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INTRODUCTION |
The cystic fibrosis transmembrane conductance regulator
(CFTR)1 protein is a
cAMP-regulated chloride channel located in the apical membrane of
epithelial cells. CFTR is predicted to consist of twelve putative
membrane-spanning segments (TM), two nucleotide-binding domains, a
regulatory domain, and four cytoplasmic loops (CL) connecting the TMs
on the cytoplasmic side of the protein (1). Mutations in the
CFTR gene cause cystic fibrosis (CF), the most common
genetic disease in Caucasians.
The number of complex CFTR genotypes identified, including
double-mutant alleles where two missense mutations are carried by the
same chromosome, has been growing (2-6). It is difficult to evaluate
the contribution of each mutation/or polymorphism to the phenotype as
mutations in cis may act in concert to alter or reverse
defective CFTR function and thus modify the CF phenotype (2).
The recent discovery of severe CF associated with a
F508/R347H-D979A
compound heterozygote genotype in two related patients suffering from
pancreatic insufficiency and severe respiratory symptoms suggests that
the R347H-D979A mutation has an important influence on CFTR processing
and/or function (7). At least four CF-associated mutations have been
identified in isolation at position 347 (R347C, R347H, R347L, and
R347P) and two at position 979 (D979A and D979V), suggesting that
Arg-347 and Asp-979 are important for CFTR structure and/or function.
The mutation D979A was found in isolation in a patient with a
congenital bilateral absence of the vas deferens (8) and the R347H
mutation in CF patients with pancreatic sufficiency, congenital
bilateral absence of the vas deferens, and no or mild pulmonary
symptoms (7). As the R347H mutation is mostly associated with mild CF,
it was suggested that the D979A mutation has a significant effect on CFTR function when combined in cis with R347H. Arg-347 lies
within TM6 and is believed to line the pore (9, 10), whereas Asp-979 is
located in CL3 connecting TM8 and TM9 in the C-terminal half of CFTR.
The mutations R347H and D979A replace positively charged (Arg) and
negatively charged (Asp) residues with ones that are uncharged (His and
Ala, respectively) at physiological pH.
The present study investigates the structure-function relationships of
the R347H-D979A double mutant, and as charged residues are involved in
this complex genotype, single and double mutants with different charge
combinations at residues 347 and 979 were constructed. All these
mutants were transiently expressed in HeLa cells, and we analyzed CFTR
processing by immunoprecipitation and chloride channel activity using
the whole-cell patch-clamp technique.
Our data show that R347H is associated with defective chloride channel
activity and that the D979A defect leads to misprocessing. The mutant
R347H-D979A combines both defects for a dramatic decrease in
Cl
current. These studies also revealed that residue 979 is a critical location for both CFTR processing and Cl
channel activity.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis, Cells, and CFTR Expression--
CFTR
mutants were constructed in the expression plasmid pTCFwt, a vector
designed for the visual detection of transfected mammalian cells by the
green fluorescent protein (11), using the GeneEditor kit (Promega).
HeLa cells (7.5 × 105) were grown on 60-mm diameter
dishes at 37 °C with 5% CO2 in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum, 100 units/ml
penicillin, and 100 g/ml streptomycin (all from Life Technologies,
Inc.). Confluent cells (60%) were transfected by lipofection using 12 µl of LipofectAMINE and 8 µl of Plus reagent (Life Technologies,
Inc.) with 2 µg of plasmid according to the manufacturer's
instructions. Confluent monolayers were harvested and used 48 h
post-transfection for functional assays or immunoprecipitation.
Immunoprecipitation/cAMP-dependent Protein
Kinase Assay and Pulse-Chase Experiments--
The CFTR protein
immunoprecipitation using the monoclonal antibody (mAb) 24-1 (R&D
System), which recognizes the C terminus of CFTR, was already described
(12). Briefly, cell lysates were mixed with 0.4 µg of mAb 24-1 and
Pansorbin (Calbiochem). The resulting proteins were
phosphorylated in vitro with 5 units of the catalytic
subunit of cAMP-dependent protein kinase (Promega) and 10 µCi of [
-33P]ATP (Amersham Pharmacia Biotech),
separated by 5% SDS polyacrylamide gel electrophoresis, dried, and
autoradiographed. Radioactivity was quantitated by radioanalytic
scanning (using a Molecular Dynamics PhosphoImager).
Pulse-chase experiments were performed by incubating cells for 30 min
in Dulbecco's modified Eagle's medium lacking cysteine and methionine
and then for 15 min in the same medium containing 100 µCi/ml
[35S]-labeled methionine and [35S]-labeled
cysteine (Redivue Pro-mixTM [35S];
Amersham Pharmacia Biotech). CFTR was immunoprecipitated from cell
homogenates with the same mAb 24-1.
Electrophysiology--
Whole-cell patch-clamp recordings were
performed at room temperature (20-25 °C) on isolated cells, 24 h after trypsinization and replating on plastic dishes at a low
density. Before recording, the culture medium was replaced by the
external solution to be used during the recording. This solution
contained the following (in mM): 140 NaCl, 2 CaCl2, 1 MgCl2, 10 Hepes, 10 glucose, and 25 sucrose; its final pH value was adjusted to 7.3 with NaOH. Patch-clamp
micropipettes were made from hard glass (Kimax 51); their shank was
covered with Sylgard, and their tip was fire-polished. They were filled
with an internal solution containing the following (in mM):
30 CsCl, 113 NaCl, 10 Hepes, 1 MgCl2, and 0.5 EGTA. In some
experiments the Cl
concentration of bath and pipette
solutions was reduced to 32 mM by equimolar replacement
with glutamate. The cells were voltage-clamped by an EPC7 List
amplifier, controlled by a TANDON 38620 computer, via a Cambridge
Electronic Design 1401 interface, using patch- and voltage-clamp
software. The current monitor output of the amplifier was filtered at
0.5 kHz before being sampled on-line at 1 kHz, as described previously
(13). The bath was connected to the ground via an agar bridge. The zero
indicated on current traces and current plots is the absolute zero
current level. To establish I-V curves of the response, a series of 9 voltage jumps (of 1-s duration each, separated by 10 s) of
incrementing amplitude was applied from the holding potential (0 mV)
toward a variable membrane potential between
80 and +80 mV.
CFTR Cl
currents were activated with 200 µM
8-(4-chlorophenylthio)-cAMP sodium salt (CPT-cAMP).
Statistics--
Results are expressed as the means ± S.E.
of n observations. We used the Student's t test
to compare mean values. Differences were considered statistically
significant when the p value was <0.05.
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RESULTS |
Processing of CFTR Mutants--
Many CF-associated mutations cause
a loss of CFTR Cl
channel function because of
misprocessing of the mutant protein so that it does not reach the cell
membrane. We first studied the maturation of CFTR in HeLa cells to
determine why patients with the R347H-D979A-CFTR allele suffered from
severe CF. HeLa cells were transiently transfected with cDNA encoding
the wild-type and mutated CFTR proteins. The processing of CFTR can be
assessed by examining its glycosylation. In our experimental
conditions, electrophoresis of immunoprecipitated wild-type CFTR gave
two bands (Fig. 1A). One was a
diffuse band of an approximate molecular mass of 170 kDa (band
C) that represented mature, fully glycosylated protein that has
migrated through the Golgi complex to the cell membrane. The second was
a thin band of about 140 kDa (band B) that represented the
core-glycosylated protein located in the endoplasmic reticulum.

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Fig. 1.
Production of mature protein by wild-type and
mutants assessed by CFTR glycosylation states. A,
typical immunoprecipitation of cAMP-dependent protein
kinase-phosphorylated (with [ -33P]ATP) CFTR proteins
using mAb 24-1. Bands B and C are indicated by
arrows. WT, wild-type. B, quantitation
of CFTR maturation efficiency calculated as the amount of mature CFTR
(%C) relative to the total amount of CFTR produced
(B+C). Radioactivity was quantitated by radioanalytic
scanning. Data are the means ± S.E. of at least three independent
experiments. *, significantly different from wild-type (WT;
p < 0.01). C, pulse-chase experiments
showing the turnover of the immature and mature forms of wild-type and
R347H-D979A-CFTR (results are representative of two independent
experiments).
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Immunoprecipitation experiments show that both wild-type and
CF-associated mutants R347H, D979A, and R347H-D979A-CFTR cells produced
mature, fully glycosylated protein (Fig. 1A; band
C), whereas none of the mock-transfected cells produced CFTR (data not shown). However, the D979A and R347H-D979A mutants produced significantly less band C (59 and 56% of the total CFTR; ratio C/(B+C)) than wild-type and R347H-CFTR (92 and 91%; see Fig.
1B). This indicates that the R347H-D979A mutation caused a
misprocessing defect. The turnover of immature and mature forms of
wild-type and R347H-D979A-CFTR proteins was further investigated by
pulse-chase experiments (Fig. 1C). The kinetics of wild-type
and R347H-D979A core-glycosylated and mature forms of CFTR were
identical, whereas the efficiency of conversion to mature band C was
lower for the mutant. Similar results were obtained with the D979A
mutant (data not shown). This indicates a defect in the biosynthetic
pathway, which accounts for the decreased amount of band C observed in the steady-state measurements.
Other mutants were generated to further characterize the D979A defect.
Asp-979 was changed to Val (small hydrophobic residue; D979V), Arg
(positively charged residue; D979R), or Glu (negatively charged
residue; D979E). D979V (also a naturally occurring mutant) and D979R
had impaired processing similar to D979A, whereas D979E permitted the
complete maturation of the protein (Fig. 1B).
Altogether, these results indicate that D979A is responsible for the
defective processing of R347H-D979A-CFTR and that a negative charge at
979 residue is necessary for proper CFTR processing.
Cl
Channel Function of CFTR Mutants--
We tested
the cAMP-activated chloride channel activity of transfected HeLa cells
using the whole-cell patch-clamp technique. None of the
mock-transfected cells displayed a Cl
current in either
basal or cAMP-stimulated conditions (data not shown). By using 140 mM Cl
(symmetrically) and 200 µM cAMP agonist, cells expressing wild-type CFTR produced
saturating whole-cell currents for clamp potentials above/below ± 40 mV. We therefore reduced Cl
(32 mM
symmetrically by replacement with glutamate) and cAMP agonist (50 µM) to characterize wild-type CFTR current. As shown in
Fig. 2A, there was little or
no current under basal conditions, and application of CPT-cAMP
activated large currents that reversed at the Cl
equilibrium potential (close to 0 mV with the solutions used). The
currents were glibenclamide-sensitive and DIDS- and
TS-TM-calix[4]arene-insensitive as expected for CFTR
Cl
current (data not shown). These currents were
activated a few seconds after stimulation and were slowly reversible
after washout of CPT-cAMP. The current-voltage relationship exhibited a
slight outward rectification (Fig. 2B), as described when
internal Cl
were replaced by large impermeant anions
(14).

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Fig. 2.
Whole-cell current properties of naturally
occurring CFTR proteins. A, representative traces of
whole-cell currents from HeLa cells transiently expressing the
wild-type CFTR before (control) and after stimulation with
50 µM CPT-cAMP. The voltage protocol used is detailed
under "Experimental Procedures." B, corresponding
current-voltage (I-V) curve before (control) and
after stimulation with 50 µM CPT-cAMP in HeLa cells
expressing wild-type CFTR. C, current-voltage
(I-V) curve in HeLa cells expressing R347H (filled
circle), D979A (filled triangle), and R347H-D979A
(filled square) mutants. Whole-cell currents were calculated
as the difference between steady-state current amplitude after CFTR
stimulation with 200 µM CPT-cAMP and baseline current
amplitude. All values were normalized for cellular capacitance.
D, comparison of the cAMP-stimulated whole-cell currents in
HeLa cells expressing wild-type (WT) and mutated CFTR.
Whole-cell currents were measured at 20 mV and normalized for cellular
capacitance. Bars represent mean change (± S.E.) in
whole-cell Cl current calculated as the difference
between steady-state current amplitude after CFTR stimulation and basal
current amplitude. The number of cells analyzed is indicated. Mean cell
capacitance was 37.4 ± 9.9 pF (n = 42). Data were
obtained with 32 mM Cl (symmetrically) in
A and B, and 140 mM Cl
(symmetrically) in C and D (see "Results" for
explanations).
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We separated the contributions of the R347H and D979A mutations to
R347H-D979A-CFTR whole-cell Cl
current production
studying both single and double mutants. To maximize our ability to
detect differences between mutants and to compare mutants with
wild-type CFTR, we used 140 mM Cl
and 200 µM cAMP agonist, and data were analyzed at 20 mV.
Current-voltage relationships of naturally occurring mutants are shown
in Fig. 2C. Mean changes in cAMP-activated currents recorded
at 20 mV from R347H (29.4 ± 12.2 pA/pF; n = 8),
D979A (67.9 ± 18.9 pA/pF; n = 5), and R347H-D979A
(1.2 ± 0.8 pA/pF; n = 9) mutants were significantly different (p < 0.05) from wild-type
(130.2 ± 34.7 pA/pF; n = 5), corresponding to 23, 52, and 1% of the wild-type Cl
current (Fig.
2D). R347H, D979A, and R347H-D979A were also significantly different from each other (p < 0.01).
As R347H processing is similar to wild-type, the small Cl
current produced by R347H reflected defective channel properties, as
demonstrated by single-channel studies (9). The decreased whole-cell
Cl
cAMP-dependent current observed with D979A probably
reflected CFTR protein misprocessing, although we cannot firmly exclude altered channel properties. Thus these data indicate that the R347H-D979A double mutant combined at least D979A misprocessing and the
R347H Cl
channel defect to produce a very severe phenotype.
Charge-reversal Mutants--
Taking into account the functional
defects that result when Arg-347 and Asp-979 are each replaced with an
uncharged amino acid such as His (uncharged at pH 7.3) and Ala (R347H
and D979A), we constructed additional mutants with different charge
combinations at residues 347 and 979, including the R347D-D979R double
mutant in which the positive and negative charges were swapped.
The processing of R347D was similar to those of R347H and the wild-type
(Fig. 3, open bars). Thus,
Arg-347 is not critical for processing, as the replacement of Arg by
His or Asp permitted complete maturation of the protein. Surprisingly,
the processing of D979R, R347H-D979R, and R347D-D979R was differently
impaired (Fig. 3; gray bars). These data indicate that when
residue 979 is positively charged, different CFTR processing efficiency
occurred depending on the charge at residue 347. This suggests that 347 residue could interact directly or indirectly with Arg-979 (D979R).

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Fig. 3.
Maturation of charge mutants at 347 and 979 residues. Data are obtained as in Fig. 1. Charge is indicated
in brackets for each amino acid residue. Data are the
means ± S.E. of at least three independent experiments. *,
significantly different from the other CFTR proteins studied
(p < 0.01).
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Whole-cell measurements indicated that D979R resulted in a much greater
decrease in chloride current (4.5 ± 2.4 pA/pF; n = 6) than D979A (Fig. 2D), although both proteins were
processed similarly. These data suggest that residue 979 is also
important for the chloride channel activity in transfected HeLa cells
and that the requirements for channel processing and function are different. The Cl
current of R347D-D979R (2.8 ± 1.7 pA/pF; n = 9) was not significantly different from
those of D979R and R347H-D979A (Fig. 2D). This result is
consistent with the poor amount of R347D-D979R protein at the cell surface.
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DISCUSSION |
Complex alleles have been described clinically (R553Q-
F508,
F508-V1212I, and R334W-R1158X), and most of them are considered to
reverse the phenotype, as they are associated with milder symptoms than
the most common mutation in isolation. However, as these complex
alleles are very rare, and epistatic or environmental factors may
contribute to the CF phenotype, in vitro studies are of
value to confirm the genotype-phenotype relationships and to understand
the functional defects causing the disease.
Clinical data suggested that R347H and D979A, two mild
CF-associated mutations, can produce severe CF similar to that of
F508 homozygotes when combined in cis. We have determined
the contribution of each mutant to the double-mutant phenotype. D979A
reduces the amount of CFTR protein at the cell membrane, whereas R347H
generates a defective Cl
channel. The mutant R347H-D979A
combines both defects for a dramatic decrease in Cl
current. The magnitude of Cl
current in vitro
paralleled the severity of the disease, with D979A (congenital
bilateral absence of the vas deferens) > R347H (mild CF) > R347H-D979A (severe CF).
Manavalan et al. (15) observed significant amino acid
homology and length conservation in the CL regions between CFTR and other transporter proteins. It was then suggested that CLs may be
important for channel function and/or the folding and processing of the
CFTR protein, and extensive studies of CF-associated mutations in the
four CLs have revealed channel properties and/or processing defects
(16-20). Our data strongly suggest that mutation D979R alters the
properties of the chloride channel, and mutational analysis of 979 residue also confirmed that the requirements for channel processing and
function are different, consistent with data for other residues (17,
18, 21). Many mutations in the CLs impair CFTR processing (16-20),
suggesting that the correct folding of CLs is important for proper
maturation of the whole molecule. Our data support this view, as
replacing Asp-979 by Ala, Val, or Arg significantly impaired
processing. Based on mutational analysis of 970 residue, Seibert
et al. (18) proposed that the "influence on folding may
depend more on the location of the altered residue than the specific
residue change." We observed that the negatively charged Asp-979
could be replaced only by the negatively charged Glu-979 without
affecting processing, indicating that a negative charge is important at
that location. Moreover, Asp-979 is highly conserved throughout CFTR
evolution (22, 23) and belongs to a consensus sequence shared with
other ABC transporters (15). These observations all indicate a crucial
role for Asp-979 and thus raised the following question: how might
removal of the negative charge at position 979 affect CFTR processing?
Little is known about how the CLs contribute to CFTR processing.
Considering the highly hydrophilic composition and length of CFTR CLs
(53-61 residues) (15), they could take part in physical interactions with other cytoplasmic domains of CFTR, solvent-accessible TM residues,
or cytoplasmic proteins. First, several lines of experimental evidence
indicate that there is probably no direct salt bridge between Arg-347
and Asp-979: (i) removal of either the positive charge at position 347 (R347H and R347D) or the negative charge at position 979 (D979A, D979V,
and D979R) has different effects on CFTR processing; (ii) the
double-neutral (R347H-D979A) and reversed-charged (R347D-D979R)
replacements for Arg-347 and Asp-979 do not lead to the recovery of
wild-type processing. Second, Asp-979 might interact with an as yet
unidentified positively charged residue located either in a cytoplasmic
part of CFTR or in a solvent-accessible part of a TM domain,
stabilizing CL3 at a distance from residue 347. Removal of the negative
charge at position 979 would disrupt this interaction and could direct
CL3 to residue 347 and thus allow a charge-dependent
interaction with residue 347. This is supported by the decrease in the
processing efficiency of D979R mutants combined with positive, neutral,
and negative charges at residue 347. Furthermore, a single charge can
form salt bridges with several opposite charges. Given the highly
hydrophilic nature of CLs, multiple ion pairs could maintain a
conformation of the CLs favorable to proper maturation of the protein,
which is supported by the multiple charge-disruption mutations in CLs
affecting CFTR processing (17-20). It may thus be difficult to
identify a single partner for Asp-979. However, interactions between
charges exposed on the protein surface may do little to stabilize
proteins, unlike buried ion pairs (24). Rather than being engaged in
intramolecular interactions, Asp-979 might interact with cytoplasmic
proteins. Several proteins have recently been shown to interact
directly with CFTR cytoplasmic domains (25-28). It would be
interesting to see whether Asp-979 participates in such binding.
In conclusion, this study highlights the importance of
structure-function analysis of naturally occurring mutants for
deciphering complex genotype and identifying residues important for
CFTR processing and/or chloride channel activity. These results also
have important implications for CF, as they show that two mutations
in cis can act in concert to alter dramatically CFTR
function. This may contribute to the wide phenotypic variability of CF
disease and points to the need to screen for all mutations.