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(Received for publication, January 2, 1997, and in revised form, April 8, 1997)
From the Department of Medicine, Cystic Fibrosis/Pulmonary Research
and Treatment Center, The University of North Carolina, Chapel Hill,
North Carolina 27599-7248 and the § Universite de Lausanne,
Institut de Pharmacologie et de Toxicologie, 27 Rue du Bugnon, CH-1005
Lausanne, Switzerland
ABSTRACT Abnormal regulation of ion channels by members of
the ABC transport protein superfamily has been implicated in
hyperinsulinemic hypoglycemia and in excessive Na+
absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we
report that the cystic fibrosis transmembrane conductance regulator
(CFTR), which is defective in CF, reverses the regulation of the
activity of single epithelial sodium channels (ENaC) by cAMP. ENaC
expressed alone in fibroblasts responded to activation of
cAMP-dependent protein kinase with increased open
probability (Po) and mean open time, whereas
ENaC co-expressed with CFTR exhibited decreased
Po and mean open time under conditions optimal
for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at
the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a
decrease.
INTRODUCTION Recent studies (1, 2) have identified ENaC as the channel that
mediates amiloride sensitive Na+ absorption in mammalian
airways. In cystic fibrosis (CF),1
ENaC-mediated Na+ absorption is increased 200-300% in
airway epithelia and, abnormally, further stimulated by raising
intracellular cAMP (3). Because most CF mutations result in little if
any functional CFTR in the apical cell membrane of affected epithelia
(4), we inferred that normal CFTR must either down-regulate the number
of active Na+ channels or decrease the activity of
individual Na+ channels. In the present study we have
studied the effects of cAMP-dependent
protein-phosphorylating conditions on the single channel kinetics of
ENaC expressed alone or together with CFTR in NIH 3T3 fibroblasts.
EXPERIMENTAL PROCEDURES RESULTS The single channel conductance (4-5 picosiemens) of ENaC
expressed in NIH 3T3 fibroblasts, as well as cation selectivity
(Li+ > Na+ > K+), amiloride
inhibition (Ki
ENaC present in excised membrane patches exhibited a variable
degree of rundown following excision. Rundown was partially reversed
(Fig. 2A, panel i) or prevented (Fig.
2A, panel ii) by exposure of the cytoplasmic surface to PKA
catalytic subunit and 2 mM ATP (CS + ATP). Fig.
2A, panel iii, summarizes the results from both paradigms,
revealing positive regulation of ENaC activity by PKA. One explanation
for a range of basal activity, for rundown following excision, and for
variable degree of activation by CS + ATP is that the resting
phosphorylation state differs from patch to patch. Moreover, it seemed
possible that water-soluble reagents, such as PKA catalytic subunit,
might have poor access to hydrophobic compartments within the membrane
patch. We tested these possibilities with a specific peptide inhibitor
of PKA (mPKI) that had been modified by myristoylation to promote its
association with biologic membranes (10, 11). mPKI was effective in
(6/6) inside out membrane patches, reversing the effects of exogenous
CS + ATP (Fig. 2A) by inhibiting Po
(Fig. 2A, panel iii) and MOT (not shown) to levels lower
than "basal." This observation suggests that the level of basal
phosphorylation in the system influences the gating of ENaC in the
absence of external manipulation.
The presence of CFTR caused a dramatic change in the regulation of ENaC
in excised patches by CS + ATP. Whereas the gating and rundown of ENaC
in patches excised from CFTR expressing cells were not obviously
abnormal under nonstimulated conditions, exposure to CS + ATP routinely
inhibited ENaC activity in two different paradigms (Fig.
2B). First, in 4/5 excised inside out patches, CS + ATP
decreased Po (Fig. 2B, panel i).
Second, ENaC in 5/5 patches excised from CFTR expressing cells directly
into CS + ATP demonstrated low Po (Fig.
2B, panel ii) and MOT (not shown). mPKI further decreased
Po of ENaC co-expressed with CFTR (Fig. 2B, panels i and iii). Fig. 2B, panel
iii, summarizes the very different pattern of regulation of ENaC
by PKA in the presence of CFTR (compare with Fig. 2A, panel
iii).
To study PKA and CFTR regulation of ENaC in the absence of
excision-induced rundown, we exposed cells to permeant PKA activators (cpt-cAMP + forskolin (cpt-cAMP/FSK)) during cell-attached
recording (Fig. 3). In ENaC-only cells cpt-cAMP + forskolin increased ENaC Po (Fig.
3A), whereas in ENaC + CFTR-expressing cells PKA activators routinely decreased Po (Fig. 3B).
This result, coupled with the effects of CS + ATP in excised patches,
strongly indicates that the CFTR-mediated regulation of whole cell
amiloride-sensitive Na+ current observed previously (5)
reflects modulation by CFTR of ENaC single channel gating.
The results in Figs. 2 and 3 suggest that negative regulation of ENaC
by CFTR reflects an effect on ENaC activity rather than ENaC number.
Additional analyses of our data support this conclusion. First,
co-expression of CFTR with ENaC did not affect the number of ENaC
channels observed per patch (2.17 ± 0.29 (n = 28)
without CFTR and 2.29 ± 0.29 (n = 26) with CFTR).
Second, the MOT of unambiguous single channel openings in excised, and
cell-attached patches under optimal conditions of PKA activation were
markedly decreased by the presence of CFTR (Fig. 4).
Thus, CFTR negative regulation of ENAC can be explained by decreased
activity of individual ENaC channels.
DISCUSSION Our data reveal a surprisingly strong positive regulation of ENaC
alone by PKA. The low Po recorded in the
presence of mPKI (Fig. 3) and the high Po and
long MOT measured during PKA activation (Fig. 4) indicate that
increasing protein phosphorylation increased the time ENaC occupied a
stable open conformation. This result differs from the
cAMP-dependent increase of the number of endogenous amiloride sensitive Na+ channels seen in A6 epithelial
cells (9), which are reported to regulate surface expression of
transport elements by membrane insertion and retrieval (12), but is
similar to cAMP-dependent regulation of
Po of partially purified renal (13) and lung
alveolar type II cell Na+ channels (14). Studies of
heterologously expressed ENaC in oocytes (15) and of reconstituted ENaC
in lipid bilayers (16) detected no effect of PKA activation on single
channel gating. In intact oocytes (15), or in ENaC reconstituted in lipid bilayers
after expression in oocytes (16), the presence of CFTR decreased whole
cell currents or single channel open probability. Thus, CFTR appears to
exert a negative modulatory regulation of ENaC in several distinct cell
types, including human airway epithelia, mouse fibroblasts, and
amphibian oocytes.
The present findings help explain the long standing observation that
Na+ absorption across CF airway epithelia is increased and
inappropriately further stimulated by cAMP (3). In CF airways, the
abnormally high rate of basal Na+ absorption reflects the
absence of negative regulation of ENaC by CFTR under basal
phosphorylating conditions, and increased PKA activity leads only to
further absorption. In contrast, CFTR function in normal airways
converts the activation of PKA into a stimulus for both inhibition of
ENaC-mediated Na+ absorption and stimulation of
CFTR-mediated Cl A general mechanism of regulation of ion channels by ABC proteins is
yet to be identified (23), but it is clear that CFTR regulates ENaC at
the level of single channel gating. This observation is an important
consideration for understanding the mechanism by which ABC proteins,
including not only CFTR but also SUR and MDR (23), can influence other
ion channels. Potentially, ABC proteins regulate the activity of other
ion channels through transported substrates, as proposed for
CFTR-mediated ATP release (24, 25). Alternatively, ABC proteins may
regulate the activity of other ion channels by direct or indirect
protein-protein interactions.
FOOTNOTES REFERENCES
Volume 272, Number 22,
Issue of May 30, 1997
pp. 14037-14040
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,
-, 
-, and 
-ENaC subunits were stably expressed in NIH
3T3 cell lines that had been previously transduced with a truncated (inactive) interleukin-2 receptor (ENaC alone cells) or with human CFTR
(ENaC + CFTR cells) (5). ENaC-mediated single channel currents were
recorded from cell attached and excised membrane patches as described
in the figure legends.
0.3 µM) and the
slow gating pattern (MOT 1 s), are similar to what has
been reported for the cloned channel expressed in oocytes (6, 7) and
for endogenously expressed ENaC in rat cortical collecting tubule (8)
or A6 cells (9) (Fig. 1). These similar results in very
different cells suggest that cell specific cytoskeletal or other
elements are not critical determinants of the basic biophysical
characteristics of ENaC. The basal conductance and amiloride
sensitivity of ENaC were not affected by co-expression with CFTR (Fig.
1).
Fig. 1.
Characteristics of ENaC expressed
heterologously in NIH 3T3 fibroblasts. A, representative
pipette currents at a range of clamp voltages from a patch excised from
an ENaC-expressing cell. B, IV plots, including
the data from A, and similar data obtained from patches
excised from cells expressing ENaC alone (
) and cells expressing
CFTR and ENaC (
). Clamp voltage is plotted as
Vpipette. Downward (inward) currents represent
cations leaving the pipette. C, effect of amiloride on ENaC
contained in an outside out patch. D, amiloride
concentration versus inhibition of initial Po ( and as above). The approximate
ED50 was 0.3 µM. Methods: NIH 3T3 fibroblasts
that were previously stably transduced with CFTR or the inactivated
interleukin-2 receptor (26) were infected with a tricistronic
retrovirus containing -, -, and -rENaC cDNAs as reported
(5) and maintained in Dulbecco's minimal essential medium-H
supplemented with 10% fetal bovine serum, 1 µM
amiloride, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 250 µg/ml G418 at 37 °C in an atmosphere of 95% air and 5% CO2. Cells were plated for patch clamp experiments on 35-mm
culture dishes and incubated for 24 h in growth medium containing
2 mM sodium butyrate to stimulate ENaC expression by the
long terminal repeat promoter, washed thrice with bath solution
containing (in mM) 150 Tris aspartate, 2 MgSO4,
1 calcium gluconate, and 5 TES (buffered to pH 7.2 with CsOH), and
studied on the stage of an inverted microscope at 22 °C using
standard patch clamp configurations (27). Pipette solution, unless
noted, was 280 lithium aspartate, 2 MgSO4, 1 calcium
gluconate, and 5 TES. Current was recorded on VCR tape (Vetter
Instruments). Selected recorded currents were filtered (50 Hz, Ithaco)
and digitized (1000 Hz, Labmaster 1200) for analysis using PClamp 6.0 Software (Axon Instruments). Single channel amplitude at each voltage
was determined by the separation of peaks in amplitude histograms or by
the mean of at least 12 openings measured with the cursor of a digital
oscilloscope. The means of two to three separate experiments are
plotted in B. Selectivity was demonstrated by lack of
reversed currents against Tris aspartate or potassium aspartate in the
bath with 280 mM lithium aspartate (B) or 140 mM lithium aspartate or sodium aspartate in the pipette (not shown). Amiloride was exposed to outside out patches while recording active channels by diluting the bath by 1/2 with 200% of the desired final concentration. Po was
calculated as the ratio of area under open peaks to total area of
amplitude histograms.
[View Larger Version of this Image (31K GIF file)]
Fig. 2.
Effect of CFTR on regulation of ENaC by PKA
in excised patches. A: panel i, current recorded from an
inside out patch of ENaC only cell, starting just after excision.
"c" indicates all channels closed. The probability of
one channel being open decreased from 0.72 in the first 60 s
following excision to 0.42 in the 60 s before addition of CS + ATP
(rundown) and increased during exposure to CS + ATP to 0.65 in the last
60 s before addition of mPKI. mPKI completely inhibited ENaC.
Panel i is representative of six experiments carried out
with this paradigm). Panel ii, experiment illustrating the
excision of an ENaC only cell attached patch directly into bath
solution containing CS + ATP. Up to six ENaC remain active until
exposed to mPKI by addition to the bath. Panel ii is
representative of five patches excised into CS + ATP. Panel
iii, summary of Po calculated from data
recorded (minimum duration of 60 s) from inside out patches
exposed to different bath solutions. Basal (n = 11)
includes the six patches from panel i and five patches
studied under basal conditions only; CS + ATP (n = 11)
includes all patches from panels i and ii; and
mPKI (n = 6) includes five patches from panel
i and one patch from panel ii. *, different from basal
by unpaired t test, p < 0.05). **, different from CS + ATP by unpaired t test,
p < 0.01). B: panel i, similar experiment
as in A (panel i) but paradigm carried out on a
patch excised from a ENaC + CFTR cell. Panel ii, effect of excision into CS + ATP on ENaC in a patch made from an ENaC + CFTR
cell. Panel iii, summary of Po of
ENaC + CFTR patches, as described for A, panel ii. Basal,
n = 10, CS + ATP (n = 10), mPKI (n = 5). Methods: membrane patches were excised in the
inside out mode. Basal refers to stationary channel activity following excision or just before exposure to CS and ATP. "CS + ATP" refers to the highest Po observed
during a minimal interval of 60 s in the period 3-10 min
following exposure to 100 units/ml CS (Promega) + 2 mM ATP
to the bath. mPKI refers to the Po recorded in
the period from 15 to 75 s following exposure to 1 µM mPKI (Biolmol) in the bath. Po
was determined from amplitude histograms. For multichannel patches,
nPo was calculated and Po
derived assuming independent and equal gating of each channel and
observation of maximal number of channels in the patch during
recording.
[View Larger Version of this Image (32K GIF file)]
Fig. 3.
Effects of cAMP on open probability of ENaC
studied on cell. A, cell-attached patch of ENaC only
expressing cell. Pipette current was recorded at 30 mV
(Vpipette). Cell-permeant cAMP (cpt-cAMP) (500 µM) and forskolin
(FSK, 10 µM) were added (as indicated by the
arrow). The second and third traces
were recorded 90 and 180 s later, respectively. For analysis, the
Po during basal conditions (Basal,
n = 8) and after stimulation (Stim, n = 8)
were compared. (Histogram; p < 0.05, n = 8). B, effect of cpt-cAMP and forskolin (FSK)
on ENaC activity in a cell attached patch from an ENaC plus CFTR
expressing cell. Analyzed as in A. (Histogram;
n = 8 in each condition). Methods: cell attached
recordings were carried out under basal (Basal, prior to
additions) and stimulated conditions (Stim, 3-8 min
following 500 µM cpt-cAMP and 10 µM forskolin), at Vpipette of 20 to 40 mV. A
minimum of 60 s of data was analyzed from each experiment.
Po was determined as above.
[View Larger Version of this Image (33K GIF file)]
Fig. 4.
CFTR alters cAMP regulation of ENaC kinetics
(Po and MOT). Excised inside out patches or
cell-attached patches that demonstrated only single ENaC during the
entire experiment or patches with two channels that exhibited
infrequent coincident openings were selected from the experiments
presented in Figs. 2 and 3 to determine the effect of CFTR on ENaC
gating in the presence of maximal PKA activity. Methods:
Po was calculated as above, and lists of the
durations of unambiguous openings were compiled from each experiment,
with the events list feature of PClamp 6 (Axon Instruments). Very long
openings precluded sufficient observations for conventional analysis of
the distribution of open time durations. Accordingly, the arithmetic
average of all openings greater than 40 milliseconds was calculated as
an estimate of mean open time (MOT), for each experiment
(minimum 60 s or 40 openings analyzed). *, ENaC + CFTR
(n = 7) different from ENaC (n = 9) by
unpaired t analysis (p < 0.02).
[View Larger Version of this Image (27K GIF file)]
-rENaC used in our study contains two consensus PKA
phosphorylation sites, but these are not highly conserved across
species (6, 7). Thus, PKA regulation of ENaC gating may well involve
the phosphorylation and function of an additional protein or proteins, including cytoskeletal components such as actin (17). Cell-specific expression of these proteins could explain why fibroblasts reproduce the defect in CF airways better than oocytes (15).
secretion. Despite previous reports of
abnormal regulation of Na+ channel activity in CF (18-20),
this conclusion was in doubt until now, because PKA has been reported
to regulate only the number of active amiloride-sensitive
Na+ channels in A6 cells (9), and because another genetic
disease associated with excessive Na+ reabsorption
(Liddle's syndrome) has been attributed solely to increased ENaC
number (21). More recently, the mutations associated with Liddle's
syndrome have been shown to act predominantly by increased ENaC
Po and MOT (22). This observation, coupled with the present results, make it clear that regulation of ENaC single channel kinetics is broadly implicated in the control of epithelial sodium absorption.
To whom correspondence should be addressed: Dept. of Medicine,
Cystic Fibrosis/Pulmonary Research and Treatment Center, 6023 Thurston-Bowles Bldg., The University of North Carolina, Chapel Hill,
NC 27599-7248. Tel.: 919-966-1077; Fax: 919-966-7524; E-mail: hoopster{at}unc.med.edu.
1
The abbreviations used are: CF, cystic fibrosis;
CFTR, cystic fibrosis transmembrane conductance regulator; MOT, mean
open time; ENaC, epithelial sodium channel(s); PKA, protein kinase A;
CS, catalytic subunit; mPKI, myristoylated protein kinase A inhibitor;
TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. Schreiber, R. Nitschke, R. Greger, and K. Kunzelmann The Cystic Fibrosis Transmembrane Conductance Regulator Activates Aquaporin 3 in Airway Epithelial Cells J. Biol. Chem., April 23, 1999; 274(17): 11811 - 11816. [Abstract] [Full Text] [PDF]
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D. C. GADSBY and A. C. NAIRN Control of CFTR Channel Gating by Phosphorylation and Nucleotide Hydrolysis Physiol Rev, January 1, 1999; 79(1): 77 - 107. [Abstract] [Full Text] [PDF]
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E. M. SCHWIEBERT, D. J. BENOS, M. E. EGAN, M. J. STUTTS, and W. B. GUGGINO CFTR Is a Conductance Regulator as well as a Chloride Channel Physiol Rev, January |
ABSTRACT