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INTRODUCTION |
Of the 1480 amino acids in human cystic fibrosis transmembrane
conductance regulator
(CFTR),1 only ~59 are
predicted to be exposed at the exterior surface of the cell membrane
(1). These few residues are distributed among six extracytoplasmic
loops that join successive pairs of membrane-spanning sequences (TMs).
Most of these putative loops are extremely short; all except the first
(EL1) and the fourth (EL4) are five residues or less in length (1). EL1
and EL4 are only 15 and 31 residues in length, respectively,
with two N-linked oligosaccharide chains attached to the
latter. This relative lack of exposure to the cell exterior is not
unexpected because the CFTR ion channel is not regulated from this side
of the membrane but from the cytoplasmic side where most of the mass of
the protein resides. In addition to the large nucleotide binding
domains and R-domain even the cytoplasmic loops joining the TMs with
lengths of ~60 residues are much longer than the ELs. Hence little
attention has been paid to these short external sequences in studies of the structure-function relationships of CFTR. However, one relatively common missense mutation in EL1, R117H, was shown earlier to reduce chloride conductance (2). Subsequently several other disease-associated single residue substitutions in the ELs have been reported (CF Genetic
Analysis Consortium). To determine what influence they may have
on the biosynthetic processing and function of the molecule that might
lead to the disease phenotype, we have reconstructed these mutants in
an expression plasmid and established them in stable cell lines. In
contrast to 18 of 30 disease-associated missense mutations that were
found to prevent maturation (3-5), none of the 13 EL mutations
examined in this study had that effect. Instead, they had strong
effects on the stability of the CFTR Cl
channel.
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MATERIALS AND METHODS |
Cell Culture and Stable Expression of CFTR--
The BHK cells
expressing wild-type CFTR have been described in our earlier
publications (6, 7). The mutated CFTR sequences described below in the
pNUT expression plasmids were employed to establish stably transfected
BHK cell lines by the same methods.
In Vitro Mutagenesis of CFTR cDNA--
The human CFTR
cDNA had been inserted previously into the pNUT expression vector
(6) and the Bluescript plasmid (Stratagene). CF-associated point
mutations were reconstructed in the Bluescript cloning vector
containing the CFTR cDNA using the Quick Exchange kit from
Stratagene with the oligonucleotide primers listed in Table I.
To allow efficient stable transfection, the mutated fragments
were transferred to pNUT-CFTR, utilizing
XmaI and DraIII for mutations in EL1 and EL2 or
the two endogenous DraIII restriction sites of CFTR for
mutations in EL4 and EL5. The correct sequence of the inserted
fragments was confirmed by sequencing.
Metabolic Labeling with [35S]Methionine--
After
a 30-min starvation of methionine, cells were labeled metabolically
with [35S]methionine for 30 min as described in Loo
et al. (8). For the following chase, the
[35S]methionine-containing medium was replaced by
complete medium containing 5% fetal bovine serum and 1 mM methionine.
Surface Labeling of Cells--
Cells were grown to confluency in
10-cm plates. Plates were placed on ice for 30 min and then washed
twice with 10 ml of ice-cold PBS containing 0.1 mM
CaCl2 and 1 mM MgCl2. All
subsequent steps were performed at 4 °C. Surface proteins were
labeled by incubation for 30 min in 1 ml of PBS, pH 8.0, containing 1 mg/ml sulfo-NHS-SS-biotin (Pierce). Plates were washed four
times with PBS containing 1% bovine serum albumin and four times with
PBS. Cells were solubilized in 1 ml of Nonidet P-40 lysis buffer as
described below for immunoprecipitation. After centrifugation the
supernatant was transferred to fresh centrifuge tubes containing
50 µl of prewashed streptavidin-agarose beads (Pierce) and incubated
overnight with gentle mixing. Beads were washed four times with
radioimmune precipitation buffer (1% Triton X-100, 1% deoxycholic
acid, 0.1% sodium lauryl sulfate, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4) and eluted with 50 µl of 2×
sample buffer. After SDS-polyacrylamide gel electrophoresis (6%
acrylamide) and transfer to nitrocellulose, Western blots were probed
with M3A7 (19).
Cell Lysis and Immunoprecipitation--
Cells were lysed in
Nonidet P-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 20 mM NaMoO4, and 0.09%
Nonidet P-40 plus a mixture of protease inhibitors (E64 (3.5 µg/ml),
benzamidine (100 µg/ml), aprotinin (5 µg/ml), leupeptin (10 µg/ml), and Pefabloc (50 µg/ml)). After centrifugation at 4 °C
at 15,000 × g for 15 min, the supernatant was
incubated overnight with the monoclonal antibody M3A7 followed by a 4-h
incubation with Protein G-agarose (Life Technologies, Inc.). After four
washings with radioimmune precipitation buffer, attached complexes were
dissolved in electrophoresis sample buffer.
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Total cell lysates, streptavidin precipitates, or
immunoprecipitates were separated on 6% acrylamide gels. For Western
blotting, proteins were transferred to nitrocellulose (Bio-Rad) and
probed with M3A7 as primary antibody and detected by enhanced
chemiluminescence (Pierce). After electrophoresis of radiolabeled
immunoprecipitates, gels were analyzed by fluorography and electronic
autoradiography using a Packard Instrument Co. Instant Imager.
36Cl
Efflux Assay--
Cells were
grown to confluency in 6-well culture dishes. After two washes with
efflux buffer (20 mM HEPES, pH 7.4, 11 mM
glucose, 2 mM Ca2NO3, 2 mM Mg2NO3, 2 mM
KNO3, 135 mM NaNO3), cells were loaded for 1 h at room temperature with 0.5 ml of efflux
buffer containing 1 µCi of 36Cl
. Loaded
cells were washed three times with 1 ml of efflux buffer at 1-min
intervals. 0.5-ml samples were collected into 24-well Topcount plates
and replaced with the same volume of efflux buffer at 1-min intervals.
Starting at time 0, the efflux buffer contained 10 µM
forskolin, 1 mM isobutylmethylxanthine, 100 µM dibutyryl cAMP to stimulate CFTR activity. To each
sample 1 ml of Microscint scintillation fluid (Packard Instrument Co.)
was added. Samples were analyzed in a Topcount scintillation counter
(Packard Instrument Co.).
Single Channel Records--
Microsomal membrane vesicles were
isolated from BHK cells expressing the different CFTR variants and
phosphorylated with protein kinase A as described previously (9). CFTR
single channels were incorporated into the lipid bilayer by fusion of
the microsomes with a preformed planar bilayer. Single channel records
were collected in a buffer containing 300 mM Tris-HCl, pH
7.2, 1 mM EGTA, 3 mM MgCl2 on both
sides of the bilayer. 2 mM Na2ATP was present
also on the cis side. Single channel currents were recorded using an Axopatch 200A amplifier (Axon Instruments).
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RESULTS |
Expression of Disease-associated EL Variants--
Fig.
1A sketches the positions of
the EL substitutions. Although most are in EL1, three other loops are
represented also. One mutation reported in a patient in EL6, G1127E,
was not expressed. Fig. 1B indicates that although there is
considerable variability in the level of protein expression among
representative clones of each of the CFTR variants, they are all
expressed and mature. In fact, the ratios of the intensities of the
larger mature bands to the smaller immature bands are relatively
similar to that of the wild type. Confirmation that the mature
molecules that have acquired complex oligosaccharide chains are
transported to the cell surface is provided by their labeling with a
membrane-impermeant amino-reactive biotinylation reagent from the
exterior (Fig. 1C). The immature core-glycosylated forms are
not labeled because they remain intracellular. The relative intensities
of the surface labeled bands (Fig. 1C) are generally
proportional to those of the Western blots (Fig. 1B).
However, in the case of E116K the surface signal is disproportionally
weaker relative to the immunoblot indicating that it could be somewhat
impeded in transport to the cell surface.

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Fig. 1.
Disease-associated EL mutations are
expressed, mature, and reach the cell surface. A, CFTR
was drawn according to Ref. 1. Mutated residues in the enlarged ELs are
depicted as shaded circles. Each mutation is designated as
the original amino acid residue (first letter), its location
(number), and the amino acid to which it was changed
(second letter). NBF, nucleotide binding fold;
R, regulatory domain. B, cells stably expressing
wild-type and the different CFTR variants as well as untransfected host
BHK cells were lysed in sample buffer. 15-µg amounts of total protein
were analyzed by Western blotting using M3A7 as the primary antibody
(19). C, cell surface proteins were covalently labeled with
biotin. After cell lysis, biotinylated proteins were precipitated with
streptavidin beads (Pierce), separated by SDS-polyacrylamide gel
electrophoresis (5%), and blotted as described above.
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The detection of the mature form of each of these variants in
Western blots and at the cell surface showed that biosynthesis and
intracellular transport occurred. To better evaluate how efficiently this occurred, pulse-chase experiments were performed (Fig.
2). As widely demonstrated previously
(10) conversion of the wild-type core-glycosylated precursor to the
mature complex-glycosylated product reached a maximum of ~40% after
~2 h of chase. After that, the mature protein decays with a
t1/2 between 15 and 20 h. The efficiency of
conversion for most of the EL variants seems further reduced so that
the maximum proportion of [35S]methionine incorporated
into the precursor during the pulse, which is detected in the
mature form, is as low as 0.2. Exceptions are S108F, which matures
as efficiently as the wild type, and T908N, which matures even more
efficiently. Although the processing of the nascent forms of most of
these mutants occurs less well than the wild type, the turnover of the
mature form is little changed and hence a reasonable steady-state level
is achieved reflecting largely the amount that does mature.

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Fig. 2.
Biosynthesis and stability of EL variants as
assessed in pulse-chase experiments. Cells were labeled
metabolically with 100 µCi of [35S]methionine for 20 min. After this pulse, the [35S]methionine-containing
medium was replaced by complete medium supplemented with 1 mM methionine and chased for the times indicated above each
film image. 35S radioactivity in the same dried
gels was quantified by electronic autoradiography using a Packard
Instrument Co. Instant Imager and plotted relative to the amount in the
immature band after the pulse.
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36Cl Efflux from Cells Expressing EL Variants--
To
gain some initial information on how these mutations influence the
chloride channel activity of CFTR, cells were loaded with tracer
36Cl
, and its efflux followed after
stimulation to increase cAMP levels. The rates of efflux at times after
stimulation are plotted in Fig. 3.
Although appearing to be generally reduced compared with the wild type,
there were substantial rates of efflux from cells expressing all of
these variants. Thus it was feasible to undertake studies of the single
channel properties of each as described below. As far as the comparison
of the rates of 36Cl efflux goes, it has to be kept in mind
that the amount of CFTR protein in each clonal cell line is somewhat
variable. However, some significant changes are apparent. For example,
E217G in EL2 reduces the efflux rate greatly. On the other hand, P1013L
in EL5 exhibits rates as high as wild type.

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Fig. 3.
36Cl efflux form cells expressing
wild-type and CF-associated mutated CFTR variants. Cells were
incubated with loading buffer containing 1 µCi of
36Cl for 1 h at room temperature. The
cells were washed three times with chloride-free efflux buffer. From
time 0 the efflux buffer contained 10 µM forskolin, 1 mM isobutylmethylxanthine, and 100 µM
dibutyryl cyclic AMP. The efflux of 36Cl was determined
with a Topcount scintillation counter (Packard Instrument Co.). Each
point is the average of three independent samples. A,
squares, wild type; circles, S108F;
triangles, Y109C; diamonds, D110H;
crosses, wild type without stimulation. B,
squares, P111A; circles, P111L;
triangles, E116K. C, squares, R117C;
circles, R117H; triangles, R117L;
diamonds, R117P. D, squares, E217G;
circles, T908N; triangles, P1013L.
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Single Channel Properties of EL--
Fig.
4 contains representative recordings of
single channels in planar lipid bilayers with which membrane vesicles
isolated from cells expressing each of the variants and the wild type
were fused. Amplitude histograms accompany each tracing. Most of the salient features of the wild-type CFTR channel are illustrated in the
first tracing shown. At this temperature of 21 °C with 300 mM Cl
on both sides of the bilayer, the
principal mode of the wild-type channel exhibits two well defined
conductance levels of 10.1 and 11.5 pS similar to those described by
Gunderson and Kopito (11).

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Fig. 4.
Single channel kinetics of different
CF-associated CFTR variants. Phosphorylated CFTR single channels
were incorporated into lipid bilayers by fusion of the microsomes with
a preformed planar bilayer. Representative current traces were obtained
at 21 °C and amplitude histograms are shown for each. Time scales
are indicated by the horizontal bars.
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The behavior of the most N-terminal mutant in EL1, S108F, indicates
that substitution of the small hydroxyl amino acid with the larger
aromatic phenylalanine results in a channel with no stable open state.
Channel openings are detected but these are extremely brief. No peak
value for current through the open channel can be discerned in the
all-points histogram. Simply put, this mutant channel attempts frequent
opening, but the open state cannot be maintained suggesting that this
residue or the region of EL1 it occupies is crucial to the stability of
the open-state structure. If a parameter I equal to
the mean current through a single channel at
75 mV is used to
represent the total charge transported by the channel per unit of time,
one can employ a ratio of that value for the mutant to that of the wild
type to obtain a relative measure of the charge transport ability of
the mutant compared with the wild type. The value of the parameter
I is derived from the area under the channel openings
divided by the time interval. As indicated in Table
II, the
IS108F/IWT is
~11%.
The substitution of the aromatic tyrosine in the adjacent position by
the small thiol residue (Y109C) also results in a very unstable open
state, but the tracing is different from that of S108F. There is a
burstlike behavior but within the burst there are rapid closings. This
channel is capable of gating but there is large fluctuation in the
open-state structure. The mean current carried at
75 mV is
~15% of the wild type (Table II). Replacing the aspartate residue in
the next position by histidine (D110H) resulted in a much more normal
looking channel with a major mean conductance of about 8.5 pS. Although
a minor peak for the channel open state is detectable on the all-points
histogram, the duration of the openings is much less than those of the
wild type. Hence like the mutants at the previous two residues,
instability of the open state is the most pronounced feature of this channel.
Two different mutations found in patients at the next position were
analyzed at the single channel level. P111L appears very much like wild
type with a mean conductance of 10.3 pS. The Po was 0.45 with a main mean open time of 500 ms and closed time of 600 ms. There is no indication that this variant has any difficulty in
maintaining its open-state structure at this temperature (21 °C).
However, a marked difference in its behavior is revealed at higher
temperature (35 °C; Fig. 5) at which
its Po is reduced to about half that of the wild
type. Substitution of the proline with the smaller aliphatic alanine
residue (P111A) produced a channel with much more rapid kinetics and a
reduced conductance of 9.2 pS even at 21 °C (Fig. 4).

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Fig. 5.
Gating of wild-type and EL mutant channels at
35 °C. Those variants showing minimal
differences from the wild type at 21 °C were analyzed at
35 °C.
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The mutation five residues further in the C-terminal direction resulted
in a charge reversal (E116K). This mutant displays properties quite
similar to S108F; channel openings occur but cannot be maintained. No
defined peak is apparent for openings in the all-points histogram.
The R117H mutant was reported previously to exhibit a small reduction
in both current amplitude and open time (2). Our observations confirm a
conductance decrease of 1.5 pS in both of the two conductances observed
in the wild type. Otherwise the kinetic behavior at this temperature
was not very different from wild type. In fact we found this
substitution at Arg-117 to have far less effect than the other three
disease-associated replacements at this position. Both R117C and R117L
had very unstable open states like S108F and E116K with the cysteine
substitution able to maintain openings slightly longer than the leucine
substitution. R117P also displayed only transient openings. Thus the
more frequent R117H mutation seems to compromise open-channel structure
less than the other three substitutions at this position. This may be
because of the structural similarity of the arginine and histidine residues not shared by the other three substituting residues. Overall
the set of EL1 mutations examined points to an involvement of this loop
in the formation of a stable pore structure.
Strikingly, the single missense mutation (E217G) in the short EL2 also
results in channels with only transient rather than stable openings,
perhaps implying that this loop is involved also in stabilizing open
pore structure.
The T908N mutation in EL4, the second of the two larger
extracytoplasmic loops of CFTR, has a somewhat similar effect on the single channel behavior as those in EL1 that cause a very unstable open
state. Cells expressing this variant in the large glycosylated loop at
the beginning of the second half of the molecule display channels with
a noisy open state and a mean conductance of ~9.2 pS. This behavior
is distinct from the EL1 and EL2 variants in that the openings are not
as extremely transient. As seen in the T908N trace, there are in fact
relatively long openings with several brief closings within to generate
burstlike behavior. An intraburst mean open time of ~90 ms and a mean
burst duration of ~200 ms can be derived. The brief closings appeared
as intraburst closings with a time constant of ~15 ms and intraburst
time constant of ~800 ms. The overall open probability is 0.14. The
mean conductance is just a little less than the major wild-type
conductance. This mutant was studied also at the higher temperature of
35 °C (Fig. 5) at which its difference from the wild type was more
apparent in the current tracing.
The final disease-associated mutant analyzed, P1013L, in the fifth
exterior loop exhibited robust wild-type-like behavior, and consistent
with its 36Cl efflux ability, it has a
Po of 0.5, higher than the wild type. However,
because the gating of some of the other less severely impaired mutants
became more obvious at a higher temperature, this variant was assessed
also at 35 °C (Fig. 5). The conductance remained unaltered but the
Po was reduced to approximately half of that of
the wild type. Thus although replacement of this hydroxyl amino acid
does not destabilize the channel to the extent of several of the other
mutations, it does have some impact.
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DISCUSSION |
Although only a relatively small proportion of the large number of
mutant versions of CFTR detected in CF patients have been expressed in
heterologous systems, the analysis of those that have has been
informative in several ways (for reviews see Refs. 10, 14, and 16). Of
greatest impact was the finding that the
F508 mutation that is
present on at least one allele in 90% of patients caused defective
biosynthetic maturation (12). A considerable number of missense
mutations has a similar consequence (13). Other single residue
substitutions alter specific properties of the CFTR ion channel (14).
For example a nucleotide binding domain mutation, G551D, precludes
virtually all activity and R-domain mutations have a variety of effects
(15). Substitutions in membrane-spanning sequences may alter
conductance, selectivity, occupancy, or gating of the pore (16).
Missense mutations changing residues in the cytoplasmic loops
separating the transmembrane helices have relatively minor effects on
channel gating and regulation, but the majority of them prevent normal
processing and maturation of the polypeptide, as does
F508 (13). Of
missense mutations identified in codons for residues in
extracytoplasmic loops, only R117H, which occurs relatively frequently
in patients, has been studied in any detail (2). It was found to mature
when heterologously expressed but the whole-cell current it generated
was much reduced compared with wild type. Single channel conductance
was diminished only a small amount but open probability was reduced
because of altered gating. Our observations are generally in agreement
with those, although the replacement of the large positively charged
arginine residue by histidine had much less effect on gating than the
other substitutions at this position. Each of those three drastically altered gating. This effect was most extreme with the hydrophobic leucine residue at this location precisely at the junction of EL1 and
TM2. In this case, only extremely brief openings were detected. These
were only slightly longer when either a cysteine or a proline was in
this position.
The charge-reversal mutation, E116K, at the immediately preceding
position had a remarkably similar effect to that of the most perturbing
substitutions of Arg-117. Hence these two contiguous oppositely charged
amino acids seem essential for maintenance of the open state. Although
prolines often are considered crucial structure-determining residues,
replacement of Pro-111 had less impact than some of the other changes.
The P111L variant in contrast to all the other EL1 mutants differed
from wild type only at elevated temperatures. The substitutions at
residues 110, 109, and 108 were similar to Glu-116 and Glu-117 changes
in causing a very unstable open state. The E217G mutant in EL2 had a
similar effect. Hence the overwhelming consequence of
disease-associated mutations in EL1 and EL2 is destabilization of the
CFTR channel open state.
The T908N mutation in EL4, which is novel in that it results in the
introduction of an additional consensus site for
N-glycosylation that is used (17), also alters channel
gating. Thus although the P1013L change in EL5 did not seem to
compromise channel activity except at higher temperatures, all of the
others analyzed seriously detracted from the ability of CFTR to sustain
a stable open pore. This suggests that these short loops that link the
extracellular ends of the TMs that are believed to form the pore may be
crucially involved in maintaining their precise orientation or
relationship to each other. The restraint on the TMs by these short
loops, in contrast to the freedom that the much longer cytoplasmic
loops may allow, conjures up an image somewhat different from that
provided by the low resolution three-dimensional structural image of
P-glycoprotein (18). In that case, the funnel-like formation suggested
was narrow on the cytoplasmic side and wide on the exterior face of the
membrane. However, because there are as yet not even low resolution images of CFTR, extrapolation of the effects of mutagenesis on function
to structural interpretations may be misleading.
Nevertheless, analysis of this set of disease-associated mutations in
the extracytoplasmic loops has provided interesting results, some of
which might not have been expected. The ELs are the only parts of the
protein in which none of the mutations studied had strong effects on
biosynthetic processing, suggesting that recognition of mutant CFTR by
endoplasmic reticulum quality control occurs primarily on the
cytoplasmic rather than the luminal face of the endoplasmic reticulum
membrane. Second, none of the mutations prevent channel opening, but
most of them preclude formation of a stable open-state structure
implying that the ELs may play an essential role in this function.