(Received for publication, August 1, 1995; and in revised form, September 21, 1995)
From the
The cystic fibrosis transmembrane conductance regulator (CFTR)
contains two membrane-spanning domains; each consists of six
transmembrane segments joined by three extracellular and two
intracellular loops of different length. To examine the role of
intracellular loops in CFTR channel function, we studied a deletion
mutant of CFTR (19 CFTR) in which 19 amino acids were removed from
the intracellular loop joining transmembrane segments IV and V. This
mutant protein was expressed in a human embryonic kidney cell line (293
HEK). Fully mature glycosylated CFTR (
170 kDa) was
immunoprecipitated from cells transfected with wild-type CFTR cDNA,
while cells transfected with the mutant gene expressed only a
core-glycosylated form (
140 kDa). The chloride efflux rate
(measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ
fluorescence) from cells expressing wild-type CFTR increased 600% in
response to forskolin. In contrast,
19 CFTR-expressing cells had
no significant response to forskolin. Western blotting performed on
subcellular membrane fractions showed that
19 CFTR was located in
the same fractions as
F508 CFTR, a processing mutant of CFTR.
These results suggest that
19 CFTR is located in the intracellular
membranes, without reaching the cell surface. Upon reconstitution into
lipid bilayer membranes,
19 CFTR formed a functional
Cl
channel with gating properties nearly identical to
those of the wild-type CFTR channel. However,
19 CFTR channels
exhibited frequent transitions to a 6-picosiemens subconductance state,
whereas wild-type CFTR channels rarely exist in this subconductance
state. These data suggest that the intracellular loop is involved in
stabilizing the full conductance state of the CFTR Cl
channel.
The cystic fibrosis transmembrane conductance regulator (CFTR) ()consists of five distinct regions, with two putative
membrane-spanning domains, two nucleotide-binding folds, and a
regulatory domain(1) . CFTR forms a Cl
channel of linear conductance(2, 3) , which is
regulated by cAMP-dependent protein kinase phosphorylation (2, 4, 5, 6) at multiple sites in
the regulatory domain and by binding and hydrolysis of ATP by the
nucleotide-binding folds (7, 8, 9, 10) . The structure and
function of these five domains of CFTR have been extensively
studied(11, 12, 13, 14) . In
contrast, little is known about the role of intracellular loops and
their contribution to the function of the CFTR Cl
channel.
The intracellular loops of other channel proteins
appear to participate in channel function. For instance, mutations in
the second intracellular loop of the Shaker K channel
affect channel inactivation(15) . Deletion of a portion of the
putative cytosolic loops between two transmembrane repeats of the
Na
channel slows the rate of channel
inactivation(16) . To investigate the role of the intracellular
loops of CFTR in Cl
channel function, we studied a
deletion mutant of CFTR (
19 CFTR) in which 19 amino acids were
removed from the intracellular loop joining transmembrane segments IV
and V in the first membrane-spanning domain. This is the largest and
most hydrophilic loop in the first membrane-spanning domain, as
one-third of the residues in this loop are charged. These features make
this loop a candidate for electrostatic or allosteric interactions with
other cytosolic domains of CFTR (nucleotide-binding folds, the
regulatory domain, or other intracellular loops) and cellular proteins
to contribute to channel function.
Figure 5:
Subcellular fractionation and Western
blotting of CFTR. A, a discontinuous sucrose gradient with
five densities (28, 33, 36, 38.7, and 43.7%) was used to separate the
plasma membrane from intracellular membranes. B, shown is the
top of the gradient (lane I), 28/33% (lane II),
33/36% (lane III), 36/38.7% (lane IV), 38/43.7% (lane V), and pellet (lane VI). 50 µg of total
protein from each fraction were subjected to 5% SDS-polyacrylamide gel
electrophoresis; the CFTR protein was detected as described under
``Experimental Procedures.'' Protein markers are labeled in
the middle. C, plasma (PM) and intracellular membrane (IM) fractions of F508 CFTR- and
19 CFTR-expressing
cells grown at 37 °C were run on a 5% SDS-polyacrylamide gel and
detected as described for B. Protein markers are labeled on
the left. Arrowhead a, wild-type (WT) CFTR protein
(
170 kDa); arrowhead b,
F508 CFTR protein (
140
kDa); arrowhead c,
19 CFTR protein (
140
kDa).
Figure 1: Predicted topology of CFTR. The predicted membrane topology of CFTR is shown, with the deleted 19 amino acids indicated. L, loop; TM, transmembrane segment; NBF, nucleotide-binding fold; R, regulatory.
Figure 2:
Immunoprecipitation and Western blotting
of CFTR. 293 HEK cells transfected with pCEP4(WT), pCEP4(F508), or
pCEP4(
19) were immunoprecipitated and blotted as described under
``Experimental Procedures.'' A similar number of cells were
lysed for each cell line. The proteins were visualized by exposing the
blot to a Kodak X-Omat AR film for 3 min. Lane 1,
untransfected 293 HEK cells; lanes 2 and 3, wild-type
CFTR-expressing cells; lanes 4 and 5,
F508
CFTR-expressing cells; lanes 6 and 7,
19
CFTR-expressing cells. Lanes labeled 26 °C contained cells
cultured to 80
90% confluence at 37 °C (5% CO
),
followed by incubation at 26 °C (6% CO
) for 2
days.
Figure 3:
SPQ
assay of Cl transport. Cells loaded with SPQ were
preincubated with nitrate solution (126 mM NaNO
, 5
mM KNO
, 1.5 mM
Ca(NO
)
, 1.0 mM
Mg(NO
)
, 20 mM Hepes, 0.1% bovine
serine albumin, 0.1% D-glucose, pH 7.2) on ice for 15 min to
allow the depletion of intracellular chloride. At the beginning of the
experiment, the cells were first perfused with nitrate solution until
the fluorescence was stable (
1 min). Perfusion solution was then
switched to Cl
-containing solution. SPQ fluorescence
was quenched as Cl
entered the cell. When
fluorescence reached its steady state, perfusion was switched to
nitrate solution until the fluorescence came back to its base line.
This cycle was repeated with 10 µM forskolin (FSK) added to the Cl
and nitrate solution.
At the conclusion of the experiment, cells were perfused with KSCN (150
mM) plus valinomycin (VAL; 10 µM)
solution to quench all the fluorescence. Each trace represents the
fluorescence of a single cell from the beginning to the end. A, untransfected 293 HEK cells; B, wild-type (WT) CFTR-transfected cells; C,
19
CFTR-transfected cells. Fluorescence of five to six cells was plotted
for each of the above three cases.
Figure 4:
Forskolin stimulation of the rate of
Cl efflux. The Cl
efflux rate was
calculated as described under ``Experimental Procedures'' and
is plotted as the relative fluorescence units/minute (A). Open bars indicate the rate of basal Cl
efflux; hatched bars indicate the rate of Cl
efflux upon forskolin (FSK; 10 µM)
stimulation. The percentage of forskolin-stimulated Cl
efflux over basal Cl
efflux is also plotted (B). The untransfected cells (Control) have a
forskolin-stimulated efflux rate of 157 ± 7% of the base line
(198 cells at 37 °C, n = 7). The wild-type (WT) CFTR-transfected cells have a forskolin-stimulated efflux
rate of 618 ± 37% of the base line (297 cells at 37 °C, n = 8). The
19 CFTR-expressing cells have a
forskolin-stimulated efflux rate of 178 ± 15% of the base line
(170 cells at 37 °C, n = 10), which is not
statistically different from the control. Data labeled with 26
°C (wild-type CFTR: 453 ± 144% forskolin stimulation,
40 cells, n = 3;
19 CFTR: 90 ± 10%
forskolin stimulation, 65 cells, n = 4) were obtained
from cells incubated at 26 °C for 15-48 h before the
experiment.
Figure 6:
Reconstitution of CFTR Cl channel in lipid bilayer membranes. Represented traces are
selected single channel currents at the given test potential from a
wild-type CFTR channel (A) and a
19 CFTR channel (B). Both wild-type and
19 CFTR channels exhibited a
linear current-voltage relationship with a slope conductance of 8.2
± 0.6 pS and an extrapolated reversal potential of +22.2
± 1.4 mV.
, wild-type CFTR (n = 76);
,
19 CFTR (n =
14).
The
reconstituted CFTR channels had slow kinetics of gating. The open
lifetime histogram contained at least two exponentials, with mean open
lifetimes of = 23.6 ms and
= 111.9 ms (Fig. 7A). The relative
occurrence of
was y
/(y
+ y
) = 0.38. An average open probability (p
) of 0.318 ± 0.028 was measured at
-80 mV. In separate studies, we found that the wild-type CFTR
channel contained two distinct subconductance states with conductances
of 6 pS (O2) and 2.7 pS, in addition to the full conductance state (8
pS, O1). (
)The occurrence of O2, however, was low for
wild-type CFTR and accounted for <10% of the open events (see Fig. 10A).
Figure 7:
Open
time histograms at -80 mV. Open events were calculated at
-80 mV with wild-type (WT; A) and 19 (B) CFTR channels. The histograms were constructed with a
total of 4040 open events obtained in three separate experiments for
wild-type CFTR and of 4843 open events obtained in four separate
experiments for
19 CFTR. The solid lines represent the
best fit according to the following equation: y = y
/
exp(-t/
) + y
/
exp(-t/
), where
= 23.6 ms,
= 111.9 ms, and y
/(y
+ y
) = 0.38 (A) and
= 30.6 ms,
= 115.8 ms, and y
/(y
+ y
) = 0.48 (B).
Figure 10:
Open
current histogram at -80 mV. The current amplitudes of the
individual open events were calculated with the 50% threshold detection
method using the pClamp software. The currents were sorted at a
resolution of 0.0025 pA/bin. The histogram in A contained
three separate experiments with the wild-type (WT) CFTR
channel, and that in B contained four separate experiments
with the 19 CFTR channel. The solid lines represent the
best fit with the sum of two gaussian distribution functions: y = W
/(&cjs3484;(2
)
)
exp(-(x -
µ
)
/
) + W
/(&cjs3484;(2
)
)
exp(-(x -
µ
)
/
), where W
= 5.04, µ
=
-0.63 pA,
= 0.07 pA, W
= 0.49, µ
= -0.45 pA, and
= 0.06 pA (A) and W
= 8.32, µ
= -0.66 pA,
= 0.06 pA, W
=
1.97, µ
= -0.47 pA, and
= 0.07 pA (B).
When 19 CFTR was incorporated into
the lipid bilayer membrane, functional Cl
channel
activity was identified. The full conductance state (O1) of the
19
CFTR Cl
channel was identical to that of the
wild-type CFTR channel (Fig. 6C and Fig. 8).
Similar to the wild-type CFTR channel, the
19 CFTR channel had an
average open probability of 0.308 ± 0.043 and mean open
lifetimes of
= 30.6 ms and
= 115.8 ms at -80 mV (Fig. 7B). The
relative occurrence of
was y
/(y
+ y
) = 0.48. Thus, gating of the
19
CFTR channel is not significantly different from that of the wild-type
CFTR channel at a time resolution of 100-Hz cutoff frequency in the
bilayer system.
Figure 8:
Subconductance states of 19 CFTR.
Consecutive traces for wild-type (WT) CFTR and
19 CFTR
were measured at a test potential of -80 mV. In both cases, the
bilayer contained three active CFTR channels. O1 corresponds to the
full open state (
8 pS), and O2 corresponds to the subconductance
state of the channel (
6 pS).
A significant difference between 19 CFTR and
wild-type CFTR was found in the distribution of channel subconductance
states. Unlike the wild-type CFTR channel, the
19 CFTR channel
displayed a prominent subconductance state (O2; Fig. 8). Fig. 9shows the amplitude histogram of two separate experiments
with the wild-type and
19 CFTR channels. The histograms were
different from each other, particularly in the first open level. The
frequent occurrence of subconductance state resulted in an asymmetric
distribution of the first open level of the
19 CFTR channel
(indicated by O1 and O2). Similar phenomena were observed in 13 other
experiments with
19 CFTR channels. There was a relative paucity of
instances of two channels in these recordings, especially for the
19 mutant, in records in which two channels are clearly present (e.g.Fig. 8and Fig. 9). The reasons for this
are unclear, and detailed analysis is beyond the scope of this paper.
Figure 9:
Amplitude histogram of CFTR channel. Each
histogram was constructed with 16 episodes of data shown in Fig. 8. All acquisition points were included. The three open
levels of wild-type (WT) CFTR could be fitted by gaussian
distributions (first open level amplitude of -0.68 pA, second of
-1.41 pA, and third of -2.11 pA). The first open level of
the 19 CFTR channels could be fitted by two gaussian distribution
functions with amplitudes of -0.66 pA (O1) and -0.45 pA
(O2).
Fig. 10shows the amplitude histograms of open channel current
pooled from multiple experiments in which a single CFTR channel was
incorporated into the bilayer membrane. Clearly, the 19 CFTR
channel contained more openings to the O2 state than the wild-type CFTR
channel (Fig. 10, compare A with B). The
histograms could be fitted with the sum of two gaussian distribution
functions, with mean currents of -0.65 pA (O1) and -0.46 pA
(O2), at -80 mV. The relative occurrence of the O2 state was
estimated at 9% for the wild-type CFTR channel and at 23% for the
19 CFTR channel.
In this study, we examined the function of a CFTR deletion
mutant, 19 CFTR. This deletion mutation results in incomplete
glycosylation and intracellular retention of CFTR based on the
following observations. (i) An immunoprecipitation/Western blot assay
identified a core-glycosylated form of
19 CFTR (
140 kDa),
different from the fully mature glycosylated protein (
170 kDa);
(ii) subcellular fractionation showed the
19 CFTR protein
localized predominantly in the intracellular membranes; and (iii) an
SPQ assay of cells expressing
19 CFTR showed no
forskolin-stimulated Cl
transport. However, this
processing mutant maintained functional Cl
channel
activity, presumably in the intracellular organelles, when
reconstituted into lipid bilayer membranes. The most notable difference
between wild-type and
19 CFTR channels lies in the distribution of
conductance states. The deletion mutation caused frequent occurrence of
a subconductance state within the Cl
channel.
The
most common disease-causing mutation of CFTR is the deletion of a
single phenylalanine residue at position 508 (F508 CFTR). Most of
the
F508 CFTR protein is retained in the endoplasmic reticulum and
fails to reach its intended site of action in the plasma membrane. With
regard to processing at 37 °C,
19 CFTR appears to be similar
to
F508 CFTR. However,
F508 CFTR appears in the fully mature
glycosylated form and can traffic to the plasma membrane following
incubation at lower temperature (26-30 °C) (Fig. 2)(22, 23) . Unlike
F508 CFTR, the
misprocessing of
19 CFTR is temperature-insensitive: the protein
remained in the core-glycosylated state in intracellular organelles
even at 26 °C. The
F508 CFTR proteins, once at the plasma
membrane, give rise to functional Cl
channels similar
to those of wild-type CFTR in terms of open probability (26) and conductance state(24, 26) .
A
cardiac-specific isoform of CFTR lacks 30 amino acids in the
intracellular loop between transmembrane segments II and III. It is
caused by alternative splicing of exon 5 of the CFTR mRNA(27) .
Cardiac CFTR functions as a cAMP-dependent protein kinase-regulated
Cl channel with conduction properties similar to
those of epithelial CFTR(9, 28) . However, when
expressed in HeLa cells, human CFTR lacking exon 5 failed to generate a
cAMP-mediated chloride transport by the SPQ assay, apparently due to
defective intracellular processing(29) . The strategy applied
here to the study of CFTR channel function can be used to study even
misprocessed or mislocalized channels and therefore ought to be
applicable to the study of the effects of deleting exon 5 on CFTR
channel function as well.
The 19 amino acids deleted in 19 CFTR
span a major part of the intracellular loop joining transmembrane
segments IV and V. This segment is highly hydrophilic, with about
one-third of the residues being charged (two lysines and four
glutamates). Several factors could account for the subconductance state
associated with the
19 CFTR channel.
One possibility is that
retention of the 19 CFTR protein in intracellular membranes alters
the function of the Cl
channel. Maturation of CFTR
from the endoplasmic reticulum through the Golgi apparatus to the
plasma membrane involves several processing and post-translational
modifications (e.g. folding and glycosylation). The
endoplasmic reticulum membrane contains other proteins, including
various types of chaperons, that could interact with CFTR and alter the
function of the Cl
channel. However, the gating
kinetics and open probability of the
19 CFTR channel in the
intracellular membrane are similar to those of the wild-type CFTR
channel in the plasma membrane, so the deletion of these 19 amino acids
did not alter the essential function of the CFTR Cl
channel. Moreover, the wild-type CFTR channel occasionally enters
the O2 subconductance state characteristic of the
19 CFTR channel.
These observations suggest that the subconductance state associated
with the
19 CFTR Cl
channel is probably an
intrinsic property of the CFTR protein, but the deletion affects CFTR
in such a way as to favor the O2 conductance state.
A second
possibility is that this loop interacts with other domains
(nucleotide-binding folds, the regulatory domain, and other
intracellular loops) in CFTR or with other cellular proteins that are
involved in the conductance state transition. Deletion of part of this
loop might then affect the channel structure allosterically by changing
these inter- or intramolecular interactions and thus induce a
quasi-stable open configuration in addition to the full open state.
Understanding the distribution of subconductance states associated with
the single Cl channel should provide valuable
information about structure-function relationships in the CFTR
Cl
channel.
The approach reported here allows
functional characterization of CFTR in any intracellular membranes
besides the endoplasmic reticulum membrane(25) . By preparing
vesicles from intracellular membranes separated from the plasma
membrane, in principle, one can study any processing mutant in the
lipid bilayer system. Further experiments are necessary to test the
role of specific amino acids of the intracellular loops (e.g. positively and negatively charged and hydrophobic amino acids) in
the regulation of the conductance state of the CFTR Cl channel.