From the * Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada; Department of Genetics, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G1X8; and § S.C. Johnson Medical Research Center, Mayo Clinic
Scottsdale, Scottsdale, Arizona 85259
Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel
to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using
the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (PX/PCl) sequence NO3 > Cl
> HCO3
> formate > acetate. The same selectivity sequence but
somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate,
propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06)
from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ~5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two
adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel
conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind
permeating anions, but may contribute to the narrowest part of the pore.
The cystic fibrosis transmembrane conductance regulator (CFTR)1 is a tightly regulated chloride channel that
mediates Cl transport across epithelia and is mutated
in cystic fibrosis (Welsh et al., 1992
; Riordan, 1993
;
Hanrahan et al., 1995
). The preceding paper examined halide permeability ratios under biionic conditions and described the unique behavior of I
. In this
paper, we examine permeation by polyatomic anions
having different dimensions and use the permeability
ratios obtained to estimate the functional diameter of
the pore.
Mutagenesis studies of CFTR selectivity (Anderson et
al., 1991; Tabcharani et al., 1997
), conductance (Sheppard et al., 1993
; Tabcharani et al., 1993
; McDonough
et al., 1994
), multi-ion pore behavior (Tabcharani et
al., 1993
), voltage-dependent block (Tabcharani et al.,
1993
; McDonough et al., 1994
; Linsdell and Hanrahan,
1996b
), and susceptibility to hydrophilic sulfhydryl reagents after cysteine substitution mutagenesis (Cheung
and Akabas, 1996
) suggest that the sixth membrane
spanning region of CFTR (TM6) lines the pore. All
TM6 mutants that have been characterized at the single
channel level have had conductances that are the same,
or lower than, that of wild-type CFTR (Sheppard et al.,
1993
; Tabcharani et al., 1993
; McDonough et al., 1994
).
Some of these low conductance mutations (R334W,
R347P, and R347H) occur in cystic fibrosis patients and have been associated with relatively mild disease symptoms. During a preliminary study of channels bearing
mutations of polar residues in TM6 that might disrupt
hydrogen bonding, we identified a mutant that had significantly higher conductance than wild-type CFTR. This mutant, with two threonine-to-alanine substitutions near the middle of TM6, was also found to have
elevated permeability to polyatomic anions, consistent
with an increase in the caliber of the narrowest region
of the pore. Preliminary reports of this work have appeared (Tabcharani and Hanrahan, 1993
; Linsdell et al., 1996
).
CFTR Mutagenesis and Expression
Chinese hamster ovary cells expressing wild-type CFTR were described previously (Tabcharani et al., 1991; Chang et al., 1993
; Tabcharani et al., 1997
). The TM6 double mutant (TT338,339AA; see
Fig. 4) was constructed by site-directed mutagenesis using the
polymerase chain reaction with Vent polymerase (New England Biolabs, Inc., Mississauga, Ontario, Canada), as described previously (Tabcharani et al., 1993
). A silent change was introduced in
the original CFTR cDNA to create a Stu1 restriction endonuclease site at nucleotide 950. Mutagenesis was carried out using a
fragment that extended from this Stu1 site to an Fsp1 site at nucleotide 1169. The manipulated portion of the construct was verified by sequencing with dideoxy chain termination and Sequenase (United States Biochemicals, Cleveland, OH). Cells were
transfected and immunoreactive mutant CFTR protein was detected in multiple cell lines after selection in methotrexate as described previously (Tabcharani et al., 1991
; Chang et al., 1993
).
Solutions
The standard recording solutions in the pipette and bath contained
(mM): 150 NaCl, 2 MgCl2, 10 Na N -tris[hydroxymethyl]methyl-2-aminoethanesulfonate, pH 7.4. Channel activity was maintained when recording from excised patches by adding 180 nM
protein kinase A catalytic subunit and 1 mM MgATP (Sigma
Chemical Co., St. Louis, MO) to the bath solution, as described
previously (Tabcharani et al., 1997). Permeation was studied under biionic conditions by replacing the chloride salts in the pipette or bath solutions with those of the appropriate anion. Bicarbonate permeation was studied under conditions of high pH 8.3 and 5% CO2 as described previously for the outwardly rectifying
anion channel (Tabcharani et al., 1989). Under these conditions,
the carbonic acid, bicarbonate, and carbonate concentrations would be ~1.4, 147, and 1.4 mM, respectively.
Single Channel Record
Pipettes and recording equipment were as described previously
(Tabcharani et al., 1997). The bath agar bridge had the same ionic composition as the pipette solution. Voltages have been corrected for liquid junction potentials measured at the agar bridge using a flowing 3-M KCl electrode as follows (mV): 1 NO3
, 3 formate, 5 acetate, 5 methane sulfonate, 6 ethane sulfonate, 6 pyruvate, 6 propanoate, 10 gluconate. Permeability ratios were calculated using the equation
![]() |
(1) |
where Erev is the reversal potential and other terms have their
usual meanings. The relationship between channel conductance and symmetrical Cl activity for both wild type and TT338,339AA
channels was fitted by a Michaelis-Menten-type hyperbolic function of the form
![]() |
(2) |
where is conductance,
max the saturating conductance of the
channel, Km the apparent affinity of the channel for Cl
ions, and
(Cl
) the Cl
activity calculated using the Debye-Hückel theory.
To estimate pore size, the permeation pathway of CFTR was
modeled as a cylinder permeated by cylindrical ions (e.g., Dwyer et al., 1980; Bormann et al., 1987
; Cohen et al., 1992b
). According to this model, ionic permeability is proportional to the ratio of the diameters of the permeating ion and the pore by an excluded volume effect (Dwyer et al., 1980
). The permeability of an
ion, relative to Cl
, is then given by
![]() |
(3) |
where a is the diameter of the ion, d is the diameter of the pore, and k is a proportionality constant.
Mean values are presented as mean ± SEM. For graphical presentation of mean values, error bars represent ±SEM; where no error bars are shown, this is smaller than the size of the symbol. Experiments were performed at room temperature (22 ± 1°C) unless otherwise indicated.
Biionic Permeability of Extracellular Anions
In the first series of experiments, single channel currents were measured using inside-out patches with Cl
solution in the bath and different polyatomic anions in
the pipette. Fig. 1 shows recordings under these conditions at 0 mV, and at large positive potentials when
measurable currents were carried by test anions. The
current-voltage (i/V) relationships are shown in Fig. 2.
Four of the nine anions tested in this paper (NO3
,
HCO3
, formate, and acetate) were clearly permeant
and gave reversal potentials within the range ±60 mV
(see Table I). When a reversal potential was not observed with a particular anion, the voltage was increased until the seal was lost, which was usually at
greater than +100 mV. The control i/V relationship in
symmetrical chloride solutions is shown at normal pH
7.4 in Fig. 2, A-D. To allow comparison with results obtained with bicarbonate on one side, Fig. 2 A also shows
the i/V relationship with symmetrical 154 mM Cl
at
pH 8.3. CFTR-mediated currents were recognizable by
their slow gating and activation by PKA plus MgATP, regardless of the anion carrying the current.
Table I. Comparison of the Permeability of Wild-Type and TT338,339AA Channels to Some Extracellular Anions |
Although reversal potentials were not observed with external pyruvate, propanoate, methane sulfonate, ethane
sulfonate, or gluconate, CFTR might still have some
permeability to these anions below the detection threshold of single channel recording. Indeed, the channel
must have some permeability to extracellular methanethiosulfonates because they reach cysteine residues when
they are engineered near the cytoplasmic end of TM6
(Cheung and Akabas, 1996). Anions that appeared to
be impermeant under the conditions used in this study
were assigned permeability ratios <0.06. Table I summarizes the reversal potentials and permeability ratios
obtained for external anions, and also the estimated
minimum unhydrated dimensions of each ion used.
Permeability to Intracellular Anions
Fig. 3 shows i/V relationships obtained when different
anions were present on the cytoplasmic side of the
membrane. Permeability ratios calculated for cytoplasmic anions were somewhat higher than when the same
anions were present extracellularly; for example, the
mean Pformate/PCl ratios were 0.18 ± 0.03 and 0.25 ± 0.01 with external and internal formate, respectively (Tables
I and II). Acetate displayed a similar asymmetry, with
Pacetate/PCl being 0.09 ± 0.00 from the extracellular side
and 0.19 ± 0.01 from the intracellular side. Nevertheless, the overall permeability sequence observed was
the same regardless of the direction of the anion gradients (NO3 > Cl
> HCO3
> formate > acetate). The
i/V relationships measured with internal pyruvate, propanoate, methane sulfonate, ethane sulfonate, or gluconate on the cytoplasmic side did not reverse (Fig. 3),
indicating negligible permeability to these ions (PX/PCl <
0.06). Reversal potentials and permeability ratios for intracellular anions are summarized in Table II.
Table II. Permeability of Wild-Type Channels to Intracellular Anions |
Double Mutation in TM6 Increases Single Channel Conductance
In a second series of experiments, the properties of wild-type channels were compared with those of the mutant
TT338,339AA, in which two polar threonine residues
near the middle of TM6 were simultaneously replaced
by smaller, nonpolar alanines (Fig. 4). Threonines are
important potential hydrogen bond-forming residues in the pores of both anion- and cation-selective channels
(MacKinnon and Yellen, 1990; Yool and Schwarz, 1991
;
Cohen et al., 1992a
, 1992b
; Sansom, 1992
; Villarroel and
Sakmann, 1992
; Heginbotham et al., 1994
; McDonough
et al., 1994
). Mutation of serine 341, another potential
hydrogen-bonding residue in CFTR TM6, to alanine causes pronounced rectification of the macroscopic i/V
relationship and reduced sensitivity to block by diphen-ylamine-2-carboxylate (DPC; McDonough et al., 1994
).
By contrast, mutating threonines 338 and 339 individually to alanines had no effect on the shape of the i/V relationship or DPC block (McDonough et al., 1994
). Fig.
5, A-C shows that Cl
currents carried by single
TT338,339AA channels were consistently larger than
those carried by wild-type channels. Wild-type channels had a linear current-voltage relationship over the range
±80 mV, with a mean slope conductance of 7.97 ± 0.10 pS (n = 6; Fig. 5 C), similar to values reported previously
under similar conditions (Tabcharani et al., 1993
; Tabcharani et al., 1997
). The TT338,339AA mutant also had
a linear i/V relationship over the same voltage range,
but its conductance was 9.88 ± 0.26 pS (n = 8), significantly higher than that of the wild-type channel (P < 0.05, one-tailed t test; Fig. 5 C).
Elevated conductance was also observed when
TT338,339AA channels were bathed in symmetrical solutions having different Cl activities (Fig. 5 D). The relationship between channel conductance and symmetrical Cl
activity for both wild-type and TT338,339AA
channels was well fitted by a Michaelis-Menten-type hyperbolic function (Eq. 2; see Fig. 5 D). The fits shown
in Fig. 5 D gave
max = 11.1 pS and Km = 45.9 mM for
wild type, and
max = 14.0 pS and Km = 52.9 mM for
TT338,339AA. The fact that this equation fit the data
well without correction for local changes in ion concentration suggests that fixed charges on the surface of
the channel protein do not greatly influence conductance in wild-type or TT338,339AA channels (for review see Green and Andersen, 1991
). The primary
functional effect of the TT338,339AA mutation was
to increase the saturating conductance of the channel
by ~26%. The Km may also be increased somewhat
(~15%); however, this would probably have little effect at 150 mM Cl
.
Permeability of the TT338,339AA Mutant to Different Anions
To assess whether the increase in conductance and possible decrease in Cl affinity of TT338,339AA might be
associated with a change in pore diameter, permeability of the mutant channel to a number of extracellular
anions was tested under biionic conditions as described
for wild-type channels (see above; Tabcharani et al., 1997
). Mean single channel current-voltage relationships for TT338,339AA obtained with different external anions are shown in Fig. 6. As can be seen from Table I, all permeant anions tested had higher permeability ratios in TT338,339AA than in the wild-type channel. Moreover, two anions that were not measurably permeant in wild-type channels (propanoate and pyruvate)
showed significant permeability in TT338,339AA. The
small anion F
, which has a high hydration energy and
may be unable to interact with "weak field strength"
sites in the pore (see below), was not measurably permeant in the TT338,339AA mutant, as reported previously for the wild-type channel (Tabcharani et al., 1997
).
Estimates of CFTR Pore Diameter
If the ability of large polyatomic ions to permeate depends on their size relative to that of the narrowest region of the pore (Dwyer et al., 1980), then the increased permeability of the TT338,339AA channel to
acetate, formate, propanoate, and pyruvate would suggest that this double mutation may widen the narrowest region. The pore of wild-type CFTR accommodates acetate (unhydrated molecular dimensions 3.99 × 5.18 × 5.47 Å; see Table I) but not the slightly larger ions propanoate (4.12 × 5.23 × 7.05 Å) or pyruvate (4.09 × 5.73 × 6.82 Å). Assuming that the narrowest dimensions of the pore are large enough to accommodate the
two smaller dimensions of any (unhydrated) permeant
anion, this constriction must have minimal dimensions
of 3.99 × 5.18 Å and a cross-sectional area of at least 21 Å2. By contrast, the pore of TT338,339AA is permeable
to both propanoate and pyruvate but not to methane
sulfonate (5.08 × 5.43 × 5.54 Å), suggesting minimal
dimensions of 4.12 × 5.73 Å and a cross-sectional area
of at least 24 Å2 for the narrowest region. The relative
permeability of the different extracellular anions studied as a function of their apparent diameters is shown
in Fig. 7 A. The diameter of unhydrated ions is often expressed as the geometric mean of the three minimum dimensions of the ion (e.g., Dwyer et al., 1980
;
Cohen et al., 1992b
). However, several of the anions
studied here are roughly cylindrical in shape and their
ability to pass through the CFTR channel may depend on the minimum cross-sectional dimensions of a cylinder that could contain the ion (McDonough et al.,
1994
; Cheung and Akabas, 1996
). The largest dimension of the ion (i.e., the length of the cylinder) would
therefore affect its permeability far less than the two smaller dimensions. We therefore took the geometric
mean of the two smallest dimensions for each ion (Table I) as our estimate of ionic diameter in Fig. 7 A. Plotting the permeability ratios calculated for extracellular
anions (Table I) against these apparent ionic diameters
and fitting them with Eq. 3 gave d = 5.34 Å and k = 2.61 for the wild-type channel and d = 5.83 Å and k = 3.94 for TT338,339AA mutant, again consistent with a
substantial increase in diameter of the TT338,339AA
variant. These diameters would produce cross-sectional
areas of ~22 Å2 for wild type and ~27 Å2 for
TT338,339AA if the pores were cylindrical. Small anions (Cl
, F
, Br
, I
, NO3
), the permeability of which
is dependent more on their hydration energies than
their size (see below), were excluded from these fits.
Lyotropic Selectivity
Ionic permeability through channels is thought to involve at least partial dehydration of permeating ions,
with ion-solvent interactions being replaced by interactions between the ion and polar groups lining the
channel pore. The permeability sequence described for
CFTR (I > NO3
> Br
> Cl
> HCO3
> acetate > F
) follows a lyotropic sequence (Dani et al., 1983
; Tabcharani et al., 1997
), suggesting that hydration energies
are mainly responsible for controlling anion permeability (Fig. 7 B). High iodide and thiocyanate permeabilities were reported previously (Tabcharani et al., 1992
,
1993). Thus, in CFTR, ion-channel interactions may be
relatively weak compared with ion-solvent interactions,
indicating a weak field strength selectivity site (Wright
and Diamond, 1977
). The relationship between permeability and hydration energy is maintained in TT338,
339AA (Fig. 7 B), suggesting that this mutation does not
strongly affect the selectivity filter of the channel.
This paper describes the most complete permeability
sequence of the CFTR Cl channel measured under biionic conditions, which we find to be I
> NO3
> Br
> Cl
> HCO3
> formate > acetate when these ions
are present on either side of the membrane. Propanoate,
pyruvate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06).
Our permeability ratios for NO3
(1.43-1.61), HCO3
(0.14-0.25), and gluconate (close to zero) are consistent with previous reports for CFTR in different systems
(Gray et al., 1990
, 1993
; Bell and Quinton, 1992
; Bajnath et al., 1993
; Copello et al., 1993
; Overholt et al.,
1993
; Poulsen et al., 1994
; Kottra, 1995
; Linsdell and
Hanrahan, 1996a
).
As with halide permeability (Tabcharani et al., 1997),
the permeability sequence to polyatomic anions followed a lyotropic or (inverse) Hofmeister sequence
(Fig. 7 B). This series is favored when cationic groups
or dipoles in proteins attract anions to a region of
structured water, such as that found near hydrophobic groups (Von Hippel and Schleich, 1969
; Dani et al.,
1983
; Tabcharani et al., 1997
). The same lyotropic sequence has been observed in GABAA and glycine-gated
Cl
channels in spinal neurons (Bormann et al., 1987
)
and hippocampal neurons (Fatima-Shad and Barry,
1993
), in a voltage-dependent Cl
channel in hippo-campal neurons (Franciolini and Nonner, 1987
), and
in the epithelial outwardly rectifying Cl
channel (Reinhardt et al., 1986; Halm and Frizzell, 1992
). Although the physical basis of lyotropic anion selectivity has not
yet been studied in Cl
channels using mutagenesis, it
is likely that a positively charged amino acid and/or
cationic dipole within the channel pore is the anion attracting group. As discussed in the companion papers
(Tabcharani et al., 1997
; Linsdell et al., 1997
), one contributor in the CFTR pore may be arginine 347 in TM6,
since mutations that remove positive charge at this position drastically reduce selectivity between Cl
and I
(Tabcharani et al., 1997
), abolish voltage-dependent
inhibition of Cl
currents by the lyotropic anion SCN
(Tabcharani et al., 1993
), and reduce channel block
by cytoplasmic disulfonic stilbenes (Linsdell and Hanrahan, 1996b
).
Wild-type CFTR channels showed low permeability to
formate and acetate ions, and were not measurably permeant to the larger anions propanoate, pyruvate, methane sulfonate, ethane sulfonate, and gluconate. However, these large anions may be able to permeate at
rates that are too low to be resolved as single channel
currents. Relatively large, hydrophilic sulfhydryl reagents (~6 Å in diameter) are able to penetrate from
the extracellular solution to interact covalently with engineered cysteine residues at the cytoplasmic end of
TM6 (Cheung and Akabas, 1996). The irreversible nature of that reaction probably enables permeation by
the cysteine reagent to be detected when the flux rates
of similar compounds (e.g., ethane sulfonate) are too
low to generate measurable current at the single channel level. The anionic channel blockers diphenylamine-2-carboxylate and flufenamic acid are also permeant in
CFTR (McCarty et al., 1993
).
The relationship between ion diameter and permeability in CFTR (Fig. 7 A) suggests a pore diameter of
~5.3 Å and a cross-sectional area of ~21-22 Å2. Other
Cl channel types have been estimated to have pore diameters between 5.2 and 6.4 Å (Bormann et al., 1987
;
Franciolini and Nonner, 1987
; Halm and Frizzell, 1992
;
Fatima-Shad and Barry, 1993
; Arreola et al., 1995
). Our
estimate of the pore diameter is likely to be a lower
limit, since large anions may have permeabilities below our detection threshold (see above). However, our estimates for the pore diameter are less than the diameter
of ATP (Table I), which has been reported to diffuse
through CFTR channels at high rates (Reisin et al.,
1994
; Schwiebert et al., 1995
), although this has not
been observed in all laboratories (Reddy et al., 1996
; Li
et al., 1996
; Grygorczyk et al., 1996
). If CFTR can support ATP transport under certain conditions, it seems
unlikely that this would involve ATP permeation through
the pore.
The increased permeability of large anions in
TT338,339AA (Table I) indicates an increase in the dimensions of the narrowest part of the pore in this mutant. We estimate the diameter of the mutated pore to be
~5.8 Å, with a cross-sectional area of 24-27 Å2. One
possible interpretation of these results is that threonine residues 338 and/or 339 might contribute to the narrowest part of the pore, either directly or via an allosteric effect on a constricted region that is physically
located elsewhere. Threonine residues have previously
been suggested to contribute to the narrowest region
of the pore in cation-selective nicotinic acetylcholine receptor channels (Cohen et al., 1992a, 1992b
; Villarroel and Sakmann, 1992
). However, substituted cysteine
accessibility mutagenesis experiments indicate that the
R groups of these two threonine residues are not in
contact with the aqueous lumen of the CFTR pore (Cheung and Akabas, 1996
).
TT338,339AA had a larger saturating conductance
than wild-type CFTR (Fig. 5), suggesting that conductance of the wild-type channel may be limited by the
rate of Cl flux through this narrow region. Conductance could be elevated due to a reduction in nonspecific frictional interactions between the permeating ion
and the pore walls (although the smallest estimate of
the narrowest part of the pore is still much larger than
the diameter of an unhydrated Cl
ion, 3.62 Å). The
i/V relationship of TT338,339AA, like wild type, was linear, suggesting anion binding is not strongly altered in
this mutant. This agrees with the results of McDonough
et al. (1994)
, who found that mutating each of these
threonine residues individually to alanines did not affect the linearity of the macroscopic CFTR Cl
current
expressed in Xenopus oocytes. In contrast, mutating
serine 341 to alanine produced outward rectification of
the i/V relationship, consistent with its proposed role
as a binding site for permeating anions (McDonough et
al., 1994
). Subsequent cysteine mutagenesis also indicated that serine 341 lines the pore (Cheung and Akabas, 1996
). The increased conductance of TT338,339AA
is unlikely to be a nonspecific effect of mutations in
TM6 since many mutations in this region have been
studied, but none has previously been found to elevate
conductance. Moreover, the fact that the selectivity sequence and channel gating were not affected in the
mutant also argues against gross structural alterations,
although these cannot be excluded. The altered apparent pore size and conductance of TT338,339AA are
consistent with the proposed key role of TM6 in forming the CFTR pore (Anderson et al., 1991
; Sheppard et
al., 1993
; Tabcharani et al., 1993
, 1997
; McDonough et
al., 1994
; Cheung and Akabas, 1996
; Linsdell and Hanrahan, 1996b
).
A CFTR variant with increased conductance might be
useful in maximizing Cl transport in gene or protein
replacement therapy for cystic fibrosis, particularly
where the efficiency of gene or protein delivery was
low. The 24% increase in channel conductance seen in
TT338,339AA might not be therapeutically significant
and would also have to be weighed against the possibly
deleterious increased permeability to large organic anions. Nevertheless, since it is the first CFTR mutation to
increase channel conductance, it suggests that other mutations in this region may allow the development of
therapeutically advantageous forms of CFTR.
The lyotropic sequence of permeability ratios is the
same in both wild-type and TT338,339AA channels
(I > NO3
> Br
> Cl
> acetate > F
; Fig. 7 B). This
suggests that the narrow region disrupted in the
TT338,339AA mutant is not a major determinant of selectivity in CFTR, unlike voltage-gated Na+ (Lipkind
and Fozzard, 1994
) and K+ channels (Lipkind et al.,
1995
), where a selectivity filter has been proposed in
the narrowest part of the pore. Nevertheless, permeability ratios for I
, NO3
, and Br
are all increased relative to the smaller Cl
ion. Thus, in wild-type channels, the narrow region may interact preferentially with
Cl
compared with these other ions.
Address correspondence to John W. Hanrahan, Department of Physiology, McGill University, 3655 Drummond St., Montréal, Québec H3G 1Y6, Canada. Fax: 514-398-7452; E-mail: hanrahan{at}physio.mcgill.ca
Received for publication 10 October 1996 and accepted in revised form 11 July 1997.
We thank Jenny Eng and Shu-Xian Zheng for technical assistance.
This work was supported by the Canadian Cystic Fibrosis Foundation (CCFF), the Medical Research Council (MRC; Canada), and the National Institute of Diabetes and Digestive and Kidney Diseases. P. Linsdell is a CCFF postdoctoral fellow. J.W. Hanrahan is an MRC Scientist.
CFTR, cystic fibrosis transmembrane conductance regulator.
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