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INTRODUCTION |
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 Cl
channel contains three cytosolic domains as follows: a regulatory R
domain and two nucleotide-binding domains (NBDs). ATP binding and
hydrolysis by the NBDs gate the channel, and phosphorylation of the R
domain regulates activity (for reviews see Refs. 1 and 2). Although the
boundaries of the R domain are not known precisely, several lines of
evidence suggest that this domain extends from approximately residues
700 to 830 (3-6). Within this region, we found that the
cAMP-dependent protein kinase (PKA) phosphorylates three
serines (residues 737, 795, and 813) in intact cells to regulate
channel activity (7). Picciotto and colleagues (8) found that these
residues plus residue 700 were phosphorylated in vivo.
Outside this region, Ser-660 is phosphorylated by PKA and contributes
to channel regulation (7, 8). Studies of CFTR variants in which the
phosphoserines have been mutated to alanine show that phosphorylation
of multiple R domain serines contributes to regulation, although no one
serine is required for activity (for reviews see Refs. 1 and 2).
Further insight into R domain structure and function has come from
studies in which the R domain has been deleted. Such studies have
suggested that the R domain may have two functions as follows: the
unphosphorylated R domain may prevent constitutive activity
(i.e. opening in the presence of ATP alone), and the
phosphorylated R domain may stimulate activity.
Speculation that the unphosphorylated R domain prevents constitutive
activity has been based primarily on studies of CFTR in which residues
708-835 have been deleted. Expression of this variant generated a
channel that was constitutively open; only ATP was required for
activity, and PKA-dependent phosphorylation was not
required (9, 10). Larger deletions that extended the N terminus of
residue 708 failed to generate channels, likely because of a severe
disruption of structure (3). There have been only two reports of a
smaller deletion, and although the portion deleted was similar in the
two studies, the results were different (11, 12).
The R domain can also stimulate channel activity. Addition of a
phosphorylated recombinant R domain protein stimulated activity of CFTR
in which residues 708-835 were deleted. Phosphoproteins consisting of
residues 645-834, 590-858, and 708-831 each stimulated Cl
current (6, 10, 13).
Thus it appears that the R domain may both prevent constitutive
activity and, when phosphorylated, stimulate activity. However, it is
not clear whether different portions of the R domain mediate these
functions. To investigate these functions, we took two approaches; we
deleted portions of the R domain, and we translocated portions of the R
domain to the C terminus of the channel. We then asked how constitutive
and stimulated activity were affected.
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EXPERIMENTAL PROCEDURES |
Chemicals and Solutions--
The catalytic subunit of PKA was
obtained from Promega (Madison, WI) and ATP from Sigma. Protein
phosphatase 2C
was expressed and purified as described previously
(14).
For patch clamp studies, the pipette (external) solution contained (in
mM) the following: 140 N-methyl-D-glucamine, 2 MgCl2, 5 CaCl2, 100 L-aspartic acid, and 10 HEPES, pH
7.3, with HCl (Cl
concentration, 50 mM). The
bath (cytosolic) solution contained (in mM) the following:
140 N-methyl-D-glucamine, 3 MgCl2, 1 Cs-EGTA, and 10 HEPES, pH 7.3, with HCl (Cl
concentration, 140 mM).
Mutagenesis and Transfection--
CFTR deletion mutants were
constructed in the pTM1-CFTR4/S660A plasmid (in which Ser-660 is
mutated to alanine) as described previously (15). To make
R-C(709-835),
R-C(709-759), and
R-C(760-835), a
unique MluI site was inserted in the plasmid encoding
CFTR-
R/S660A using the method of Kunkel (16). The inserts encoding R
domain fragments (709-835, 709-759, and 760-835) were generated by
polymerase chain reaction from wild-type CFTR with flanking
MluI sites. These fragments were then inserted into the
MluI site in CFTR-
R/S660A, preserving the C-terminal
amino acid sequence DTRL. Constructs were verified by restriction
digest, sequencing through insertions, and by in vitro
transcription and translation assays. The amino acid sequences of the
variants are shown schematically in Fig. 1. All of the variants
contained the S660A mutation and an intact Ser-700.
We transiently expressed wild-type and mutant CFTR in HeLa cells using
the vaccinia virus/T7 hybrid expression system described previously
(15). Cells were studied 4-24 h after transfection depending on the
level of expression desired.
Immunoprecipitation and Phosphorylation of CFTR--
CFTR was
immunoprecipitated from soluble lysates of HeLa cells with an antibody
that recognizes the C-terminal amino acids (1477-1480). The
antibody-CFTR complex was labeled by phosphorylation with
[
-32P]ATP and the catalytic subunit of
cAMP-dependent protein kinase (PKA). Protein was analyzed
by SDS-polyacrylamide gel electrophoresis and autoradiography, as
described previously (17).
Electrophysiologic Methods--
The methods for excised
inside-out patch clamp recordings are similar to those described
previously (18). Wild-type and mutant CFTR channels were phosphorylated
with 1 mM ATP, and 75 nM PKA was added to the
bath solution. Bath was maintained at 35-37 °C by a
temperature-controlled microscope stage (Brook Industries, Lake Villa,
IL). Pipette resistance was 4-10 M
, and seal resistance was 2-25
G
. An Axopatch 200-A amplifier (Axon Instruments, Foster City, CA)
was used for voltage clamping and current amplification. The pClamp
6.0.3 software package (Axon Instruments) was used for data acquisition
and analysis. Data were recorded on digital audiotape (DTR-1203,
Biological Science Instruments, Molecular Kinetics, Pullman, WA).
When recording from patches containing multiple channels, data were
filtered at 1 kHz using a variable 8-pole Bessel filter (Frequency
Devices, Haverhill, MA) and digitized at 2 kHz. Membrane voltage was
held at
40 mV, referenced to the external surface of the membrane
patch. Data points during time course experiments are mean current
values during 10-s sweeps. Average current for an intervention was
determined as the average of the last 2-5 min of that intervention.
For single channel analysis, data were filtered at 1 kHz using a
variable 8-pole Bessel filter, digitized at 5 kHz, and digitally
filtered at 500 Hz. Open state probability (Po)
was determined from patches containing 1-3 channels, and the number of
channels in a patch was determined by the greatest number of
simultaneously open channels observed during the entire experiment.
Statistical Analysis--
Values are presented as means ± S.E. An unpaired Student's t test and analysis of variance
were used for assessment of statistical significance. Values were
considered statistically significant at p < 0.05.
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RESULTS |
Effect of Deleting Portions of the R Domain--
In the presence
of ATP, wild-type CFTR generates little current until it is
phosphorylated with PKA (Fig. 2A) (1, 2). Earlier studies
showed that when residues 708-835 were deleted, channels were
constitutively active, producing current without phosphorylation in the
presence of ATP alone (9). Subsequent addition of PKA further increased
current. As observed previously (15), when Ser-660 was mutated to
alanine in that construct,
708-835 (see Fig.
1), the channel functioned in a
constitutive, ATP-dependent, and PKA-independent manner
(Fig. 2C). A full-length CFTR
with only the Ser-660 mutated (CFTR-S660A) generated no constitutive current but was stimulated by PKA (Fig. 2B). Thus, Ser-660
is neither required to keep the unphosphorylated channel closed nor required for phosphorylation-dependent stimulation.
Therefore, to avoid confounding effects due to phosphorylation of
Ser-660, all of the constructs described here contained the S660A
mutation (Fig. 1). For brevity, we do not include the S660A designation in the names of the channel variants.
708-835 also retains Ser-700. One study reported that Ser-700 is phosphorylated in vivo
(8), although another did not (7). Wilkinson et al. (19)
showed a small but significant increase in the concentration of
3-isobutyl-1-methylxanthine required for half-maximal activation of
channels with an S700A mutation expressed in Xenopus
oocytes. Nevertheless, when residues 708-835 were deleted, the
presence of Ser-700 seemed to have little functional effect (3, 13),
suggesting that it plays a minor role in regulating the variants we
studied.

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Fig. 1.
Schematic diagram of CFTR variants.
Dashed line indicates deleted sequence. All variants had
Ser-660 mutated to alanine. Sites of serines at positions 660, 700, 737, 795, and 813 are indicated. The variants with portions of the R
domain translocated to the C terminus retained the DTRL sequence
(residues 1477-1480) at the C terminus.
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Fig. 2.
Effect of truncations in the R domain on
activity of CFTR. Data are examples of current from inside-out
patches of membrane containing multiple channels. Cross-hatched
bar indicates presence of ATP (1 mM) and solid
bar indicates presence of PKA (75 nM) in the cytosolic
bathing solution.
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To identify portions of the R domain that may prevent constitutive
activity and that may contribute to
phosphorylation-dependent stimulation, we deleted portions
of the R domain. Deleting the first half of the R domain (
708-759)
produced channels that were closed until PKA addition (Fig.
2D), whereas deleting the second half (
760-835)
generated channels with constitutive activity (Fig. 2E).
These data suggested that a region between residues 759 and 836 prevented constitutive activity. Therefore, we further subdivided this
region. Fig. 2, F and G, shows that prior to PKA addition,
760-783 channels were open in the presence of ATP alone, and
784-835 channels were closed. Thus, whenever residues 760-783 were deleted (
708-835,
760-835, and
760-783), the channels were constitutively active, and when these residues were present (CFTR-S660A,
708-759, and
784-835) the channels were closed. These results suggest that this portion of the R domain contains sequences that prevent constitutive activity.
Because a consensus site for PKA-dependent phosphorylation,
Ser-768, lies within residues 760-783, we wondered if Ser-768 might be
important in preventing constitutive activity. Phosphorylation of
Ser-768 has not been detected in cells stimulated with cAMP agonists
(7, 8). However, a CFTR-S768A mutant expressed in Xenopus
oocytes showed half-maximal stimulation with a lower concentration of
3-isobutyl-1-methylxanthine than wild-type CFTR, raising the
possibility that Ser-768 might have an inhibitory function (19).
Therefore, we mutated Ser-768 to alanine in the
708-759 and
784-835 constructs (Fig. 1). Neither of these channels showed
significant constitutive activity (Figs. 2H and 3). These data suggest that although residues between 760 and 783 were important in keeping the unphosphorylated channel closed, this function was not
dependent on the presence of Ser-768.
Constitutive activity and the response to PKA from multiple patches are
shown in Fig. 3. With the exception of
708-835, PKA stimulated all the variants. Because each of these
other channels retains at least a portion of the R domain and at least
one of the in vivo consensus phosphorylation sites, these
data suggest that phosphorylation of any one or two of these serines
can stimulate activity.

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Fig. 3.
Current from CFTR with truncations in the R
domain. Data are currents from patches containing multiple
channels. Shaded bars indicate amount of current in the
presence of ATP (1 mM), and black bars indicate
current in presence of ATP (1 mM) and PKA (75 mM). Asterisk indicates significant difference
compared with current in the presence of ATP alone, p < 0.05. n = 4 or more for each variant.
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Phosphorylation-dependent Regulation of
R-C(709-835)--
To understand better how portions of the R
domain determine constitutive and phosphorylation-stimulated activity,
we moved residues 709-835 from their normal location to the C
terminus, producing
R-C(709-835) (Fig. 1). Fig.
4A shows that this protein was
phosphorylated by PKA. In the presence of ATP alone,
R-C(709-835) was constitutively active (Fig. 4, B and C).
Addition of PKA further increased current. Earlier studies showed that
protein phosphatase 2C
dephosphorylates and inactivates wild-type
CFTR (14, 20). Consistent with phosphorylation-dependent
regulation by the C-terminal R domain, addition of protein phosphatase
2C
reduced current to prestimulation values (Fig. 4, B
and C).

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Fig. 4.
Function of CFTR with the R domain
translocated to the C terminus. A, SDS- polyacrylamide
gel electrophoresis and autoradiography of immunoprecipitated CFTR
variants phosphorylated with [32P]ATP. Band B
represents the core glycosylated and band C the fully
glycosylated protein. Wild-type (WT) and R-C(709-835)
but not 708-835 were phosphorylated. B, Cl
current from membrane patch containing multiple R-C(709-835)
channels. ATP (1 mM, cross-hatched bar), PKA (75 nM, solid bar), and PP2C (0.1 unit/ml,
open bar) were present during the time indicated.
C, current from membrane patches containing R-C(709-835)
channels. Data are mean ± S.E. current normalized to current
present with 1 mM ATP. n = 9 for ATP alone
and ATP plus PKA and n = 5 for PP2C .
Asterisk indicates significant difference from basal and
phosphatase-treated patches (p < 0.05).
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We also studied patches containing 1-3 channels. Fig.
5A shows that
R-C(709-835)
was constitutively active and phosphorylation further increased
activity. The Po of this unphosphorylated
channel was similar to that of unphosphorylated
708-835 (Fig.
5B). These results suggest that an R domain translocated to
the C terminus does not prevent constitutive activity. However, because
PKA increased Po (Fig. 5, A and
B), the translocated, phosphorylated R domain retained its
stimulatory function.

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Fig. 5.
Single-channel analysis of CFTR with the R
domain translocated to the C terminus. A, recording
from an excised membrane patch containing two R-C(709-835)
channels. Dotted line indicates closed state (C) and
downward deflection indicates channel opening. The
upper panel shows activity in the presence of ATP (1 mM); lower panel shows activity after addition
of ATP and PKA (75 nM). Data were obtained at-80 mV. Single
channel conductance for the variants was: wild-type 11.5 ± 0.3 pS, 708-835 11.3 ± 0.3 pS, and R-C(709-835) 11.2 ± 0.3 pS (n = at least 3 for each). There was no
significant difference between these values. B, open state
probability (Po) of CFTR-S660A, 708-835, and
R-C(709-835), before and after addition of PKA. Data are mean ± S.E., n = 4. Asterisk indicates
significant difference compared with the presence of ATP alone.
Cross indicates significant difference in
Po obtained with ATP alone compared with
CFTR-S660A (p < 0.05).
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Effects of Smaller Portions of the R Domain Translocated to the C
Terminus--
Several studies have suggested that various portions of
the R domain might differentially influence activity. For example, mutation of the various phosphoserines produces qualitatively and
quantitatively different functional effects (13, 19, 21). Therefore, we
attached smaller portions of the R domain to the C terminus (Fig. 1);
this also reduced the number of phosphoserines. Fig.
6A shows that
R-C(760-835)
was constitutively active, that PKA increased current, and that
dephosphorylation with PP2C
decreased current.
R-C(709-759) also
showed constitutive and phosphorylation-stimulated activity (Fig.
6B). Thus both the first and second halves of the R domain
retained PKA-dependent stimulatory activity. However, all
three channels with R domain translocations were constitutively active
(Fig. 7). These results suggest that,
when translocated, the R domain loses the ability to prevent
constitutive activity.

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Fig. 6.
Current recordings from membrane patches
containing R-C(760-835) channels
(A) and R-C(709-759)
channels (B). ATP (1 mM), PKA (75 nM), and PP2C (0.1 unit/ml) were present during time
indicated by bars.
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Fig. 7.
Current from CFTR with portions of the R
domain translocated to the C terminus. Data are from membrane
patches containing multiple CFTR channels. Data were obtained in the
presence of ATP alone (shaded bars) or in the presence of
ATP plus PKA (black bars). Data are mean ± S.E.,
n = 7-10 for each variant. Asterisk
indicates significant increase following addition of PKA
(p < 0.05).
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Fig. 7 shows that all three C-terminal variants were regulated by
PKA-dependent phosphorylation. These results suggest that residues 709-759 and residues 760-835 both contributed to PKA-induced current in
R-C(709-835). As an additional test that it was serines 737, 795, and 813 that were responsible for PKA stimulation of
R-C(709-835), we mutated them to alanine (
R-C(709-835)/S3A, Fig. 1). Mutation of these three serines abolished
PKA-dependent stimulation (Fig. 7).
Current-Voltage Relationship of
R-C(709-835)--
Phosphorylation will change the charge of the R
domain. If the C-terminal R domain is influenced by the electrical
field across the membrane, then voltage might affect the activity of
R-C(709-835) channels. Like wild-type CFTR (22) and
708-835
(15), the current-voltage relationship of
R-C(709-835) was linear,
both when unphosphorylated and phosphorylated (Fig.
8). These results suggest that the
function of the C-terminal R domain is not affected by the membrane
electrical field.

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Fig. 8.
Current-voltage relationship of
R-C(709-835). Data were obtained in the
presence of ATP alone ( ) and after phosphorylation with PKA ( )
(n = 3).
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DISCUSSION |
Previous studies have suggested that in the unphosphorylated
state, the R domain may prevent constitutive activity, and when phosphorylated, it may stimulate channel activity. In the Introduction, we reviewed some of the evidence that supports these conclusions. In
this study, we investigated these functions by deleting portions of the
R domain and by translocating portions of the R domain to the C terminus.
An advantage of this study is that we were able to examine multiple
CFTR variants under well defined conditions, i.e. in excised inside-out patches of membrane with addition of ATP and PKA to the
cytosolic surface. Moreover, for the variants in which portions of the
R domain were translocated to the C terminus, the stoichiometry of
channels to R domains was fixed at 1:1. Conversely, a limitation of our
study is that the number of channels in a patch was not defined, and
for only a few constructs did we measure Po.
This limits our ability to compare activity between the various
constructs. However, our interpretation does not depend on such an analysis.
Prevention of Constitutive Activity by a Portion of the R
Domain--
Earlier studies showed that deleting residues 708-835
generated a constitutively active channel (9, 10, 15). Here we show
that the N- and C-terminal portions of the R domain (residues 708-759
or residues 784-835) could be deleted without producing constitutive
activity. However, residues 760-783 were critical for preventing
constitutive activity; when they were deleted, channels opened without
phosphorylation. These results suggest that a relatively small and
specific portion of the R domain may keep the unphosphorylated channel
closed. It is interesting that this small region did not contain a
serine that is phosphorylated in vivo (7, 8). Although
Ser-768 is contained within this region, mutating it to alanine in
either
708-759 or
784-835 was not sufficient to either produce
constitutive activity or to prevent phosphorylation-stimulated activity.
Two other studies each examined the effect of a single short deletion.
Vankeerberghen et al. (11) studied channels in whole-cell patches and found that deletion of residues 780-830 generated channels
that were closed under basal conditions but opened when phosphorylated.
This result is similar to our findings with
784-835. In contrast,
Xie et al. (12) studied channels in planar lipid bilayers
and reported that deleting residues 817-838 caused channels to be open
without phosphorylation. Despite the presence of serines 660, 737, 795, and 813, they found that phosphorylation did not stimulate activity of
this channel. The reason for the differences between these studies is
not clear, but they may relate to differences in methodology
(whole-cell and excised patches of membrane versus planar
bilayers) or to differences in the constructs studied (the construct
studied by Xie et al. (12) deleted three residues, 836-838,
that were not deleted in the other two studies).
Because the data described above suggested that a portion of the R
domain prevents constitutive activity, it seemed surprising that when
we translocated residues 709-835 or shorter portions of the R domain
to the C terminus, constitutive activity persisted. This was the case
even though the translocated C-terminal R domain constructs were able
to stimulate activity, suggesting that they had access to the rest of
the channel. These data are also consistent with earlier findings that
adding exogenous unphosphorylated portions of the R domain (residues
645-834, 590-858, or 708-831) did not close the
708-835 channel
(6, 10, 13).
It has been reported that an unphosphorylated R domain protein
(residues 590-858) inhibits activity of wild-type CFTR studied in
lipid bilayers (23). However, the significance of this observation is
unclear based on the results of two different types of studies. First,
an isolated unphosphorylated R domain protein encompassing residues
645-834 did not reduce activity of wild-type CFTR studied in excised
membrane patches (13). Second, as indicated above, addition of
unphosphorylated R domain protein (either residues 645-834, 590-858,
or 708-831) did not reduce activity of CFTR in which residues 708-835
were deleted (6, 10, 13). Perhaps an explanation for the ability of
residues 590-858 to inhibit wild-type CFTR (23) is that portions of
the protein corresponding to NBD1 may have inhibited activity. Further
work is needed to understand the mechanism.
It is possible that R domain deletions might lead to a constitutively
active channel by inducing nonspecific structural changes. This might
explain why an R domain translocated to the C terminus did not close
the unphosphorylated channel. However, nonspecific structural changes
seem unlikely given our finding that much of the domain can be deleted
without causing the channel to open. The observation that the region
responsible for keeping the channel closed may be fairly small
(involving residues 760-783) suggests a more specific mechanism.
Future studies focused on this small region of the R domain may help
elucidate the molecular mechanism.
Phosphorylation-dependent Stimulation by Portions of
the R Domain--
Our data extend the earlier observations that
exogenous addition of phosphorylated R domain proteins, either residues
645-834, 590-858, or 708-831 (6, 10, 13), stimulated channel
activity. Here we found that the R domain retained its capacity for
phosphorylation-stimulated activity when it was moved from its normal
location to the C terminus. This ability to translocate a domain that
regulates activity from one place in a channel protein to another has
also been observed in the CIC2 channel (24). We also found that when we
split the R domain into halves (residues 709-759 and 760-835) and
attached them to the C terminus, PKA continued to stimulate the
channel. These data indicate that the R domain contains more than one
region that can stimulate activity; no one specific phosphoserine or unique sequence is required. It is also interesting that the regions responsible for phosphorylation-mediated stimulation may be different from those that prevent constitutive activity, perhaps suggesting different mechanisms. This suggests that the mechanisms that prevent constitutive activity may differ from those by which phosphorylation stimulates activity.
A Model of R Domain Structure--
We recently showed that the R
domain is primarily random coil in solution (6). Our current work is
consistent with the conclusion that the R domain does not form a region
with a highly ordered tertiary structure. We found that it was not
necessary to add the entire R domain to stimulate activity; portions of the R domain (residues 709-759 and 760-835) attached to the C terminus stimulated activity on addition of PKA. Moreover, portions of
the R domain (residues 708-759, 708-783, and 760-835) remaining in
our deletion mutants retained the capacity to mediate
phosphorylation-stimulated activity. If the R domain had a well ordered
tertiary structure that was required for activity, it seems likely that
such alterations would disrupt its stimulatory activity. We speculate
that short regions of sequence around the phosphoserines interact with
and stimulate the rest of the channel. In addition, it is possible that
sequences within the R domain associate with the rest of CFTR and in so
doing adopt local regions of ordered tertiary structure. Recent data
also suggest that an interaction between the R domain (residues
708-835) and the N terminus of CFTR has a stimulatory effect (25). It
is possible that some or all of the variants we constructed retain such
an interaction. However, if this interaction is required for
stimulation, the data suggest that the N terminus can interact with
multiple regions within the R domain.
A domain that is predominantly random coil is also consistent with our
data on constitutive activity. We found that despite deletions of large
portions of the R domain, the unphosphorylated channels remained
closed. Only when residues 760-783 were missing did unphosphorylated
channels open. These observations, pointing to a small region within a
larger domain, can be explained by an R domain that exhibits
considerable flexibility, although they do not exclude an R domain that
folds into a well ordered tertiary structure.
These data and the observation that the R domain is composed
predominantly of random coil are both consistent with a model in which
net phosphorylation-induced channel activity reflects the integrated
effect of interactions with multiple sites in the R domain. In this
way, graded phosphorylation could generate graded Cl
channel activity to control precisely transepithelial Cl
transport. Further understanding these interactions and identification of specific R domain binding sites in CFTR may yield additional insights into the complex regulation of CFTR.