(Received for publication, October 19, 1995; and in revised form, December 22, 1995)
From the
Cystic fibrosis airway epithelia exhibit enhanced Na reabsorption in parallel with diminished Cl
secretion. We tested the hypothesis that the cystic fibrosis
transmembrane conductance regulator (CFTR) directly affects epithelial
Na
channel activity by co-incorporating into planar
lipid bilayers immunopurified bovine tracheal CFTR and either
heterologously expressed rat epithelial Na
channel
(
,
,
-rENaC) or an immunopurified bovine renal
Na
channel protein complex. The single channel open
probability (P
) of rENaC was decreased by 24% in
the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR,
but did not have any effect on rENaC. CFTR also decreased the extent of
elevation of the renal Na
channel P
following PKA-mediated phosphorylation. Moreover, the presence of
CFTR prohibited the inward rectification of the gating of this renal
Na
channel normally induced by PKA-mediated
phosphorylation, thus down-regulating inward Na
current. This interaction between CFTR and Na
channels occurs independently of whether or not wild-type CFTR is
conducting anions. However, the nonconductive CFTR mutant, G551D CFTR,
cannot substitute for the wild-type molecule. Our results indicate that
CFTR can directly down-regulate single Na
channel
activity, thus accounting, at least in part, for the observed
differences in Na
transport between normal and cystic
fibrosis-affected airway epithelia.
Cystic fibrosis (CF) ()is a genetic disease in which
the ion secretory and absorptive functions of several epithelia,
including airway mucosa (nasal and tracheal), pancreas, salivary
glands, and sweat glands, are seriously
impaired(1, 2, 3, 4) . Among these
tissues, airway epithelial dysfunction leads to chronic clinical
manifestations that ultimately result in morbidity. In the airways of
CF patients, fluid and electrolyte secretion is inhibited because of
the defect in the cystic fibrosis transmembrane conductance regulator
(CFTR), and absorption due to an increased rate of Na
transport is enhanced(5) . These events result in the
accumulation of mucus, pulmonary congestion, bronchiectasis, and
excessive dryness of the epithelial surface. The relationship between
the pulmonary etiology of CF and the primary physiological disturbances
in ion transport is not well defined.
Boucher and
co-workers(5, 6, 7, 8, 9, 10) initially
found that CF nasal epithelia display rates of amiloride-sensitive
Na reabsorption 2-3-fold greater than epithelia
obtained from normal individuals. Moreover, both the
-adrenergic
agonist isoproterenol and the adenylate cyclase activator forskolin,
agents that act via the cAMP second messenger system, stimulate airway
Na
reabsorption. Aerosolized amiloride, an inhibitor
of epithelial Na
channels, has been found to inhibit
excessive Na
reabsorption, to improve mucociliary
clearance, and to reduce the decline in pulmonary
function(11) . In cell-attached patches of cultured human nasal
epithelial cells, Chinet et al.(12) recorded
amiloride-sensitive cation channels. These channels had a single
channel conductance of 21 pS and a Na
/K
permeability ratio of 6. However, upon excision, these channels
lost their ability to discriminate between Na
and
K
. The same channels were observed in cultured CF
nasal tissue(13) . However, in cell-attached patches, the
single channel open probability (P
) was found to
be twice as high compared with that in normal cells, consistent with
the earlier results of Duszyk et al.(14) . Grubb et al.(15) found that freshly excised nasal epithelia
from CFTR
``knockout'' mice also
displayed Na
hyperabsorption. These electrophysiologic
results contrast with studies on the development of Na
transport in the neonatal rat lung, which indicate that increased
Na
transport results in an increase in absolute levels
of Na
channel mRNA(16, 17) .
Furthermore, Northern analysis of human airway epithelia revealed that
all three subunits of a recently cloned epithelial Na
channel (ENaC) (18) were present in both normal and CF
tissues, but with a lower relative amount in CF compared with normal
tissue(19) . Thus, while it is clear that Na
reabsorption is increased in CF-affected airway epithelia, the
mechanism underlying this enhanced Na
transport is not
known.
Recent evidence that CFTR can regulate the function of other
ion channels (20, 21, 22) prompted us to
examine the hypothesis that CFTR may also regulate epithelial
Na channels. We co-incorporated immunopurified CFTR
and either immunopurified bovine renal Na
channel
protein or
,
,
-rENaC into planar lipid bilayer membranes.
The intimate relationship between ENaC (18) and immunopurified
bovine renal Na
channel (23) has yet to be
determined. However, recent experimental evidence suggests that ENaC is
one of the components, most likely the core conduction unit, of the
immunopurified complex(24) . We examined basal Na
channel activity in the absence and presence of CFTR as well as
the influence of CFTR on protein kinase A regulation of renal
Na
channel function. In the presence of CFTR, the
single channel activity of both ENaC and the immunopurified renal
Na
channel was decreased. In addition, the inward
rectification of the renal channel induced by PKA-mediated
phosphorylation (25) was prevented by co-incorporation of this
channel with CFTR. In the absence of CFTR, PKA-mediated phosphorylation
increased the activity of the immunopurified renal Na
channel, but had no effect whatsoever on ENaC activity either in
bilayers or following expression in Xenopus oocytes. (
)Our results are consistent with the hypothesis that CFTR
exerts a tonic inhibition of Na
transport through
amiloride-sensitive channels, as suggested by the recent observations
of Stutts et al.(27) .
The total number of channels incorporated into any
given bilayer membrane was determined by PKA-induced phosphorylation or
by imposition of a hydrostatic pressure gradient as described
previously in detail (30, 31) . Briefly, PKA +
ATP act by increasing the P of immunopurified
amiloride-sensitive renal Na
channels and CFTR in
planar lipid bilayers rather than by recruiting new channels because no
channel-containing vesicles are present in the solutions bathing the
bilayer. Imposition of a hydrostatic pressure gradient across a
channel-containing bilayer also increases P
, but
only for the ENaC channels (31, 32) . Thus, these
maneuvers reveal ``silent'' channels already resident in
planar lipid bilayers.
The existence
of conductance sublevels in both ENaC (32) and immunopurified
renal amiloride-sensitive Na channels (30, 33) makes the definition of P
ambiguous because it depends upon whether the unitary current is
assumed to be the main state current (I) or a substate current (i
). Mean current (I) is the product of
unitary current (I), number of functional channels (N), and their P
: I = I N P
. However, P
calculated
using the same value for unitary conductance within the same set of
experiments can represent accurately the changes in activity of the
channel. For our purposes (namely, current/voltage (I/V)
curves and as a descriptor of channel activity), we employed this
approach of calculating P
, using the most
frequently observed main transition state as the unitary current. We
have previously shown that using either I or i
results in the same P
as long as the
subconductance levels (m) are equally spaced(24) .
Figure 1:
Single channel records of
,
,
-rENaC in planar lipid bilayers and effect of
subsequent incorporation of CFTR and PKA-mediated phosphorylation on
rENaC activity. Both rENaC and CFTR were expressed in Xenopus oocytes, and the plasma membranes were fused to a bilayer.
Bilayers were bathed with symmetrical solutions of 100 mM NaCl
and 10 mM MOPS/Tris (pH 7.4). Additions were made sequentially
as shown. PKA (1.85 ng/ml), ATP (100 µM), anti-CFTR
antibodies (50 ng/ml), and DPC were added to both bathing compartments,
while amiloride was added only to the trans bathing solution.
The holding potential was +100 mV. Traces are representative of 12
separate experiments.
As described above, in 26
of the 38 experiments in which incorporations of
,
,
-rENaC and CFTR coexpressed in oocytes were attempted,
only
,
,
-rENaC was detected. Because CFTR channel
activity could not be recorded in the absence of PKA-mediated
phosphorylation and because CFTR by itself oriented in the bilayer in a
random fashion (22, 34) , PKA + ATP were first
added to the trans solution, followed by addition to the cis bathing solutions. In the 12 successful experiments, PKA
+ ATP addition to the cis compartment resulted in
activation of CFTR, whereas trans addition had no effect.
Three CFTR channels are evident in the experiment shown. The addition
of anti-CFTR-(505-511) antibodies (34, 35) subsequent to the addition of PKA + ATP
inhibited CFTR, leaving
,
,
-rENaC unscathed (P
= 0.39 ± 0.04; p >
0.1) (Fig. 1, fourth trace, left), thus
providing a way to discern ENaC activity. These
anti-CFTR-(505-511) antibodies did not have any effect on
,
,
-rENaC in the absence of co-incorporated CFTR (n = 5) (data not shown). PKA + ATP had no effect on
,
,
-rENaC activity in the absence of CFTR (n = 5).
The subsequent addition of 5 µM amiloride or 300 µM DPC completely inhibited
,
,
-rENaC and CFTR, respectively (Fig. 1, bottom trace). These results indicate that CFTR, in
combination with
,
,
-rENaC, preferentially oriented in
the bilayer in the same direction as the Na
channel,
namely, with its presumptive cytoplasmic side facing the cis compartment. Consequently, amiloride or DPC was effective only
from the trans compartment, while the
anti-CFTR-(505-511) antibodies worked from the cis side.
Amiloride and DPC had no effect on CFTR and Na
channels, respectively, at these concentrations (n = 6 for each). Therefore, the increase in total membrane
current seen upon PKA + ATP addition was due to activation of CFTR
alone.
A similar sidedness to PKA action was noted in the series of
experiments performed in which CFTR was incorporated into a bilayer
already containing an active ,
,
-rENaC. The total
membrane current was increased in three experiments (out of a total of
67) when PKA + ATP were added to the cis side of the
bilayer chamber. In these same three experiments, the P
of
,
,
-rENaC was decreased after the presumed
insertion of CFTR, but before PKA + ATP addition. In 63 of the
remaining 64 experiments in which the activity of
,
,
-rENaC was not altered upon the presumed insertion of
CFTR, the cis addition of PKA and ATP did not result in any
change of membrane current, suggesting that CFTR was not present.
However, in 2 of these 64 experiments, the subsequent addition of PKA
and ATP to the trans side revealed increased CFTR channel
activity, with no concomitant alteration of
,
,
-rENaC P
. Also, an increase in CFTR channel activity upon
PKA + ATP addition to the cis compartment was noted in a
single experiment. In this experiment, the activity of
,
,
-rENaC was not initially altered upon the insertion of
CFTR, nor was it altered after CFTR activation with PKA + ATP. One
explanation for these observations is that while both
,
,
-rENaC and CFTR are present in the membrane, they are
not in close enough proximity to interact. These results are consistent
with our previous observations of a directed orientation of ENaC
channels incorporated into bilayers held at -40 mV (32) and a random orientation of CFTR channels incorporated
into bilayers by themselves (34) . These results also suggest
that a parallel orientation of CFTR and ENaC may be critical for their
interaction.
To ensure that the anti-CFTR-(505-511) antibodies
did not alter the activity of ,
,
-rENaC in the presence
of wild-type CFTR, a series of anion replacement experiments was
performed with CFTR-impermeable MOPS
anion
substituted for Cl
(Fig. 2). Incorporations of
,
,
-rENaC and CFTR were performed using vesicles isolated
from oocytes expressing either channel. In 27 experiments, no CFTR
channel activity was observed when PKA and ATP were added to either or
both bathing solutions. This result was expected because CFTR, although
activated by phosphorylation, could not conduct MOPS
.
However, at the end of each experiment, dry CsCl was added to both
compartments to a final concentration of 100 mM (note: in five
separate experiments, we found that ENaC in bilayers was essentially
impermeable to Cs
). In 7 of the 27 experiments, CFTR
channel activity became evident after the addition of CsCl. In five of
these seven experiments, the initial P
of
,
,
-rENaC was lower subsequent to the putative
incorporation of CFTR. This decrease in P
averaged
23.1 ± 2.0%. There was no change in the P
of
,
,
-rENaC in two of these experiments. This lack
of effect of CFTR on
,
,
-rENaC presumably occurred
because CFTR was not oriented properly in the bilayer, as evidenced by
the inability of DPC to inhibit CFTR when applied to the same
compartment as amiloride. The addition of PKA and ATP did not affect
,
,
-rENaC, similar to what was observed in
Cl
-containing solutions. All of these results on ENaC
are summarized in the current/voltage curves shown in Fig. 3. Fig. 3demonstrates that CFTR decreased current through ENaC at
all potentials independent of whether or not CFTR was conductive.
Figure 2:
Effect of CFTR and PKA-mediated
phosphorylation on single channel activity of ,
,
-rENaC
in planar lipid bilayers. Bilayers were bathed with symmetrical 100
mM MOPS and 10 mM MOPS/Tris (pH 7.4) solutions. All
other conditions were the same as described for Fig. 1. The
membrane was held at +100 mV. Results are representative of five
experiments.
Figure 3:
Mean current versus applied
voltage relationships of ,
,
-rENaC in presence or absence
of wild-type CFTR before and after PKA + ATP activation in planar
lipid bilayers. Experimental conditions are indicated by the symbol
legend on the figure. Each point represents the mean
± S.D. for at least three separate experiments under each
condition. Ionic conditions were identical to those described for Fig. 1.
Given the usual variability in the gating of ion channels over long
periods of time, we have performed a rigorous analysis of
,
,
-rENaC activity in the presence and absence of CFTR
(before and after the addition of PKA + ATP (Fig. 4, A and B, respectively). P
was
calculated for each minute of recording (total of 10 min for each
experimental condition) starting at the third minute after the addition
of reagents. Clearly, the P
of
,
,
-rENaC was significantly higher in the absence of CFTR
than in its presence. p values for these unpaired measurements
were less than 0.01 and 0.05 over the time course of these experiments.
Figure 4:
Bar graphs of single
,
,
-rENaC P
versus time in
absence (white bars) and presence (hatched bars) of
CFTR. This analysis was performed in the absence of (A) or
following (PKA + ATP)-mediated phosphorylation (B). The vertical bars represent the means ± S.D. of 17 separate
experiments.
Figure 5:
Channel activity of immunopurified renal
Na channel before and after co-incorporation of
wild-type CFTR in planar bilayer. The bathing solution in the trans compartment contained 100 mM NaCl and 10 mM MOPS/Tris (pH 7.4), and that in the cis compartment
contained 10 mM NaCl, 90 mMN-methyl-D-glucamine chloride, and 10 mM MOPS/Tris (pH 7.4). The holding voltage was ±80 mV. This
experiment is typical of 21 such
experiments.
Figure 6:
Mean current versus applied
voltage relationships of immunopurified renal Na channel in presence or absence of wild-type CFTR before and after
PKA + ATP activation in planar lipid bilayers. Conditions are
stipulated by the symbol legend on the graph. Each symbol, and the error bars indicate the mean ±
S.D. for at least six separate experiments for each condition. Ionic
conditions were the same as described for Fig. 5.
The presumed incorporation of CFTR
did not change NaCh P, although it affected the
NaCh gating pattern in a way that the channel resided predominantly in
its main 40-pS conduction state. In addition, the
Na
/K
permeability ratio of the
phosphorylated renal Na
channel was unaltered by CFTR,
namely, 20:1 (n = 7). Given a negligible change in P
, this effect on gating by itself could not
account for the extent of the increase in Na
transport
observed in CF airway epithelia. Moreover, it was not clear if this
effect was due to the presence of CFTR in the bilayer membrane.
Therefore, we used PKA and ATP to clarify this issue. The success of
co-incorporation, as with
,
,
-rENaC, was good (21/59,
where 59 represents the total number of successful incorporations of
NaCh) when proteoliposomes containing both NaCh and CFTR were fused
with the bilayers. The success rate of co-incorporation was
significantly lower when attempts to insert CFTR were made subsequent
to the successful incorporation of NaCh (4/96, where 96 represents the
total number of successful incorporations of NaCh).
With regard to the orientation of these two channels in the membrane, the situation was complex. In 58 out of the 59 experiments in which proteoliposomes presumably containing both NaCh and CFTR were fused with the bilayer, the subsequent addition of PKA + ATP to the cis compartment activated NaCh. CFTR was simultaneously activated, however, only in 13 of these experiments. Moreover, in seven experiments, CFTR channels were seen following the trans addition of PKA + ATP. In a single experiment, the cis addition of PKA + ATP activated CFTR, whereas NaCh was activated by PKA + ATP added to the trans side.
The cis addition of PKA + ATP activated NaCh in 92 out of the 96 experiments in which CFTR was incorporated into a bilayer already containing NaCh, while in the remaining four, NaCh was activated by the trans addition of PKA + ATP. In only 2 of these 92 experiments did the cis addition of PKA + ATP simultaneously activate CFTR. The subsequent addition of the phosphorylating mixture to the trans compartment revealed CFTR channels only twice more. Therefore, in total, the shift in gating of NaCh to the main conductance mode occurred in 15 experiments and only when NaCh and CFTR were oriented in the membrane in the same fashion (i.e. when PKA + ATP activated both channels simultaneously when added to the same side of the bilayer). The identity of these channels was established using their respective specific inhibitors (anti-CFTR-(505-511) for CFTR and amiloride for NaCh). The effects of amiloride were reversible, thus permitting further identification of the CFTR channel. These pharmacological tests independently confirmed the sidedness of the incorporation.
In the
presence of anti-CFTR-(505-511), the characteristic pattern of
(PKA + ATP)-mediated stimulation of NaCh activity was similar to
that reported earlier for NaCh in the absence of CFTR(33) , i.e. a shift to long-lived, 13-pS conductance sublevels (Fig. 5, bottom two traces). However, certain
biophysical properties of these renal Na channels in
the presence of CFTR were altered compared with those measured in its
absence. First, as indicated earlier, the presence of wild-type CFTR
promoted a shift of NaCh gating into its largest (40 pS) conduction
state. Second, the P
of the renal Na
channel, PKA-phosphorylated in the presence of CFTR, was
significantly lower (P
= 0.44 ±
0.05) than observed under the same experimental conditions in the
absence of CFTR (P
= 0.62 ± 0.07; p < 0.005). Third, the amiloride sensitivity of this
purified renal Na
channel decreased following
phosphorylation, from an apparent K
of 0.7 ± 0.1 µM in controls (n = 19) to 2 ± 0.3 µM in the presence of
CFTR (n = 15), rather than to 20 µM previously observed in the absence of CFTR(33) . Fourth,
in the presence of CFTR, PKA-mediated phosphorylation did not produce
an inward rectification of NaCh gating, unlike what occurred in the
absence of CFTR(25, 33) . The elimination of the
phosphorylation-induced voltage-dependent gating of NaCh in the
presence of CFTR can be more clearly seen in the mean current (i.e. the current averaged over the entire period of observation at each
applied voltage) versus applied voltage (I/V) curves
shown in Fig. 6. It is apparent from this graph that CFTR
effectively prohibited the PKA-mediated induction of inwardly rectified
gating of NaCh, significantly lowering its P
at
negative applied potential and thus lowering inward current. Again, the
use of control liposomes devoid of CFTR left unaltered the properties
of the renal Na
channel with regard to PKA-mediated
phosphorylation (n = 100).
Figure 7:
Effect of G551D CFTR on single
immunopurified renal Na channel before and after PKA
+ ATP phosphorylation in planar lipid bilayer. Bilayers were
bathed with symmetrical 100 mM NaMOPS and 10 mM MOPS/Tris (pH 7.4) solutions. All other conditions were the same
as indicated for Fig. 1. G551D CFTR and an outwardly rectified
chloride channel were contained in the same proteoliposome
preparation(29) . The holding potential was ±80 mV. This
experiment is representative of four such
experiments.
Figure 8:
Mean current versus voltage
relationships of immunopurified renal Na channel in
presence or absence of G551D CFTR before and after PKA + ATP
activation in planar lipid bilayers. Each symbol represents
the mean ± S.D. (n = 4). Ionic conditions were
the same as indicated for Fig. 7.
The results presented in this paper demonstrate a direct
interaction between CFTR and amiloride-sensitive epithelial
Na channels. Our findings are consistent with those
observed in native epithelia, namely, that in the absence of cell
membrane CFTR (i.e. cystic fibrosis), basal Na
transport rate is
enhanced(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
Furthermore, our experiments demonstrate that CFTR can directly
modulate single Na
channel P
,
again consistent with previous patch-clamp
data(12, 13) . Na
channels of normal
upper airway epithelia are not responsive to elevations in
intracellular cAMP (7) . When CFTR is absent, these airway
Na
channels can be activated by cAMP(7) . The
results with PKA-sensitive renal NaCh suggest that its interaction with
CFTR may be more complex than that of
,
,
-rENaC. The
effect of CFTR on NaCh channels occurred primarily at the level of the
gating of these channels following PKA-induced phosphorylation,
resulting in an inhibition of Na
current in the
physiologically relevant direction (inward). If ENaC subunits are part
of the native Na
channel complex of upper airway
epithelia, cAMP sensitivity must be conferred by additional protein
components because ENaC itself cannot be activated by PKA + ATP
either in the presence or absence of CFTR (Fig. 1).
Moreover, in native epithelia, CFTR appears to uncouple the
airway Na
channel's sensitivity to cAMP,
opposite to CFTR's effect on outwardly rectified chloride
channels (34) . The fact that CFTR does not totally uncouple
PKA sensitivity of the renal Na
channel studied here
is consistent with the minimal renal function impairment in patients
with CF (37, 38) and suggests major differences in PKA
regulatory pathways between renal and airway Na
channels. Moreover, distal lung Na
channels
studied in mammalian alveolar type II cells are activated by
PKA-mediated phosphorylation(39) . We speculate that the
hormonal regulation of amiloride-sensitive Na
channels
will thus be regional and specifically designed to accommodate the
physiological needs of the specific tissue in which the channels are
located.
Stutts et al.(27) recently reported that
transfection of ,
,
-rENaC into Madin-Darby canine kidney
cells resulted in the appearance of an inward Na
current following induction by an overnight incubation with
dexamethasone and butyrate. Moreover, this induced Na
current was amiloride-sensitive and could be further augmented by
forskolin. Cotransfection of these cells with CFTR significantly
lowered basal amiloride-sensitive Na
current and
obliterated the forskolin response. Likewise, in similar experiments
using 3T3 fibroblasts, permeable cAMP analogs stimulated whole cell
Na
currents in cells expressing rENaC only, but
inhibited them in cells coexpressing rENaC and CFTR. Thus, these
authors concluded that CFTR acts ``as a cAMP-dependent negative
regulator of Na
channels.'' While the results
presented in the present paper are consistent with their conclusion,
they are not as dramatic as those reported by Stutts et al.(27) . We suggest that the interaction between CFTR and
amiloride-sensitive Na
channels is complex and may
involve many other factors in addition to direct protein-protein
associations. For example, in many Na
-transporting
epithelial tissues, particularly renal distal tubules in which both
Na
channels and CFTR are simultaneously
present(40) , amiloride-sensitive Na
transport
can still be activated by cAMP(41) . Our observations are also
consistent with the hypothesis that ENaC channels represent the core
conduction element of NaCh, and its responsiveness to regulatory inputs
is due to associated
polypeptides(24, 26, 42, 43) .
There is no apparent theoretical explanation for how a CFTR channel
in a relatively large two-dimensional artificial membrane can influence
the function and regulation of a single Na channel. It
is likely that energetically favorable protein-protein interactions
occur, especially between molecules that are predisposed for such
interaction. Similar interactions between CFTR and an outwardly
rectified anion channel in bilayers have been
demonstrated(22) , thus establishing precedence for the
phenomenon. Moreover, in four experiments, the conduction mutant G551D
CFTR was not able to substitute for CFTR in producing diminution of the P
of ENaC and prohibiting the PKA-induced inward
rectification of the renal Na
channel. We cannot rule
out the possibility that in these four experiments, G551D CFTR and NaCh
did not establish protein-protein interaction in the bilayer (e.g. because of the presence of ORCC). However, we did not observe any
effect of G551D CFTR in any of the other 51 experiments performed
without ORCC. In fact, in only one experiment did we observe both
wild-type CFTR and the renal Na
channel present in the
same membrane and oriented in the same direction, but apparently
non-interacting. Taken together, these observations suggest that, while
wild-type CFTR does not have to be functional in order to exert
influence over Na
channels, the first
nucleotide-binding domain region of CFTR is critical for the
interaction to occur because that is the locus of the G551D mutation.
Moreover, in experiments in which G551D CFTR was inserted into
NaCh-containing bilayers, PKA and ATP were present on both sides of the
membrane without eliciting any inhibitory effect of G551D CFTR on NaCh
function. This should be contrasted with the situation in which the
addition of ``extracellular'' ATP activated ORCC when G551D
CFTR was present(34) .
In summary, we have shown that
functional CFTR can directly influence amiloride-sensitive
Na channel activity in planar bilayers. CFTR not only
decreases single ENaC P
, but also decreases the
extent of elevation of renal Na
channel P
following PKA-induced phosphorylation. Moreover,
CFTR can prohibit the induction of voltage-dependent gating of the
immunopurified renal Na
channel by (PKA +
ATP)-mediated phosphorylation. Thus, at physiologically relevant
negative voltages, the mean current through these Na
channels would be reduced in the presence of CFTR, thus
accounting for the observations made in native epithelia (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .