENaC-containing channels (
= 5.95 ± 1.66 s (n =
10); p = 0.5), confirming that Trp513 is at the basis of
differential amiloride affinity, but not of differential speed of
self-inhibition.
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DISCUSSION |
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xENaC is already the sixth ENaC subunit identified in
Xenopus. All subunits are expressed in kidney
(13,
14) and could, assuming that
channels contain an
-like, a
, and a
subunit,
theoretically assemble in eight different combinations. Channels formed by
only two different subunits would increase the variety of ENaCs in
Xenopus even more. Why this remarkable diversity evolved is unclear.
Mammalian ENaCs seem to be less variable.
2ENaC and
2ENaC subunits do not appear to be present in mammalian
genomes. Moreover, the closest
xENaC homolog in the draft version of the
human genome is
ENaC, suggesting that there is no
ENaC subunit in
humans.
Na+ Self-inhibitionNa+
self-inhibition had been best described in frog tissues
(5). It has, however, also been
indirectly shown for native ENaC in rat cortical collecting tubules
(24). Here, an apparent
Na+ affinity of 9 mM was measured for the whole cell
Na+ current (INa), whereas the current through
single channels (iNa) saturates with an apparent
Km of 48 mM
(24). Because
INa = (iNa)NPo,
this implies that either the channel density (N) or the open
probability (Po) decreases as
[Na+]o increases
(24). Thus, Na+
self-inhibition seems to be a general characteristic of ENaCs. Strikingly,
however, most of the studies characterizing recombinant ENaC in heterologous
expression systems did not reveal self-inhibition. It was only very recently
that Chraibi and Horisberger
(15) convincingly showed that
human ENaC, which is formed by subunits, shows
self-inhibition when expressed in Xenopus oocytes. The
self-inhibition was observable only after fast amiloride washout or a change
in the extracellular Na+ concentration, and the current relaxed to
a quasi-steady state with a time constant of 3 s. Thus, self-inhibition is
fast and can be observed only with a high time resolution. At room
temperature,
40% of the initial current was inhibited at steady-state
with human ENaC and
20% with rat ENaC. For
xENaC,
self-inhibition was too fast to be reliably determined
(15).
Due to the speed of self-inhibition, the time constants we determined
should be taken only as estimates. Indeed, it seems that for all of the native
channels, self-inhibition is faster than for xENaC-containing channels.
The time course of self-inhibition has been reported in detail for ENaC from
Rana skin and is on the order of 24 s
(9,
11,
18). However, the solution
flow rate in these experiments was as fast as 40 ml/s
(9). Such fast flow rates and
ensuing step changes in ionic concentrations can, unfortunately, not be
applied to Xenopus oocytes. Considering that the time constant we
determined is probably limited by a comparably slow flow rate, the time course
of self-inhibition of
xENaC is in reasonable agreement with that of ENaC
from Rana skin. However, self-inhibition of
xENaC seems to be
considerably faster. Moreover, self-inhibition of ENaC from Rana skin
can be relieved by 1 mM BIG
(11,
18), which has a dominant
inhibitory effect on channels composed of
subunits (Ref.
15 and this study). The time
course of self-inhibition, the stimulatory effect of BIG, and the low
amiloride affinity suggests that
xENaC is the molecular correlate of the
channel from Rana skin. Because
xENaC is not expressed in
Xenopus skin (Fig. 2),
this would then imply species differences in
ENaC tissue expression
patterns.
Molecular Mechanism of Na+ Self-inhibitionIt appears that upon exposure to a high extracellular Na+ concentration, ENaC passes into an inactive state. According to a model proposed by Palmer and Frindt (25), ENaC exists either in a gating mode characterized by a high Po or in a gating mode characterized by a low Po. It may be that an increase in the extracellular Na+ concentration favors the low Po mode. In this model, Na+ self-inhibition endows ENaC with a regulatory mechanism to respond to high extracellular Na+ concentrations. Because other regulatory mechanisms also act on Po, the extracellular Na+ concentration cannot, however, be the sole determinant of the open probability of ENaC.
Most likely, ENaC possesses a site in the extracellular loop that senses
the extracellular Na+ concentration. Interaction of Na+
with this site would entail a conformational change, which would then reduce
channel Po, leading to the apparent decrease of the whole
cell current. The comparatively slow self-inhibition with
xENaC-containing channels probably reflects a slower inactivation rate
constant for the transition from the active (A, high
Po) to the inactive (I, low
Po) state. This may be due to a higher activation energy
for this transition and indicates less favorable conformational changes during
self-inhibition in
xENaC-containing channels. Our approach using subunit
chimeras has identified crucial determinants for the speed of self-inhibition
in the extracellular loop, indicating conformational transitions during
self-inhibition in this region.
Because self-inhibition could also be observed after amiloride washout,
amiloride binding to the channel apparently interferes with self-inhibition.
Either it masks the Na+ sensor, which should then be located distal
to the amiloride-binding site, or it stabilizes the active (high
Po) state. This would also explain why amiloride analogs
with a short dwell time such as BIG release the channel from self-inhibition.
1 mM BIG led to partial macroscopic blockade of the current
mediated by xENaC-containing channels. But BIG activity would also
interfere with self-inhibition, leading to the paradoxical increase in the
macroscopic current amplitude.
Using an exponential fit, we calculated a time constant for the rising
phase of the current after amiloride washout that was significantly larger for
ENaC-expressing oocytes than for
ENaC-expressing oocytes (3.6 ± 2.5 s compared with
0.9 ± 0.3 s; p < 0.05)
(Table I). This suggests that
the dissociation rate constant (koff) for amiloride for
channels containing the
subunit is significantly higher than for those
containing the
subunit. A higher off-rate could account for the
reduced apparent affinity of
ENaC-containing channels for amiloride.
Moreover, because amiloride washout was slower than self-inhibition for
ENaC-containing channels (Table
I), this could explain why self-inhibition could not be observed
for these channels after amiloride washout. In agreement with this notion,
amiloride affinity was increased and self-inhibition could no longer be
observed for
xENaC containing the single substitution W513L. Thus,
Na+ self-inhibition can be more easily observed with
xENaC-containing channels for two reasons: self-inhibition is slower,
and amiloride washout is faster probably due to a higher off-rate.
Our analysis showed that the majority of xENaC-containing channels
passed into the inactivated state at room temperature, suggesting a lower
energy level for the inactive state compared with the active state. Due to the
speed of self-inhibition, the proportion of
xENaC-containing channels
that passed into the inactive state could not be determined with precision.
However, chimeric channels that contained the intracellular N terminus and the
first transmembrane domain of
xENaC showed fast self-inhibition and a
high proportion of inactive channels at steady state (C2)
(Fig. 5), suggesting that
Isteady-state/Ipeak is not
overestimated for
xENaC-containing channels. Moreover, channels
containing human or rat
ENaC also show a low proportion of inactive
channels during steady state at room temperature
(15). Thus, it is likely that,
compared with
xENaC-containing channels, a significantly larger
proportion of
xENaC-containing channels undergo self-inhibition. Our
approach using subunit chimeras suggests that the proximal part of the
extracellular loop is most crucial in determining the ratio of the active and
inactive states and consequently in determining the energy levels of these
states. Therefore, it seems that the same regions in the extracellular loop
control the rate constant for inactivation (ki)
as well as that for activation (ka).
Only 1040% of human or rat
ENaC passes into
the inactive state at room temperature
(15), compared with 70% of
xENaC. However, due to a strong temperature dependence of
the inactivation rate, a fraction of the mammalian channels comparable to
xENaC pass into the inactive state at 35 °C
(15). Thus, because
Xenopus is an ectothermic organism with a temperature preference of
20 °C, both channel types have comparable equilibria of active and
inactive states at the respective body temperatures, suggesting similar
physiological implications for Na+ self-inhibition by
xENaC and mammalian
ENaC.
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FOOTNOTES |
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* This work was supported by Grant FG 1-0-0 from the Attempto Research Group
Program of the University of Tübingen (to S. G.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology II, University
of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. Tel.:
49-7071-29-77357; Fax: 49-7071-29-5074; E-mail:
stefan.gruender{at}uni-tuebingen.de.
1 The abbreviations used are: ENaC, epithelial Na+ channel; xENaC,
X. laevis ENaC; RACE, rapid amplification of cDNA ends; BIG,
benzimidazolylguanidine.
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ACKNOWLEDGMENTS |
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REFERENCES |
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