(Received for publication, July 26, 1995; and in revised form, October 10, 1995)
From the
A cloned rat epithelial Na channel (rENaC) was
studied in planar lipid bilayers. Two forms of the channel were
examined: channels produced by the
subunit alone and those formed
by
,
, and
subunits. The protein was derived from two
sources: either from in vitro translation reaction followed by
Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found
that either
-rENaC alone or
- in combination with
- and
-rENaC, produced highly Na
-selective (P
/P
= 10),
amiloride-sensitive (K
= 170
nM), and mechanosensitive cation channels in planar bilayers.
-rENaC displayed a complicated gating mechanism: there was a
nearly constitutively open 13-picosiemens (pS) state and a second 40-pS
level that was achieved from the 13-pS level by a 26-pS transition.
-,
-,
-rENaC showed primarily the 13-pS level.
-rENaC and
,
,
-rENaC channels studied by patch clamp
displayed the same gating pattern, albeit with >2-fold lowered
conductance levels, i.e. 6 and 18 pS, respectively. Upon
treatment of either channel with the sulfhydryl reducing agent
dithiothreitol, both channels fluctuated among three independent 13-pS
sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude
between all three conductance levels. Different combinations of
-,
-, and
-rENaC in the reconstitution mixture did not produce
channels of intermediate conductance levels. These findings suggest
that functional ENaC is composed of three identical conducting elements
and that their gating is concerted.
An epithelial amiloride-sensitive Na channel
has recently been cloned from rat distal colon (1, 2, 3) and, subsequently, from other
tissues(4, 5, 6, 7) . This channel,
termed rENaC for rat epithelial Na
channel, consists
of three homologous subunits
,
, and
. While the precise
function of any these subunits has not yet been determined, it appears
that the conductive portion of the channel resides in the
subunit
and that
and
are necessary for enhanced ion channel
activity in the Xenopus oocyte heterologous expression
systems(2, 6, 7) . It is also apparent that
the channel's gating properties are influenced by
and
, because truncations in the C-terminal region of these subunits
produce a constitutive activation of the channel by increasing single
channel open probability (P
; Refs. 8 and 9). It is
these
subunit mutations that underlie the autosomal dominant
genetic hypertensive disorder, Liddle's
disease(10, 11, 12, 13, 14) .
Moreover,
-,
-, and
-ENaC expression has been localized
to aldosterone-responsive, Na
-reabsorbing epithelial
tissues in the rat by in situ hybridization and
immunocytochemistry using subunit-specific
probes(15, 16) . While amiloride-sensitive
Na
channels have been shown also to be responsive to
antidiuretic hormone in many epithelia(17, 18) , the
rat distal colon is refractory to the influence of antidiuretic
hormone(19) . Thus, the
,
, and
subunits may
comprise only a portion of the amiloride-sensitive Na
channels expressed by other cell types.
Amiloride-sensitive
Na channels studied by the patch clamp technique in
native epithelia have displayed a wide variety of single channel
properties. For example, single channel conductances ranging from 1 to
over 50 pS (
)have been reported(17) . Patch clamp
studies of Xenopus oocyte plasma membranes following
coexpression of
,
, and
cRNA reveal a channel of
5-8 pS in size(2, 8) , comparable with
Na
channels present in renal cortical collecting
tubules(18) . However, a recent paper by Burch et al.(16) report that the message levels of
,
, and
subunits in superficial human proximal airway epithelia are not equal
as they are in rat colon, kidney, and salivary gland (15) but
exist in a relative ratio of
>
.
Interestingly, the single channel conductance of the
amiloride-sensitive Na
channels found in this
epithelium is 19-20 pS(19) . Thus, the possibility exists
that different combinations of these subunits could produce channels
with different unitary conductances. Another possibility is that the
unitary conductances are tissue-specific, depending upon the physical
state of the membrane, which in turn is dependent upon ionic conditions
and membrane composition.
The purposes of the present work were
2-fold. First, we wished to develop a reconstitution system in which
the single channel properties of -,
-, and
-rENaC could
be studied, unencumbered by problems inherent in heterologous
expression systems. Second, we tested the hypothesis that variations in
relative levels of
,
, and
have functional consequences
on single channel behavior. Our results indicate that either
alone, or
in combination with one or both of the other subunits
of ENaC produce mechanosensitive, amiloride-inhibited,
Na
-selective ion channels following incorporation into
planar lipid bilayers.
-rENaC channels showed a complex kinetic
pattern: there were two main conductance transitions, one of 13 and the
other of 26 pS. In contrast,
,
,
-rENaC revealed only the
13-pS state. Different combinations of
,
, or
did not
produce channels of intermediate conductance. Gating of
-rENaC was
cooperative, with transitions to a 40-pS level only occurring after the
13-pS level was open. Moreover, upon treatment with the reducing agent
dithiothreitol (DTT), both
-rENac and
,
,
-rENaC
fluctuated among three independent 13-pS sublevels. We conclude that
these channels are composed of three identical conduction elements and
that the differences in the activity of the channels are modulated by
the presence of the
and
subunits within the complex.
RNA was in vitro translated using
a rabbit nuclease-treated cell-free lysate system (Promega) according
to the manufacturer's instructions and as described
previously(20) . 1.5 units of canine microsomal membranes
(Promega) were added to the translation reaction. This resulted in the
core glycosylation of the de novo synthesized
protein(7) . In vitro translated proteins were
purified on a G-75 Sephadex (Pharmacia Biotech Inc.) column as
described previously(20) . Fractions enriched in the
appropriate ENaC subunits were reconstituted into phospholipid
liposomes as described earlier(20) . For liposomes containing
,
,
-rENaC, subunits were in vitro translated
separately, and each was purified over a gel filtration column.
Identical elution fractions were collected and assayed for total
S incorporation. The relative ratios of
,
, and
subunits reconstituted into vesicles were determined by these
S measurements, assuming similar methionine compositions
for each subunit. Control liposomes were also prepared from a mock in vitro translation/Sephadex column purification run,
following an identical protocol. In this case, ENaC RNA was simply
omitted from the reaction mixture. These liposomes were used as control
material for the bilayer experiments.
Single channel recordings were acquired and analyzed using pCLAMP
software as described previously(22, 23) . The
recordings were acquired and stored unfiltered. For analysis, they were
filtered at 300 Hz with an 8-pole Bessel filter and acquired at 1
ms/point. The 50% threshold-crossing technique was employed to produce
events lists. Open and closed dwell time histograms were
logarithmically binned and fitted by a sum of exponential functions
using maximum likelihood. All data analysis was performed in bilayers
containing a single active channel. Application of a hydrostatic
pressure difference across a bilayer containing reconstituted ENaC
increased open probability to near 1 (20) . This effect was
independent of the direction of the hydrostatic pressure gradient, i.e. it was equally effective when the bathing solution level
was lowered or raised on either side of the channel-containing bilayer.
In multichannel membranes, hydrostatic pressure could thus reveal the
total number of channels present in the membrane. Therefore, at the
beginning of every experiment, 1 ml of the trans bathing
solution was removed to determine the total number of Na channels present. If more than one channel was detected, the
membrane was broken and the incorporation procedure repeated.
Figure 1:
SDS-polyacrylamide gel electrophoresis
of in vitro translated ,
, and
ENaCs. Lane
1 represents in vitro translated
-rENaC
electrophoresed alone in the absence of DTT or heating (nonreducing
conditions). Lane 2 represents in vitro translated
-rENaC in combination with equal volumes of unlabeled (cold)
and
rENaC, in the absence of heating or DTT. 0.2% Triton X-100
was used in both cases to solubilize the translated proteins and to
allow for incubation with the
and
subunits (lane
2). Note the presence in both lanes 1 and 2 of a
band that migrated at a relative molecular mass of 180 kDa, consistent
with a dimerization of
-rENaC. Lane 3 contains in
vitro
-rENaC in the presence of 20 mM DTT and after
heating for 3 min at 80 °C. Note the disappearance of the dimer
form. Molecular mass markers were thyroglobulin and ferritin (330 and
220 kDa, Pharmacia) and phosphorylase B and bovine serum albumin (92
and 70 kDa, Amersham Corp.). Lane 4 shows a control in
vitro translation performed in the absence of exogeneously added
ENaC transcripts.
Figure 2:
A, single channel records of in vitro translated -rENaC and
,
,
-rENaC at different
applied voltages in planar lipid bilayers. Bilayers were bathed with
symmetrical solutions of 100 mM NaCl, 10 mM MOPS-Tris
(pH 7.4). Records were filtered at 300 Hz. B, single channel
current-voltage curves of
-rENaC and
,
,
-rENaC in
bilayers. Each point represents the single channel current of the main
state transition versus applied potential averaged over seven
separate experiments (± S.D.).
Examination of the single channel records over several
minutes for both -rENaC and
,
,
-rENaC indicate
continuous activity with no run-down (n = 16; Fig. 3). These channels were kinetically different from
-bENaC in bilayers, in that
-bENaC channel activity was
punctuated by long closed periods lasting up to several
minutes(20) . Long (>1-s) closures were never observed for
the rENaC channels in over 2 h of recording.
-rENaC channels
displayed a specific gating pattern; with symmetrical 100 mM NaCl, the largest conductance level was 40 pS, but the channel
appeared to fluctuate among 0-, 13-, 26-, and 40-pS levels (see
associated all points amplitude histograms, Fig. 3B).
These histograms did not fit the binomial algorithm for independent
gating, indicating that these conductance levels cannot be produced by
three independent channels. Neither the 13- nor 26-pS current
transitions were ever observed independently of each other in over 300
separate experiments. In the small number of cases (19/330) in which
multiple channels were incorporated into the bilayer, the pattern shown
at the top of Fig. 3was simply repeated, i.e. for two channels in the bilayer, 4, 5, and 6 additional states
were seen. Moreover, the single channel records indicated that the
gating properties of the
-rENaC channel were not independent.
Openings of the 26-pS level were only observed after the 13-pS state
was open, never before. However, albeit infrequently, the 13-pS state
would close prior to closure of the 26-pS transition (see Fig. 2A, top).
Figure 3:
Single channel records of in
vitro-translated -rENaC and
,
,
-rENaC in planar
lipid bilayers. A, records are shown for +100 mV holding
potential and are representative of nine separate experiments. Record
was filtered at 300 Hz using an 8-pole Bessel filter prior to the
acquisition and were sampled at 1000 Hz using a Digidata 1200
interface. Bathing solutions contained symmetrical 100 mM NaCl
plus 10 mM MOPS-Tris (pH 7.4). Dotted lines indicate
zero current. B, all point amplitude histograms. Histograms
were generated by pCLAMP software from a record of 5 min in length. C, single channel events dwell time histograms. Time constants
were calculated from single exponential fits for each state. This
experiment is representative of nine and seven separate trials for
-rENaC and
,
,
-rENaC, respectively. Bin width was 1
ms.
When
,
,
-rENaC, in a 1:1:1 (w/w/w) combination, was
incorporated into bilayers, only a 13-pS conductance level was observed
( Fig. 2and Fig. 3, bottom). However, brief
openings of <250 ms in a duration to 40 pS were occasionally seen.
Again, these openings occurred on the top of 13-pS conductance level
that was, in essence, constitutively open. Event dwell time histograms
were constructed for all the conductance levels of
-rENaC and
,
,
-rENaC channels by setting a threshold at 50% of the
open level of each substate (Fig. 3C). The dwell
histograms in each sublevel for each channel were all fitted by a
single exponential function. The closed state time constants were 70
± 9 and 40 ± 5 ms for
-rENaC and
,
,
-rENaC, respectively. The open state time constants
for
-rENaC and
,
,
-rENaC, respectively, were (in ms)
77 ± 10 and 72 ± 3 (13-pS state), 35 ± 8 and 51
± 3 (26-pS state), and 91 ± 11 and 52 ± 3 (40-pS
state). From this analysis, the time constant for exit from the closed
state is nearly twice as long for
-rENaC than for
,
,
-rENaC.
Because ,
,
-rENaC expressed
in Xenopus oocytes displays a Na
-selective,
5-pS single channel conductance with relatively long lived open and
closed conductance states as measured by patch
clamp(2, 8) , we wanted to assess directly whether
bilayer reconstitution protocol utilizing in vitro translated
polypeptides could affect conductance and/or open and closed times.
Therefore, we compared rENaC single channel properties determined from
patch clamp measurements of rENaC-expressing oocytes with those made in
bilayers using in vitro translated proteins or subsequent to
fusion of rENaC-expressing oocyte plasma membranes. Oocytes were
injected either with
-rENaC or
,
,
-rENaC cRNA and
then either they were patch-clamped or their plasma membranes were used
for fusion to planar bilayers. Fig. 4shows the results of these
maneuvers. Patch clamp recordings of oocytes expressing
-rENaC
revealed channels with a large conductance of approximately 18 pS.
Interposed among the large transitions were two additional conductance
levels of 6 and 12 pS each. In recordings made from three separate
oocytes, these
-rENaC channels behaved in this manner. Except for
the absolute values of the conductance states, this kinetic behavior
was very similar to that observed for
-rENaC in the bilayer (Fig. 2). However, in contrast to the bilayer, channel activity
occurred in bursts rather than continuously. Patch clamp recordings
made from oocytes expressing
,
,
-rENaC typically showed
long lived 6-pS channels, similar to what was previously
reported(2) . Native oocyte membranes from Xenopus oocyte expressing either
-rENaC or
,
,
-rENaC
fused to bilayer membranes revealed channels with similar kinetic
behavior to those measured by patch clamp, but only with larger
conductance states. In all other respects, however, these channels
behaved identically to those observed for in vitro translated
protein incorporated into bilayers. We conclude, therefore, that the
bilayer is an appropriate system in which to study rENaC.
Figure 4:
Single
channel records of -rENaC and
,
,
-rENaC expressed in Xenopus oocytes. A, patch clamp recordings. Traces are representative of data obtained from three separate
oocytes for each ENaC. Oocytes were bathed in high K
medium in order to depolarize the resting membrane potential to 0
mV (see (2) ) B, oocyte membrane vesicles incorporated
into planar lipid bilayers. For the bilayer experiments, a holding
potential of 100 mV was employed in order to match the estimated
holding potential of the cell-attached patches of oocyte membranes. Traces are representative of 12 separate experiments each for
-rENaC and
,
,
-rENaC.
Figure 5:
Single channel records of in vitro translated -rENaC and
,
,
-rENaC in planar lipid
bilayers in the absence and presence of a hydrostatic pressure
gradient. These records are typical of seven individual experiments.
Conditions are the same as indicated in the legend to Fig. 2. A
0.26 mm Hg hydrostatic pressure gradient was produced by the addition
of 1 ml of bathing solution to the cis compartment.
Figure 6:
Mean current-voltage relationships of
-rENaC (A) and
,
,
-rENaC (B) in
planar lipid bilayers under biionic conditions in the absence and
presence of a hydrostatic pressure gradient. For
-rENaC under
nonstretched conditions,
P
/P
was 10:1 (n = 13). Stretch decreased
P
/P
to 3:1 (n = 11). For
,
,
-rENaC,
P
/P
was 10:1
and 4:1 under control (n = 17) and stretched (n = 13) conditions, respectively. Bilayers were bathed with
100 mM solutions of NaCl (trans) and KCl (cis), each containing 10 mM MOPS-Tris (pH 7.4). Each
point represents the mean ± S.D.
Figure 7:
Effect of DTT on -rENaC and
,
,
-rENaC in planar lipid bilayers. DTT was added at a
final concentration of 25 µM to the trans bathing
solution. All other conditions were the same as indicated in the legend
to Fig. 2. This experiment was repeated 15 times each for
-rENaC and
,
,
-rENaC, with identical
results.
To further test the hypothesis that ENaC
consists of three individual protochannels formed by subunits, we
cross-linked all of the subunits present in the functional complex
together. The sulfhydryl reactive reagent
5,5-dithiobis(2-nitrobenzoate) (DTNB) was used as the cross-linking
reagent. Again, DTNB was only effective from the trans side of
the bilayer. Fig. 5shows that DTNB treatment of either
-rENaC or
,
,
-rENaC produced channels that
fluctuated between a 0- and 40-pS level. Thus, the kinetic behavior of
the channels indicated that the three putative individual
subunit
proto-channels may operate in concert. However, the complete opening of
this channel complex when in its native form occurred in two steps, the
second twice the size of the first (Fig. 3A). Thus, we
hypothesized that one of the
subunit protochannels was anchored
to the complex by a noncovalent interaction. To test this idea, we
exposed rENaC to elevated salt concentrations in the hope of minimizing
electrostatic interactions between subunits (the bulk of the amino
acids comprising each ENaC subunit lies in a large extracellular loop (24, 25, 26) ). Thus, the prediction was that
both
-rENaC and
,
,
-rENaC should gate in a very
similar manner, with one of the protochannels behaving as an
independent lower conductance channel and the two disulfide-linked
protochannels operating in effect as a single higher conductance unit.
This experiment has been performed a total of six times each for
-rENaC and
,
,
-rENaC, and the results are summarized
in Fig. 8. Raising the NaCl concentration of the bilayer bathing
solution from 0.1 to 1.5 M resulted in the appearance of three
conductance levels for both
-rENaC and
,
,
-rENaC.
The conductance levels of rENaC were increased to 19, 38, and 57 pS by
the elevated [Na]. Moreover, the current transitions appeared
to fluctuate randomly from the zero conductance level to each of the
three higher levels, unlike the kinetic behavior of the channels at 0.1 M salt. In addition, both
-rENaC and
,
,
-rENaC responded identically to elevated salt and to
DTNB treatment, suggesting that the core conduction elements of both
channels are identical.
Figure 8:
Composite figure showing the
effects of high salt concentration and DTNB on -rENaC and
,
,
-rENaC in planar lipid bilayers. Each perturbation was
performed a minimum of six times for each channel type. Other
conditions were as indicated in legend to Fig. 2.
To better understand the biophysical
consequences of DTT and high salt treatment of rENaC, amplitude
histogram analyses of current records made under these experimental
conditions were performed. All points and events amplitude histograms
are presented in Fig. 9. The overall distribution of the all
points amplitude histograms was binomial, thus suggesting equal
probability of the channel residing in any conductance level (cf., Fig. 3B). The events amplitude histogram
revealed a disruption of concerted gating following DTT or high salt
treatment in that transitions to all conductance levels occurred
independently. Such an outcome of events amplitude histogram may be due
to counting channel openings ``from'' a fixed zero current
level ``to'' a conductance sublevel. In order to overcome
this limitation we have constructed amplitude histograms with a
gradually sliding zero level, the level that channel resides in becomes
an ``apparent zero-current level'' for the next transition.
This maneuver permits the construction of a histogram of absolute
values of amplitudes of transitions. The resulting histograms (Fig. 10) show that the predominant transition in the case of
DTT-treated rENaC was 13 pS, while high salt treatment produced, in
equal probability, transitions of 19 and 38 pS. Taking into account
that the increased conductance of the channels in this latter case was
due to elevated [Na], these results support
the hypothesis that rENaC consists of a minimum of three conductive
elements, two of which may be linked by disulfide bonds and the third
noncovalently anchored to the covalently linked complex. Histograms for
DTT and high salt-treated
-rENaC and
,
,
-rENaC were
almost identical ( Fig. 9and Fig. 10), indicating that
the conductive pore of these channels was formed by
-rENaC.
Figure 9:
All points and events amplitude histograms
of DTT and high salt-treated -rENaC and
,
,
-rENaC.
Experimental conditions were the same as described in the legends to Fig. 4and Fig. 5, respectively. Events lists were
produced by pCLAMP software using 50% amplitude threshold technique
with a minimum event duration. All points amplitude histograms are
shown in gray, while the events amplitude histograms are shown
in black.
Figure 10:
Events amplitude histograms of DTT and
high salt-treated -rENaC and
,
,
-rENaC, using a
``sliding'' zero-current level. Experimental conditions were
the same as described in the legends to Fig. 4and Fig. 5, respectively. Events lists were produced by pCLAMP
software using 50% amplitude threshold (with 3-ms duration) technique.
Transitions from 0-pS level, from 13-pS level, from 26-pS level, and
from 40-pS level were sorted manually in a pStat events list
spreadsheet session and processed by the pStat routine to produce
events amplitude histograms.
Figure 11:
Amiloride dose-response curves of in
vitro translated -rENaC and
,
,
-rENaC in planar
lipid bilayers under different experimental conditions. Points in plots are mean ± S.D. for at least six
experiments under each condition. Amiloride was sequentially added in
increasing concentrations to the trans bathing
solution.
In this work, we report the successful incorporation of
-rENaC and
,
,
-rENaC into planar lipid bilayers.
ENaC protein was obtained either from a rabbit reticulocyte lysate in vitro translation system or following expression in Xenopus oocytes and isolation of oocyte plasma membranes. The
results obtained using either of these preparations were identical. Our
experiments also indicate that the
-rENaC subunit alone or in
combination with other
subunits acts as the conductive element of
the channel complex. However, a high degree of concerted gating occurs
between these putative conduction elements and those covalently linked
by disulfide bonds. The kinetic behavior of ENaC suggests that a
functional channel unit is comprised of a minimum of three conductive
elements formed by
subunits. ENaCs are highly
Na
-selective, are inhibited with high affinity by the
diuretic amiloride, and are mechanosensitive.
Comparison of
rENaC in Bilayers and by Patch Clamp-A number of biophysical
experiments were performed on -rENaC and
,
,
-rENaC
to compare their properties when expressed in Xenopus oocytes
and when purified and reconstituted into planar lipid bilayers. For
-rENaC, the overall kinetic behavior of the channel was similar in
patch clamp and bilayer experiments, namely a small conductance level
on top of which another conductance level (twice the size of the small
one) would open. The absolute value of the small conductance level
differed (13 pS in the bilayer and 6 pS in the patch). For
-rENaC
and
,
,
-rENaC, a 6-pS (patch) or 13-pS (bilayers) level
was routinely measured. That these amiloride-sensitive channels
expressed in oocytes are ENaCs is supported by the fact that they were
never observed in water-injected oocytes. We conclude, therefore, that
the microenvironment in which ENaC resides determines in large measure
its conductance and mean open and closed times (31, 32, 33) . Aside from these changes, the
channels displayed comparable amiloride sensitivities, ion
selectivities, and gating patterns.
The existence of subconductive
levels within a single ion channel has been reported for many ion
channels including the acetylcholine receptor(34) , the
glycine, GABA, and glutamate
receptors(35, 36, 37) , the
dihydropyridine-sensitive Ca channel(38) ,
inwardly rectifying K
channels(39, 40) , the ryanodine receptor cation
channel(41) , and gramicidin(33) . It is not clear why
subconductive behavior has not been observed in patch records of
,
,
-rENaC channels. One possible explanation is that
these channels have not yet been analyzed at high time resolution.
Another reason may be that upon drawing the oocyte membrane into the
tip of a patch electrode, sufficient tension may have already been
applied to produce what appear to be three independent, small
conductance channels (cf., Fig. 3and Fig. 4; (2) and (8) ). The fine details of channel conductances
appear to be influenced by the methods of observation.
Both high salt and DTT disrupt protein-protein interactions. Thus,
the change in biophysical properties associated with these treatments
implies that a multimeric form of -rENaC underlies channel
behavior. Because these three levels represent subconductance states of
a single channel entity(23) , this kinetic behavior strongly
suggests that ENaC is composed of a minimum of three conductive
elements and that a pore is formed within each one of these elements.
The observations that the same kind of channel activity following DTT
treatment is seen for ENaC composed of only
or of
,
,
and that
and
cannot form ion channels by
themselves (Table 1) suggest that the conduction element is the
subunit of ENaC. Whether a monomer or dimer (or higher form) of
-ENaC acts as the unit conduction element cannot be deduced from
these experiments.
As a first approximation, a simple kinetic model of ENaC can be described as follows:
where C represents the closed state and O,
O
, and O
the 13-, 26-, and 40-pS open states,
respectively. As indicated above, there were only a few transitions to
26 pS that were observed, and these only occurred from the 40-pS
conductance level and had a time constant of 35 ± 8 ms (Fig. 2, B and C). Three possible explanations
can account for these data: 1) if the 26-pS transition is comprised of
two concertedly linked 13-pS openings, it may be that there is simply a
short-lived half-closed state associated with the closing and opening
of this 26-pS level; 2) if opening of the 13-pS level is required for
the subsequent opening of the 26-pS level, it may be that a brief
transient closing of the 13-pS level triggers the closing of the 26-pS
level; and 3) if a 13-pS level transiently dissociates from the complex
in the lipid bilayer, a 26-pS level may be observed. The first
possibility would predict zero residence in the 26-pS level, assuming
that closure of the double protochannel was reversible. This second
explanation does not account for the transition from 13 through 26 to
40 pS. If the third possibility were true, a transition from 0 to 26 pS
would be expected, but the data do not support this. Thus, we
simplified the scheme to contain two predominant transitions: one of 13
pS and the other 26 pS in size. When both are open, the conductance of
the channel is 40 pS.
There is certain probability P that the channel will reside in any of the given states, and because at any given time the channel must be in one of them, the sum of these probabilities must equal one.
Also, for a system in equilibrium the percentage of channels in
any given state must remain constant. Therefore, the rate of transition
out of one state must equal the rate of transition into it. The
constants k, m, k, and m
are measures of transition rates between these
states. Therefore, the net transition rate out of a state is the
product of the rate constant and the probability of the channel being
in that
state.
If the values for each of the rate constants are determined, it
will be possible to calculate the values for the probabilities P, P
where T equals one divided by the sum of all of the rate constants leading away from the state(42) . Thus,
Substituting with the experimental data (Fig. 2C), we can calculate the following: k = 14.5 s; k
+ m = 13.5 s
; and m
= 11.5 s
.
This gives a unique
solution for the rate constants k and m.
However, to complete the model, we need values for k
and m. The appropriate equation can be obtained by
calculating the probability of the channel proceeding to the 40-pS
state from the 13-pS state (i.e. P
Thus, we calculate the values of the rate constants k = 4.95 s
and m = 9.0 s
. Using these calculated values,
we can now solve equations 3, 4, and 6 to calculate the probability of
the channel being in any of the possible three states: P
= 0.14; P
From the all points amplitude
histograms (Fig. 3B), we can compute the probability of
finding a channel in any given state by calculating the area under each
individual curve and dividing by the total area under the histogram.
This analysis yields the following results: P = 0.14 ± 0.05; P
A comparison of the values for the probabilities calculated from the
histogram analysis and those derived from the kinetic simulation are in
good agreement. This simulation thus formalizes the kinetic behavior of
a triple-barrel model for ENaC. This triple-barrel model of ENaC is
similar to that proposed for inwardly rectifying K channels(39, 40) . Interestingly, while there is
little homology between ENaC and IRK1 or ROMK1 at the nucleotide and
amino acid levels(43, 44) , the membrane topology of
both classes of ion channel are similar in that they each have only two
putative membrane-spanning
domains(24, 25, 26) .
Although expression of -rENaC in
oocytes produced an amiloride-sensitive current(1) , the
absolute magnitude of this current was greatly augmented by
co-expression with
- and
-rENaC(2) . The roles that
each subunit plays in channel formation are unknown as is the
stoichiometry of
,
,
comprising the functional rENaC.
Nonetheless, our results indicate that co-reconstituting different
relative quantities of
,
,
-rENaC does not produce
amiloride-sensitive Na
channels with altered kinetic
signatures. The large diversity in kinetic and conductance properties
of these native channels in cells as measured via patch clamp (17, 18) is thus likely to result, at least in part,
from tissue-specific factors such as auxillary or regulatory proteins.
In fact, biochemical purification studies of amiloride-sensitive
Na
channels have revealed a different pattern of
polypeptide composition of the channel complex, depending upon the
source material(47, 48, 49) . However, a
thorough analysis of variations in
,
, and
subunit
ratios on single channel properties will only be achieved once the
functional significance of any channel-associated proteins are
elucidated.