(Received for publication, October 23, 1995; and in revised form, November 15, 1995)
From the
The common F508 mutation in the cystic fibrosis
transmembrane conductance regulator (CFTR) interferes with the
biosynthetic folding of nascent CFTR polypeptides, leading to their
retention and rapid degradation in an intracellular compartment
proximal to the Golgi apparatus. Neither the pathway by which wild-type
CFTR folds nor the mechanism by which the Phe
deletion
interferes with this process is well understood. We have investigated
the effect of glycerol, a polyhydric alcohol known to stabilize protein
conformation, on the folding of CFTR and
F508 in vivo.
Incubation of transient and stable
F508 tranfectants with 10%
glycerol induced a significant accumulation of
F508 protein
bearing complex N-linked oligosaccharides, indicative of their
transit to a compartment distal to the endoplasmic reticulum (ER). This
accumulation was accompanied by an increase in mean whole cell cAMP
activated chloride conductance, suggesting that the glycerol-rescued
F508 polypeptides form functional plasma membrane CFTR channels.
These effects were dose- and time-dependent and fully reversible.
Glycerol treatment also stabilized immature (core-glycosylated)
F508 and CFTR molecules that are normally degraded rapidly. These
effects of glycerol were not due to a general disruption of ER quality
control processes but appeared to correlate with the degree of
temperature sensitivity of specific CFTR mutations. These data suggest
a model in which glycerol serves to stabilize an otherwise unstable
intermediate in CFTR biosynthesis, maintaining it in a conformation
that is competent for folding and subsequent release from the ER
quality control apparatus.
Cystic fibrosis (CF), ()a lethal hereditary
exocrinopathy affecting approximately one in two thousand live births
among populations of Caucasian or northern European descent, is caused
by the functional absence of a plasma membrane chloride channel,
designated cystic fibrosis transmembrane conductance regulator
(CFTR)(1) . The vast majority of severe CF cases in these
populations is linked to a single genetic lesion, deletion of a
phenylalanine codon (
F508)(2, 3) , which
interferes with the folding of newly synthesized CFTR polypeptides.
Nascent
F508 molecules fail to traffic to the plasma membrane (4) but rather are retained by the ER quality control mechanism
that prevents unfolded or misfolded proteins and unassociated subunits
from exiting the ER. Instead, these retained immature
F508
molecules are rapidly degraded (5, 6) in a pre-Golgi
compartment by a process that appears to require covalent modification
by ubiquitin(7) . Moreover, plasma membrane CFTR-like
Cl
channel activity can be detected when
F508 is
overexpressed (8) or synthesized at reduced temperature (9) , suggesting that Phe
does not play an
essential role in CFTR function and raising the possibility of
therapeutic intervention in CF by increasing the efficiency of
F508 folding.
Glycerol and other polyols are known to stabilize
protein conformation (10) , increase the rate of in vitro protein refolding(11) , and increase the kinetics of
oligomeric assembly(12) . We report here that treatment of
F508-expressing cells with glycerol dramatically stabilizes newly
synthesized
F508 polypeptides and leads to the accumulation, in
the plasma membrane, of stable, functional CFTR Cl
channels.
The effect of glycerol treatment on steady-state expression
of F508 was initially evaluated by immunoblot analysis of
detergent extracts of HEK 293 cells transfected with
F508 cDNA (Fig. 1). In untreated cells, only the immature (core
glycosylated, 140 kDa) form was detected in cells incubated at 37
°C (Fig. 1A), as previously observed(5) .
However, a diffuse immunoreactive band, corresponding to the mobility
of mature (complex glycosylated, 165 kDa) CFTR, was apparent in
extracts of cells treated with glycerol for 24 h. The steady-state
levels of mature
F508 induced by glycerol or by incubation at
reduced temperature in these cells were similar, but the effects appear
to be additive. The small difference in mobility between mature
F508 rescued by reduced temperature and that rescued by glycerol
is probably due to differences in terminal glycosylation(15) .
The effect of glycerol on the maturation of
F508 was also observed
in a stable line of C127 mammary carcinoma cells expressing
F508
cDNA (16) (Fig. 1B), indicating that this
phenomenon is not unique to transiently transfected HEK cells. Glycerol
significantly increased expression of mature
F508 in these cells
above basal levels and above the levels induced by incubation at 26
°C. The effect on steady-state expression of mature
F508 in
C127 cells was maximal at 10% glycerol; concentrations above or below
this level did not support
F508 maturation. By contrast, similar
levels of
F508 maturation were observed in HEK cells between 8 and
15% (data not shown). Glycerol did not appear to be acutely toxic to
either cell type. Cell viability (determined by trypan blue exclusion)
after 24-h exposure to 10% glycerol was between 75% in HEK cells and
90% in C127 cells. Mature
F508 accumulated to clearly detectable
levels in C127 cells 6 h following the addition of glycerol and
continued to accumulate up to 48 h (Fig. 1C). This
effect was reversible; the level of mature
F508 in
glycerol-treated C127 cells decreased with time following glycerol
removal at a rate consistent with the half-life of mature
F508
protein estimated from pulse-chase experiments (see below). Taken
together, these data demonstrate that exposure of
F508-expressing
cells to 10% glycerol partially rescues the ``misprocessing''
phenotype of the mutant protein.
Figure 1:
Effect of
glycerol on steady-state expression of F508. A, HEK cells
expressing
F508 cDNA were treated with 15% glycerol at either 26
or 37 °C for 24 h prior to lysing and analyzed by immunoblotting
for steady-state levels of mature (m) or immature (i)
F508. B, concentration dependence of glycerol effect in
C127 cells. Cells expressing
F508 (lanes 2-6) were
incubated for 24 h with media supplemented with the indicated
concentrations of glycerol and evaluated by immunoblotting for mature
or immature
F508. Wild-type (wt) CFTR expressed in C127
cells (lane 1) is included for reference. In lane 7 (asterisk) cells were incubated in the absence of
glycerol for 24 h at 26 °C. C, time course of mature
F508 accumulation in cells incubated in the presence of 10%
glycerol.
The effect of glycerol on F508
processing could not be replicated by incubating the cells with similar
concentrations of other structurally related polyols (1,2-propanediol
or 1,3-propanediol), perhaps because of lower permeability of cell
membranes to these agents. Similarly, dimethyl sulfoxide (2%) did not
support
F508 maturation; higher concentrations were not tested
because of its toxicity. Glycerol, a small (M
= 98) polyhydric alcohol, is highly permeant across the
plasma membrane of animal cells(17, 18) . In HEK cells
incubated at 7.5% glycerol, [
H]glycerol
equilibrated rapidly (t
,
5 min) across
the plasma membrane (data not shown). As glycerol is uncharged its
distribution across the plasma membrane is independent of membrane
potential. Thus, it is likely that intracellular and extracellular
glycerol concentrations rapidly equilibrate in HEK cells and that
glycerol's effect on
F508 processing is due to its high
intracellular concentration.
At least two mechanisms could account
for the effect of glycerol on the accumulation of mature F508. One
possibility is that glycerol stabilizes a
F508 folding
intermediate, which is normally rapidly diverted to the degradation
apparatus. The stabilized folding intermediate would remain competent
to fold into a conformation resistant to proteolysis and permissive for
maturation beyond the ER. A second possibility is that glycerol acts by
increasing the stability of a mature but unstable
F508 polypeptide
that has escaped ER retention. To discriminate between these models,
the kinetics of
F508 maturation and degradation were evaluated by
pulse-chase labeling and immunoprecipitation in
F508-transfected
HEK cells (Fig. 2). In control cells not treated with glycerol,
label in the band corresponding to immature
F508 decayed rapidly
and was nearly undetectable after 6 h of chase (Fig. 2A). The t
of this decay was
estimated to be 45 min (Fig. 2, A and D),
similar to previously reported values(5) . No label was
detected at the mobility corresponding to the mature protein. By
contrast, in the presence of glycerol, the kinetics of immature CFTR
degradation were significantly slowed (t
=
87 min); some of this label was clearly chased into mature
F508 (Fig. 2, A and D). The fractional conversion
of immature
F508 in glycerol-treated (Fig. 2E)
cells ranged between 3 and 8% in separate experiments, which is
considerable when compared with the
20-25% efficiency of
wild-type CFTR processing. The kinetics of wild-type CFTR degradation
were also slowed by glycerol, suggesting that the effect of glycerol is
not unique to the folding of
F508 molecules (Fig. 2, C and D). Glycerol had no measurable effect on the
stability of mature
F508 or CFTR (Fig. 2, B and E). These data suggest that accumulation of mature
F508
in glycerol-treated cells is not the result of an effect on the mature
protein and that glycerol stabilizes the immature form of
F508.
However, inhibition of
F508 degradation, either directly with
protease inhibitors or indirectly by blocking ubiquitination, also
stabilizes the immature form of the protein but, unlike glycerol
treatment, does not result in any accumulation of mature
forms(7) . This disparity in the fate of stabilized immature
F508 molecules suggests that glycerol maintains immature
F508
in a maturation-competent state, either by inhibiting reactions that
are off pathway or by enhancing reactions that are on the folding
pathway.
Figure 2:
Effect of glycerol on degradation and
maturation of CFTR and F508. HEK cells expressing
F508 (A and B) or CFTR (C) were pulse-labeled and chased
in the presence or absence of 10% glycerol for the times indicated. m, mature; i, immature. D, kinetics of
immature
F508 (squares) or CFTR (circles)
degradation in the presence (closed symbols) or absence (open symbols) of glycerol. Inset, semilogarithmic
plot of data in D. E, kinetics of
F508 (squares) or CFTR (circles) maturation in the
presence (open symbols) or absence (closed symbols)
of glycerol. Data shown are derived from two experiments and are
representative of 3-5 independent
trials.
To rule out the possibility that the effects of glycerol on
F508 processing and degradation are due to a general disruption of
the ER quality control machinery, we examined the effect of glycerol on
the maturation and degradation of other CFTR mutants that, like
F508, are unable to escape the ER (Fig. 3). Cells
expressing the missense mutants D572A and S1251A and a mutant harboring
a deletion of exon 13 (
EX13) were pulse-labeled with
[
S]Met and chased for 5 h in the presence or
absence of glycerol (Fig. 3A). These mutants were
synthesized as immature polypeptides that were degraded and, unlike
F508, failed to mature even in the presence of glycerol. Thus,
some mutations in all three major cytoplasmic domains of CFTR,
including the first and second nucleotide binding domains (D572A and
S1251A, respectively) as well as the ``R'' domain, can lead
to a glycerol-insensitive ER retention phenotype, suggesting that
glycerol treatment does not induce a general suppression of ER
retention mechanisms. The efficiency of processing and the ability to
be rescued by glycerol are also highly dependent upon the nature of the
substituted amino acid in CFTR Lys
missense mutants (Fig. 3B). Processing of mutants K464R and K464A was
inefficient by comparison with wild type and was enhanced by incubation
in the presence of 10% glycerol, even after accounting for the unequal
label present in the immature precursor in the presence of glycerol (Fig. 3B). By contrast, no maturation was detectable
for the mutant K464W in the presence or absence of glycerol. These data
support the argument that glycerol rescue of CFTR maturation is not the
result of a general suppression of ER quality control and suggest a
correlation between the ``leakiness'' of the mutation and its
ability to be remediated by glycerol.
Figure 3:
The
effect of glycerol on non-F508 CFTR mutants. HEK cells expressing
D572A,
EX13, and S1251 (A) or various substitutions at
Lys
(B) were pulse-labeled (p) for 15
min and chased for 5 h (c) in the presence or the absence of
glycerol. The mobilities of the mature (m) and immature (i) forms of CFTR and immature
EX13 (i*) are
indicated for reference.
Although these data establish
that glycerol treatment facilitates the maturation of F508
molecules to a post-ER compartment, they do not establish that the
``rescued''
F508 molecules actually move to and are
functional at the plasma membrane. To test the functional surface
expression of glycerol-rescued
F508 molecules, whole cell CFTR
currents were examined in HEK cells expressing CFTR or
F508 by the
patch-clamp technique (Fig. 4). Large (56 ± 4
pA/picofarad) rapidly activated, cAMP-dependent Cl
selective currents were readily observed in cells expressing CFTR
but not in control (not glycerol-treated) cells expressing
F508
(1.54 ± 0.13 pA/picofarad). By contrast, significant (13.31
± 2.3 pA/picofarad; p < 0.002 compared with
untreated; Student's t test) Cl
currents, although slower activating than wild type, were
observed in glycerol-treated cells expressing
F508 cDNA. These
currents were not observed in the absence of cAMP, as expected of CFTR.
The difference in maximal current level and activation kinetics between
CFTR and glycerol-treated
F508 expressing cells is likely due both
to the lower steady-state expression of mature
F508 and to the
lower open probability of the mutant channels(19) .
Figure 4:
Glycerol treatment induces the expression
of plasma membrane F508 CFTR Cl
channels in the
HEK cells. A, the mean whole cell maximum current density
measured at -50 mV. The currents were divided by the cell
capacitance in order to compare results from cell to cell. The currents
in
F508 cells not exposed to cAMP (n = 8, column 1) or not glycerol-treated (n = 6, column 2) had maximum current at t = 0 after
going whole cell. The currents in glycerol-treated
F508 cells
exposed to protein kinase A and/or cAMP (n = 9, column 3) increased slowly and peaked at
3 min.
CFTR-expressing cells responded rapidly to intracellular cAMP (n = 3, column 4), peaking at less than 1 min. pF, picofarad. B, typical whole cell currents seen in
3 cells exposed to intracellular cAMP. The currents on the left are the currents seen upon break in, and the currents on the right are currents near the peak of the response. The currents
shown are in response to voltage steps from +100 mV to -75
mV in steps of -25 mV from a holding potential of -50 mV.
The currents showed linear current-voltage (IV) behavior and no time
dependence.
Collectively, these data suggest a model in which glycerol, a short
chain polyhydric alcohol, serves to stabilize an otherwise unstable
intermediate in CFTR biosynthesis, maintaining it in a conformation
that is competent for folding and its subsequent release from the
quality control apparatus. As degradation of immature CFTR and
F508 is initiated without an apparent lag following
translation(5) , we propose that glycerol serves to stabilize
nascent
F508 chains soon after or during translation. In this
role, glycerol would function as a chemical chaperone, much as HSP70
serves as a molecular chaperone. Our data suggest that these effects
are not due to a generalized breakdown of ER quality control nor to
stabilization of cell surface mature
F508 molecules that have
escaped quality control surveillance. We hypothesize that glycerol
stabilizes an early intermediate in CFTR folding that lies at a branch
point between productive folding (on pathway) and competing
non-productive (off pathway) steps. In this respect the effect of
glycerol on
F508 is similar to ``osmotic remedial''
mutants previously observed in yeast (20) and Escherichia
coli(21) . These mutations are temperature-sensitive and
can be reversed by increasing the osmotic potential of the incubation
medium causing the microorganisms to synthesize and accumulate high
concentrations of intracellular osmolytes such as
glycerol(22) . Interestingly, we observe a strong correlation
between the temperature sensitivity of CFTR mutations like
F508,
K464R, and K464A (data not shown) and their ability to be remediated by
glycerol. Glycerol may provide a useful tool to manipulate the
temperature-sensitive phenotypes of CFTR and perhaps other genes at
non-permissive temperatures.
Our data establish the precedent that
both the intracellular processing and the membrane Cl transport phenotypes of the
F508 mutation can be remediated
by chemical means. These data should stimulate a search for other small
membrane-permeant molecules, which may be more effective or more easily
delivered than glycerol at enhancing
F508 processing. Finally,
these data may have implications for the study or treatment of other
diseases, including Alzheimer's, retinitis pigmentosa, and
proteinase inhibitor deficiency that are associated with protein
misfolding.