From the Departments of Physiology and Biophysics and
Pediatrics, Case Western Reserve University School of
Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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The cystic fibrosis transmembrane conductance
regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-
and ATP-regulated chloride channel, whose gating process involves
intra- or intermolecular interactions among the cytosolic domains of
the CFTR protein. Tandem linkage of two CFTR molecules produces a
functional chloride channel with properties that are similar to those
of the native CFTR channel, including trafficking to the plasma
membrane, ATP- and PKA-dependent gating, and a unitary
conductance of 8 picosiemens (pS). A heterodimer, consisting of a
wild type and a mutant CFTR, also forms an 8-pS chloride channel with
mixed gating properties of the wild type and mutant CFTR channels. The
data suggest that two CFTR molecules interact together to form a single
conductance pore for chloride ions.
CFTR1 is a
multi-functional protein, which provides the pore of a linear
conductance chloride channel (1-5) and also functions to regulate
other membrane proteins (6, 7). Mutations in CFTR leading to defective
regulation or transport of chloride ions across the apical surface of
epithelial cells are the primary cause of the genetic disease of cystic
fibrosis (8-10). Comprehensive genotype-phenotype studies have
indicated possible contribution of protein-protein interactions to the
severity of the disease (11), but little is known on the stoichiometry
of CFTR as a chloride channel.
The native CFTR chloride channel is activated by PKA-phosphorylation of
serine residues in its regulatory or R domain and then gated by binding
and hydrolysis of ATP by the nucleotide binding folds (12). The actual
pore of the chloride channel is presumably formed by portions of the
two membrane spanning domains of CFTR, each consisting of six
transmembrane segments (13), with an ohmic conductance of ~8 pS in
200 mM KCl solution (14, 15). An early study by Rich
et al. (16) showed that deletion of amino acids 708-835
from the R domain ( Subcloning of CFTR cDNAs--
The wild type and Expression of CFTR in HEK 293 Cells--
The human embryonic
kidney (HEK 293) cells were used for expression of CFTR proteins. The
different CFTR cDNAs were introduced into the HEK 293 cells using
the LipofectAMINE reagent. Two days after transfection, the cells were
used for Western blot assay, SPQ measurement, or isolation of membrane
vesicles followed by reconstitution studies in the lipid bilayer
membranes, as described previously (14, 15).
SPQ Assay of Chloride Transport--
The CFTR-mediated chloride
transport was measured by SPQ assay with HEK 293 cells expressing the
wt, Lipid Bilayer Reconstitution of CFTR Channel--
The procedure
for single channel measurements of CFTR using the lipid bilayer
reconstitution technique has been described elsewhere (17). Briefly,
microsomal membrane vesicles were isolated from HEK 293 cells
transiently expressing either the wt-, Fig. 1A shows a Western
blot of CFTR expressed in HEK 293 cells. Both fully glycosylated
(~170 kDa) and core glycosylated (~140 kDa) proteins can be
detected in cells transfected with the wt-CFTR cDNA (lane
1). The corresponding bands for the
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ABSTRACT
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R) renders the CFTR channel PKA independent. The
open probability of
R-CFTR is approximately one-third that of the wt
channel and does not increase upon PKA phosphorylation (17, 18). Based
on these different gating properties of the wt and
R channels, we
set out to test the intermolecular interactions of CFTR by constructing tandem cDNAs between the wt-CFTR and the
R-CFTR. Our rationale is as follows. If a monomer of CFTR is sufficient to form an 8-pS chloride channel, the dimeric CFTR molecules would form either a 16-pS
chloride channel or two 8-pS chloride channels that may gate
independently or together. On the other hand, if two CFTR molecules are
required to function as a chloride channel, we expected the tandem
construct to form a single 8-pS chloride channel, provided that the
linker sequence does not affect the CFTR channel function. Furthermore,
we predicted that the wt-
R (or
R-wt) channel should exhibit mixed
properties of the wt and
R channels.
EXPERIMENTAL PROCEDURES
R
(708-835) CFTR cDNAs were cloned into the
NheI/XhoI sites of the pCEP4 expression vector
(14, 17). The tandem constructs, wt-wt, wt-
R,
R-wt, and
R-
R, were generated in three steps. First, site-directed
mutageneses were used to remove the stop codon and to introduce a
BssHII restriction site at the 3' end of the CFTR cDNA,
to create C-BssH. Second, similar approach was taken to
remove the Kozak sequence and to introduce a BssHII site at
the 5' end of the CFTR cDNA, to yield N-BssH. Third, the
entire CFTR cDNA from N-BssH was released from the pCEP4
vector through digestion with BssHII and XhoI and
ligated into the BssHII/XhoI sites of the
C-BssH, to create the wt-wt dimer. This represents a direct
head-to-tail linkage of two CFTR cDNAs. A double stranded
oligonucleotide containing the recognition sequence for thrombin
(underlined),
Arg-Ala-Ala-Ser-Leu-Val-Pro-Arg-Gly-Ser-Gly-Gly-Gly-Gly, was ligated to the BssHII site of wt-wt, to yield the
wt-e-wt construct.
R, wt-wt,
R-
R, and wt-
R CFTR proteins, following the
procedure described previously (14). Basically, the cells were loaded
with SPQ dye (Molecular Probes) using hypotonic shock, and chloride
flux across the plasma membrane was measured upon stimulation with 10 µM forskolin.
R-, wt-wt, wt-
R,
R-wt,
or
R-
R proteins and added to the cis (intracellular) solution containing 200 mM KCl, 2 mM Mg-ATP, 10 mM HEPES-Tris (pH 7.4). The trans solution
contained 50 mM KCl, 10 mM HEPES-Tris (pH 7.4).
To study the PKA-dependent regulation of the CFTR channel, 100 units/ml of the catalytic subunit of PKA was added to the cis solution. Single channel currents were recorded using an
Axopatch 200A patch clamp unit (Axon Instruments). Data acquisition and pulse generation were performed with a 486 Computer and a 1200 Digidata
A/D-D/A converter. The currents were sampled at 1-2.5 ms/point and filtered at 100 Hz. Single channel analysis were performed
with the pClamp7 software.
RESULTS AND DISCUSSION
R-CFTR run at apparent
molecular masses of ~150 and ~120 kDa (lane 2), reflecting the deletion of 128 amino acids from the R domain (amino acids 708-835). The wt-wt protein has molecular masses of ~340 and
~280 kDa (lane 4), as expected for a dimer of the wt-CFTR. Similarly, the wt-
R (lane 5),
R-wt (lane
6), and
R-
R (lane 7) proteins can all be
expressed in HEK 293 cells, with the expected size as dimers. To be
able to manipulate the oligomerization state of the CFTR proteins, we
engineered an enzymatic digestion site for thrombin in the linker
sequence of the wt-wt dimer. This construct is named wt-e-wt (Fig.
1A, lane 10). Digestion of wt-e-wt with thrombin
resulted in reduction of the apparent size from dimer to monomers
(lane 9), whereas thrombin had no effect on the wt monomer
(lane 8) or the wt-wt dimer (lane 11).
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Fig. 1.
Expression of CFTR in HEK 293 cells.
A, Western blot of CFTR expressed in HEK 293 cells. The
pCEP4 plasmids containing the different CFTR constructs were introduced
into the HEK 293 cells using the LipofectAMINE reagent. 24-48 h after
transfection, the cells were harvested and the whole cell lysate was
used in the Western blot. The proteins were separated on a 3-12%
polyacrylamide gel, and probed with a monoclonal antibody against the
COOH terminus of the CFTR protein (mAb24-1, Genzyme). Lane
1, wt; lane 2, R; lane 3, untransfected
HEK cells; lane 4, wt-wt; lane 5, wt-
R;
lane 6,
R-wt; lane 7,
R-
R; lane
10, wt-e-wt. 10 units of thrombin (purchased from Sigma) cleaved
the wt-e-wt dimer into monomers (lane 9) in a reaction run
at 37 °C for 10 min with a buffer containing 200 mM KCl,
2 mM Mg-ATP, 10 mM HEPES-Tris (pH 7.4). The wt
CFTR has no internal cleavage site for thrombin, as revealed by the
lack of effect of thrombin on the wt monomer (lane 8) and
wt-wt dimer (lane 11). B, SPQ assay of
CFTR-mediated chloride transport. HEK 293 cells loaded with SPQ were
preincubated with nitrate solution to allow depletion of intracellular
chloride ions (1). The perfusion solution is then replaced
by a buffer containing chloride to quench fluorescence as chloride ions
enter the cell (2). When fluorescence reaches a minimum,
nitrate solution is replaced to restore fluorescence (3).
This sequence is repeated with chloride and nitrate buffers containing
10 µM foskolin (4). At the completion of the
experiment, the cells are perfused with KSCN and valinomycin to quench
the fluorescence and dissipate the membrane potential (5).
Each trace represents a single cell responding over the course of the
experiment.
SPQ assays indicate that the wt-wt dimer, similar to the wt monomer,
supports chloride transport in HEK 293 cells upon stimulation with
forskolin (Fig. 1B). Those cells expressing the R and
R-
R proteins exhibit basal chloride transport activities without
stimulation with forskolin, which is consistent with the studies of
Rich et al. (16). Interestingly, the cells expressing
wt-
R have basal chloride transport activity in the absence of
forskolin, which became significantly higher upon stimulation by
forskolin (relative changes in fluorescence per minute, 0.095 ± 0.010,
forskolin; 0.203 ± 0.007, +forskolin, n = 65). These results indicate that CFTR dimers can traffic properly to
the plasma membrane of HEK 293 cells.
To study the single channel functions of the CFTR dimers, microsomal
membrane vesicles containing the wt-wt or R-
R proteins are
incorporated into the lipid bilayer membrane. Fig.
2A shows representative
current traces from the wt, wt-wt,
R, and
R-
R channels, and
their corresponding current-voltage relationships are plotted in Fig.
2B. As can be seen, all four constructs give rise to
chloride channels with unitary conductances of ~8 pS. In 9 out of 13 experiments with wt-wt, and 8 out of 12 experiments with
R-
R, we
only observed openings of a single channel (not two channels) in the
bilayer membrane. Similar to the wt channel, opening of the wt-wt
channel absolutely requires the presence of both ATP and PKA in the
cytosolic solution; and similar to
R, opening of the
R-
R
channel is independent of PKA phosphorylation. Interestingly, the
activity of the wt-wt channel appears to be significantly lower than
that of the wt channel (Fig. 2C). Studies from other
laboratories have shown that the amino- and carboxyl-terminal tails of
CFTR contribute to the overall function of the CFTR channel (19, 20).
We speculate that the head-to-tail connection in the dimeric construct
probably constrains the movement of the amino- and carboxyl-terminal
portions of CFTR, reducing activity of the wt-wt channel (see also Fig.
4).
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Thus, it appears that the dimeric constructs of CFTR form functional chloride channels with conduction properties that are indistinguishable from the monomers of CFTR, i.e. all of them have single channel conductance of ~8 pS. The dimeric constructs could in principle have two separate pores with conductance of 8 pS for chloride ions, and because of some physical constraint due to the linker sequence, opening of one pore could prevent opening of the other pore, which would result in the overall appearance of a single CFTR channel. The other possibility is that the 8-pS channel normally recorded in single channel measurements (2, 4, 5, 14, 17) actually represents dimeric complexes of CFTR that naturally assemble in the cell surface membrane. Data from the following sets of experiments support the latter hypothesis.
The heterodimers of CFTR, wt-R and
R-wt, also form functional
chloride channels with unitary conductance of 8 pS, which display mixed
gating properties of the wt and
R channels (Fig. 3). First, opening of the wt-
R and
R-wt channels exhibit bursting kinetics, but unlike either the wt or
R channels, these bursting patterns are interrupted by fast closing
transitions (compare traces of Fig. 3, A and B,
with Fig. 2A). The wt channel has an average open lifetime
of
o = 96.0 ± 9.3 ms, and the
R channel has a
o = 54.2 ± 6.5 ms (17), whereas the wt-
R
channel has a
o = 24.4 ± 5.8 ms (n = 9), and the
R-wt channel has a
o = 32.4 ± 3.4 ms (n = 7). Second, open probability of the wt-
R and
R-wt channels display a clear PKA dependence (Fig. 3C).
The channels exhibit constitutive activity in the absence of PKA, which
becomes significantly higher upon PKA phosphorylation (Fig.
2C). In contrast, open probabilities of the
R and
R-
R channels are completely independent of PKA phosphorylation
(Fig. 3D).
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Fig. 4 shows the effect of thrombin on
the wt-e-wt channel. Compared with the wt-wt and wt-R constructs,
the wt-e-wt dimer contains 14 extra amino acids in the linker sequence
corresponding to the thrombin cleavage site (underlined)
(R-A-A-S-L-V-P-R-G-S-G-G-G-G). As shown in Fig.
4A, the wt-e-wt channel opens predominantly to a single 8-pS
conductance state, with an average Po of
0.186 ± 0.045 (n = 11) at
100 mV. 3-5 min
following the addition of 10 units/ml of thrombin to the cytosolic
solution, the activity of the wt-e-wt channel increases approximately
4-fold, but the apparent number of channels in the bilayer membrane
remains unchanged. During the course of the experiment, simultaneous
opening of two channels were never observed, even though open
probability of the thrombin-treated wt-e-wt channel was as high as
p = 0.723 (Fig. 4B). Thrombin would separate
the halves of the CFTR dimer and presumably remove constraints on the
movement of the amino- and carboxyl-terminal tails of CFTR. If such
constraints limit channel openings, this may explain why the wt-wt
channel has a lower open probability than the wt channel (Fig.
2C).
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Taken together, we have shown that the dimeric constructs of CFTR can
be expressed in the plasma membrane of HEK 293 cells, and these CFTR
dimers form functional chloride channels with unitary conductance of 8 pS, similar to the native CFTR channel (2, 4). The gating properties of
the wt-wt and R-
R channels are similar to the wt and
R
channels, whereas the wt-
R and
R-wt channels exhibit mixed
properties of the wt and
R channels. The fact that both wt-
R and
R-wt channels exhibit similar PKA dependence and similar gating
kinetics (Fig. 3, A and B) suggests that both halves of the CFTR dimer are properly expressed and inserted in the
membrane of HEK 293 cells. The tandem linkage of CFTR apparently reduces the overall activity of the chloride channel due to physical constraints introduced at the junction of the two CFTR molecules, but
does not affect the conduction property of the chloride channel, since
cleavage of wt-e-wt with thrombin did not change the conductance state
of the channel. Our data suggest that two CFTR molecules are required
for the chloride channel to open to the 8-pS conductance state.
Marshall et al. (21) used co-immunoprecipitation to search for protein-protein interactions between different mutant forms of CFTR and concluded that CFTR exists predominantly in a monomeric state. It may be that the intermolecular interactions between the CFTR molecules are weak and do not survive strong detergent solubilization (i.e. SDS) or that the CFTR dimers only represent a small percentage of the total CFTR proteins that could not be detected by the co-immunoprecipitation procedure. A recent study by Eskandari et al. (22) established structural evidence for a dimeric complex with the CFTR proteins. These investigators used freeze fracture electron microscopy to investigate the oligomeric assembly of membrane proteins expressed in Xenopus oocytes, and they concluded that the intramembrane structure of CFTR was consistent with a dimeric assembly of 12-transmembrane helix of the CFTR monomers.
Besides being of fundamental importance in understanding the mechanism
by which the CFTR channel works, the concept that CFTR functions as a
dimer may have implication for persons who are compound heterozygotes
for CFTR mutations. Some mutant pairs may be able to complement each
other thereby increasing the overall CFTR function, but other mutant
pairs may not. This may explain some of the phenotypic variations in
patients with CFTR mutations, especially those with some residual
function. It will be important to know at what stage in the
biosynthetic pathway of CFTR the dimers are formed, i.e.
before leaving the endoplasmic reticulum or after trafficking to the
apical membrane. More specifically, which portions of the CFTR molecule
are involved in the contact interaction or constitute the binding
site(s) for accessory proteins that could interact with CFTR?
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ACKNOWLEDGEMENTS |
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We thank Drs. M. L. Drumm, J. W. Hanrahan, and D. C. Gadsby for helpful discussions.
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FOOTNOTES |
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* This work was supported by Grants RO1-DK51770 (to J. M.) and RO1-DK27561 (to P. B. D.) from the National Institutes of Health and by an Established Investigatorship from the American Heart Association (to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 216-368-2684; Fax: 216-368-1693; E-mail: jxm63{at}po.cwru.edu.
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ABBREVIATIONS |
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The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PKA, cAMP-dependent protein kinase; S, siemens; wt, wild type; SPQ, 6-methoxy-N-(3 sulfopropyl)quinolinium.
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