(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
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.
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.
-,
-, 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.
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 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).
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.
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. -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).
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 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.
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.