From the
Cystic fibrosis is caused by mutations in the gene encoding the
cystic fibrosis transmembrane conductance regulator (CFTR), a plasma
membrane-localized chloride channel. Some mutations in CFTR, including
one which affects most patients (
Cystic fibrosis (CF)
Three CHO lines that stably expressed wild-type CFTR,
We examined the Cl
To evaluate the significance of
The demonstration that the major disease-causing
mutant CFTR retains Cl
We thank J. Riordan and X.-B. Chang for providing
transfected CHO cells, S. Cheng for providing the CFTR antibody, D. Mak
and Y. Marunaka for valuable help in single channel analysis, L. J.
Huan and D. Wong for technical assistance, and C. Bear, M. Buchwald, D.
Mak, and S. Grinstein for comments on the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
F508-CFTR), prevent CFTR from
exiting the endoplasmic reticulum (ER) where it is synthesized. To
examine whether normal and mutant CFTRs function as chloride channels
when they reside in the ER, the patch clamp technique was used to
measure currents in the outer membrane of nuclei isolated from
mammalian cells expressing CFTR. Both
F508-CFTR as well as CFTR
were revealed to function as cAMP-regulated chloride channels in native
ER membrane. These results represent the first demonstrations of
functional activity of CFTR in the biosynthetic pathway and suggest
that conformational changes in the mutant protein, although recognized
by ER-retention mechanisms, do not necessarily affect CFTR chloride
channel properties, which may have implications for pathophysiology and
therapeutic interventions in cystic fibrosis.
(
)
is characterized by
abnormal epithelial ion and fluid transport due to mutations in the CF
gene product,
CFTR
(1, 2, 3, 4, 5) , a
cAMP-regulated, DIDS-insensitive, linear, 6-14 picosiemens (pS)
Cl
channel
(6, 7) . The most common
(affecting
90% of patients) of many mutations in CFTR is a
deletion of phenylalanine, residue 508 (
F508-CFTR)
(4) .
Mammalian cells expressing
F508-CFTR lack Cl
permeability because the mutant protein fails to exit from the
endoplasmic reticulum
(ER)
(8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and localize normally in the plasma
membrane
(2, 18) . Abnormal processing of
F508-CFTR
is believed to result from its structural misfolding, since the
processing defect is temperature-sensitive
(19, 20) ;
functional revertants have been generated by inserting point mutations
in the protein
(21) , and synthetic peptides and full-length CFTR
containing the 508 mutation have reduced
stability
(20, 22) . In insect cells and
Xenopus oocytes in which CFTR does not undergo
biosynthetic arrest, plasma membrane-localized
F508-CFTR functions
as a Cl
channel
(23, 24) , and purified
F508-CFTR from insect cells retains Cl
channel
activity when reconstituted in bilayers
(23) . Furthermore, gross
overexpression
(25) or growth of some cell types at low
temperatures
(19, 20, 24) enables some
F508-CFTR to reach the plasma membrane, where it then has
Cl
channel activity. Nevertheless, these studies do
not address whether
F508-CFTR functions as a Cl
channel when it is retained in the ER in CF patients. Because the
molecular mechanisms underlying ER retention have not been determined,
it is unknown whether the Cl
channel activity of
mutant CFTR is affected by those conformational changes that cause its
ER retention or whether the mechanisms that enable
F508-CFTR to
escape from the ER, presumably by enabling it to attain its native
conformation, also influence its Cl
channel function.
Moreover, it is also unknown whether the retention mechanisms
themselves alter or abolish the Cl
channel activity
of CFTR. The consequences of
F508-CFTR Cl
channel activity, or lack thereof, in the ER cannot be predicted
because it has also not been determined if wild-type CFTR functions
there. It has been proposed that several unexplained biochemical
abnormalities in CF result from defective CFTR Cl
channel activity in the Golgi
(26) . Lacking, however, are
measurements of CFTR activity in the biosynthetic pathway, the cellular
compartments including ER and Golgi through which proteins traverse
during their synthesis, processing, and trafficking to the plasma
membrane. Here we demonstrate that both CFTR as well as
F508-CFTR
are functional cAMP-regulated Cl
channels in native
ER membrane in mammalian cells. These channel activities may indicate
functional roles of CFTR in the biosynthetic pathway and suggest that
lack of such activities due to ER retention of mutant CFTRs could
underlie some pathophysiology of CF.
F508-CFTR, or the vector only (controls; no CFTR)
(16, 27) were used. Western blot analysis using a CFTR monoclonal
antibody (Genzyme 24-1) revealed a
160-kDa band and a lesser
140-kDa band, reflecting mature extensively glycosylated and pre-Golgi
core-glycosylated CFTR, respectively, in cells expressing wild-type
CFTR; lower levels of only the 140-kDa band in the cells expressing
F508-CFTR; and an absence of CFTR in control CHO cells, as
previously reported
(16) (data not shown). CHO cells were
cultured in 75-cm
flasks at 37 °C in
-minimum
essential medium supplemented with 7% fetal calf serum, 1% penicillin,
and streptomycin and 20 or 200 µM methotrexate (vector- or
CFTR-transfected cells, respectively). In most experiments (90%), cells
were treated with sodium butyrate (5 mM) for 18 h prior to
isolation
(14) . Confluent cells were suspended by brief
trypsinization, washed twice with the phosphate-buffered saline and
once in an isolating solution containing (in mM): 120 KCl, 5
MgCl
, 10 HEPES, and 1 phenylmethylsulfonyl fluoride, pH 7.0
with NaOH, resuspended in 1 ml of the isolating solution, and
homogenized at 4 °C with a motor-driven Teflon pestle (Glas-Col) at
1700 rpm for 30 s (
30 strokes). The integrity of nuclear membranes
of isolated nuclei was verified by electron microscopy. Nuclei that
attached to the glass bottom of a 1-ml chamber were perfused with
solutions for patch clamp experiments. Heat-polished patch pipettes
(tip, <0.5 µm in diameter) filled with the patch clamp
buffer had resistances of 20-50 megaohms. Stable (10-60
min) seals (20-100 gigaohms) of the outer nuclear membrane were
obtained in 70-80% of attempts. Thus, nuclear pores were closed
and impermeable in our nuclear membrane patches, in agreement with
patch clamp studies of isolated Xenopus oocyte
nuclei
(28) . Unless specified, in all experiments the excised
patch configuration was used and the pipette and bath contained (in
mM): 120 NMDG-Cl, 3 MgCl
, 0.1 CaCl
,
1.1 EGTA, 10 HEPES, 1 MgATP, 5 glucose, pH 7.1, with HCl, with PKA (90
nM) included in the pipette solution (cytoplasmic face of the
membrane faces into the pipette). DIDS was added directly to the bath
to achieve final desired concentration. All experiments were performed
at room temperature. The applied potential was: V
- V
=
V
- V
(in excised patches). Accordingly, positive current flowed from
pipette to bath. Currents were amplified (Axopatch-1D, Axon
Instruments, Inc.), digitized (Macintosh computer via ITC-16 interface,
Instrutech Corp.), and written directly onto the hard disk
(Pulse+PulseFit software, HEKA Electronik). Data were analyzed
using Pulse+PulseFit and MacTac (Skalar Instruments). All data
were filtered at 300 Hz unless indicated. Open probability
(P
) of single channels was determined by summing
all bursts of openings with closed times of <3 s from single channel
patches. Total burst times from all single channel patches (53 and 8 s
for
F508-CFTR and wild-type CFTRs, respectively) were combined to
calculate kinetic parameters. Data, expressed as mean ± S.E.,
were analyzed using standard statistical tests.
channel activities of
ER-localized CFTRs by patch clamping the outer membrane of the nuclear
envelope of nuclei isolated from CFTR-expressing CHO cells. Because the
outer membrane is continuous with ER
(29) , we reasoned that
ER-localized ion channels would also be present there, as confirmed in
recent single channel measurements of the inositol trisphosphate
receptor in isolated Xenopus oocyte nuclei
(28) . With
the catalytic subunit of PKA and ATP in the pipette, excised patches
from nuclei isolated from
F508-CFTR-expressing cells revealed
linear, 6-10-pS channels in 39/49 patches (Figs. 1-3). The
channels were Cl
-selective since similar channels
were observed with bath and pipette solutions containing Cs
instead of NMDG
, and they were observed at the
Cs
reversal potential with asymmetric Cs
(120 mM CsCl in bath versus 20 mM
CsCl, 100 NMDG-Cl in the pipette, or vice versa) (3 patches;
data not shown). In the absence of PKA, either no channels (8/12
patches) or only an endogenous low conductance Cl
channel (below) was observed (4/12 patches). The PKA requirement
for channel activation was confirmed by alternately patching the same
nucleus in the presence and absence of PKA (n = 5
nuclei) (Fig. 2). The PKA-stimulated Cl
channel
activities were blocked by external I
(Fig. 1B; 4/4 patches) and were DIDS-insensitive
(0.1-0.5 mM) (Fig. 1D; 7/7 patches).
Thus, the properties of the Cl
channels in the
nuclear membrane in
F508-CFTR-expressing cells are highly
reminiscent of those described for wild-type CFTR in the plasma
membrane
(6, 7) .
Figure 2:
PKA dependence of F508-CFTR
Cl
channels in the outer nuclear membrane. Currents
were recorded alternately in the absence and presence of 90 nM
PKA in the pipette at two pipette potentials as indicated. Each
trace is from a different patch excised from the same nucleus.
Similar results were obtained from 6 nuclei (17
patches).
Figure 1:
Properties of F508-CFTR
Cl
channels in excised patches of outer membranes of
nuclei isolated from CHO cells. A, single PKA-activated
F508-CFTR Cl
channel. PKA (90 nM) was
present in the pipette solution. Within bursts, P
was 0.64, and analysis of open and closed time histograms
revealed one open and one closed state (
= 42
ms and
= 20 ms) (14). Pipette potentials were
as indicated. The dashedline represents closed state
(C). This figure is representative of 5 experiments.
B, block of
F508-CFTR single Cl
channel
activities by I
. Shown is the excised patch with
pipette solution containing (in mM): 120 KCl, 3
MgCl
, 0.1 CaCl
, 1.1 EGTA, 10 HEPES, 1 MgATP, 5
glucose, and 90 nM PKA, pH 7.1, with KOH. The bath (equivalent
to extracellular side) contained a similar solution with 60 mM
Cl
replaced with I
. Pipette
potentials were as indicated. This figure is representative of 4
experiments. C, I-V relationships for A (opencircles) and B (closedcircles). The slope conductance under symmetrical
conditions (A) was 8.8 ± 0.7 pS (3-5 measurements
at each indicated voltage from 10 patches). In B with
I
on the lumenal side of patch, conductance was
reduced to 3.5 ± 0.3 pS (n = 4 patches). The
shift of extrapolated I-V curve (dashedline) toward
positive potentials indicates the channel is selective for
Cl
over I
, though currents at
positive voltages could not be resolved. D, insensitivity of
F508-CFTR Cl
channels to DIDS. DIDS (0.3
mM) was without effect on multilevel
F508-CFTR
Cl
channel activities. This figure is representative
of 7 experiments.
No channels were detected in 24/56
patches of nuclei isolated from cells transfected with the vector only,
whereas an endogenous linear 4-8-pS Cl channel
was present in the rest (not shown). However, this channel could be
distinguished from
F508-CFTR because it (a) was observed
only as a single channel, (b) displayed long closed states
(several minutes) with only occasional single openings instead of
bursts of openings seen with
F508-CFTR, and (c) was
blocked by DIDS (0.1-0.3 mM) (10/10 patches). This
channel therefore more closely resembles a cAMP-independent
Cl
channel observed in plasma membranes of the CHO
parental line
(30) .
F508-CFTR Cl
channel activity, nuclei from cells
expressing wild-type CFTR were also examined. Cl
channels were observed that had essentially identical biophysical
and pharmacological properties as ER
F508-CFTR channels
(Fig. 4). The frequency and kinetic properties of ER-localized
F508-CFTR and wild-type CFTR were also similar. True single
channels were rarely observed (5/39 and 2/14 active patches from
F508-CFTR (Fig. 1A) and wild-type CFTR nuclei (not
shown), respectively) whereas the remaining patches contained up to 20
channels (Fig. 1D and 2-4). In single channel
patches, activities appeared as bursts of openings lasting 1.3-30
s (Fig. 1) separated by long (several minutes) closings. In
contrast, multichannel patches remained continuously active
(Fig. 1D and 2-4), and channels were often
observed to ``lock'' into the open state, with no channel
gating for long periods (mean, 8.8 ± 1.3 s; 16 observations from
5 patches) (Fig. 3A). Channel activities were often
observed as simultaneous gating of two channels (Figs. 3B and
4B; 11/39 and 4/14 patches from
F508-CFTR and wild-type
CFTR nuclei, respectively). Such pronounced cooperative gating appears
to be characteristic of CFTR in the ER and may suggest that CFTR
functions as a dimer that favors pairs of channels being simultaneously
open. Cooperative gating of plasma membrane-localized CFTR has also
been observed
(31) . The very long closed times in single channel
patches and prevalence of simultaneous gating of channel pairs have not
been reported for plasma membrane-localized CFTR, but the similar
properties of wild-type and
F508-CFTR Cl
channels we observed suggest that these different kinetics are
due to ER localization rather than altered conformation induced by the
F508 mutation. Since processing of proteins in the ER is likely
mediated by protein interactions, the apparent strong cooperative
behavior of ER-localized CFTR, by suggesting CFTR-CFTR interactions,
raises the possibility that such interactions may participate in
retention or stability of normal and mutant CFTRs in the ER.
Figure 4:
Properties of wild-type CFTR
Cl channels in the outer nuclear membrane. A and B, PKA-dependent activation. The channels were
observed with PKA in the pipette (14/18 patches) but not in its absence
(9/10 patches). In the remaining patch, the endogenous Cl
channel was present. In A and B recordings
obtained alternately from the same nucleus in the presence and absence
of pipette PKA confirmed the PKA requirement for channel activation
(6/6 patches). This figure is representative of 10 experiments.
A, multichannel patch with at least 4 channels. In single
channel patches, P
= 0.42,
= 37 ms, and
= 40 ms (80% of
closures) and 80 ms (20% of closures) within bursts. B,
simultaneous gating of pairs of wild-type CFTR Cl
channels. The binomial probability of one channel open was 0.46
± 0.01, whereas the observed probability was 0.07 ± 0.03
(p < 0.003). This figure is representative of 4
experiments. Current records in A and B are from
different patches at indicated pipette potentials. Data in A were filtered at 200 Hz. C, linear I-V relationship for
currents shown in A and B. The slope conductance
under symmetrical solutions was 7.9 ± 0.7 pS (3 determinations
at each indicated voltage from 6 patches). D, insensitivity of
multilevel wild-type CFTR Cl
channels to DIDS (0.3
mM). This figure is representative of 5
experiments.
Figure 3:
Kinetic features of F508-CFTR
Cl
channels in the outer nuclear membrane.
A, long open state frequently observed in
F508-CFTR
Cl
multichannel patches. Channels became
``locked'' in the open state during the interval indicated by
the double-arrowed line. This figure is
representative of 16 observations. B, simultaneous gating of
pairs of
F508-CFTR Cl
channels. The probability
of observing both channels open simultaneously (0.49) was used to
calculate P
(39). The binomial probability of
observing one channel open was 0.42 ± 0.11, whereas the observed
probability was 0.16 ± 0.05. Thus, the probability of each
current level did not follow a binomial distribution for two
independent channels (p < 0.04). C, O1,
and O2 represent closed and two open levels, respectively.
This figure is representative of 11 patches.
CFTR
Cl channel conductance has been detected in plasma
membranes and endosomes
(32, 33) , but our evidence that
wild-type CFTR can function as a regulated Cl
channel
in the ER represents the first measurements of CFTR activity in the
biosynthetic pathway. Because both wild-type as well as
F508-CFTR
function in the ER and their half-lives there are
similar
(17, 34) , ER retention of the mutant CFTR will
not be expected to affect the ionic composition of the ER lumen. In
contrast, function of wild-type CFTR in the ER suggests that retention
of
F508-CFTR will result in the absence of normal Cl
channel activity in post-ER compartments. Abnormal trans-Golgi pH
and sialylation and mucoprotein sulfation observed in CF have been
proposed to be due to aberrant Cl
channel activity in
Golgi compartments
(26) , which may also account for altered
activities of other ion channels in CF airway
cells
(35, 36, 37) . By demonstrating that CFTR
may normally function en route to the plasma membrane, our results
provide experimental evidence consistent with such hypotheses. Lack of
CFTR function in the post-ER biosynthetic pathway may therefore
contribute to some symptoms and abnormal cellular physiology in CF.
Because our results demonstrate that CFTR can function as a regulated
Cl
channel in the biosynthetic pathway, it will now
be important to determine if it functions in this pathway in
vivo. Many other mutations in CFTR also cause its intracellular
retention
(18, 38) . The methods we have described will
now enable examination of those mutant CFTRs to determine whether they
also retain Cl
channel activities, as well as to
determine whether other plasma membrane ion channels are functional
immediately after their biosynthesis in the ER, which may have
implications for cell-specific regulation of organellar ion composition
and function.
channel activity when it is
localized in the ER in mammalian cells suggests that conformational
changes caused by the
F508 mutation, although recognized by
ER-retention
mechanisms
(8, 13, 14, 15, 16) ,
do not necessarily affect CFTR Cl
channel properties
or activation by cAMP-dependent mechanisms. The
F508 mutation
causes some loss of structure in synthetic peptides (22), but our data
indicate that
F508-CFTR is unlikely to be grossly misfolded when
it is retained in the ER in vivo. By suggesting that CFTR
domains involved in ER retention may not necessarily contribute to CFTR
Cl
channel function, our results may have
implications for therapeutic interventions based on delivery of mutant
CFTR to the plasma membrane, since they suggest that pharmacological
reagents that could promote escape of CFTR from the ER might do so
without adversely affecting its Cl
channel activity,
a prerequisite for restoring normal function.
,
N-methyl-D-glucamine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.