1 Department of Medicine, Section of Pulmonary and Critical Care Medicine, The
University of Chicago Hospitals, 5841 S. Maryland Avenue, MC 6026, Chicago, IL
60637, USA
2 Department of Neurobiology, Pharmacology and Physiology, The University of
Chicago, 947 East 58th St, MC 0926, Chicago, IL 60637, USA
3 Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis
Research Center, University of Alabama at Birmingham, 1918 University Blvd,
MCLM 985, Birmingham, AL 35294, USA
Author for correspondence (e-mail:
dnelson{at}drugs.bsd.uchicago.edu
)
Accepted 4 November 2001
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Summary |
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Key words: Membrane capacitance, FM1-43, Exocytosis, Endocytosis, SNARE proteins, Trafficking, Chloride channel, Voltage clamp
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Introduction |
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The problem of channel regulation has increased complexity in the case of
CFTR, where cAMP not only activates the channel through
protein-kinase-A-dependent phosphorylation but in some investigations appears
to mobilize heterologously expressed CFTR into the plasma membranes of
Xenopus oocytes (Takahashi et
al., 1996). The question of whether an enhancement of membrane
trafficking accompanies CFTR activation, independent of syntaxin modulation,
remains controversial. For example, CFTR activation in Calu-3 cells (a human
lung adenocarcinoma-derived line) as well as Madin Darby canine kidney (MDCK)
cells is not accompanied by changes in membrane insertion and retrieval
(Chen et al., 2001
;
Loffing et al., 1998
;
Moyer et al., 1998
). However,
a number of other investigations carried out in a variety of cell types
demonstrated that cAMP facilitates translocation of CFTR from an intracellular
compartment to the plasma membrane
(Bradbury et al., 1992
;
Howard et al., 2000
;
Peters et al., 1999
;
Schwiebert et al., 1994
;
Takahashi et al., 1996
).
Experiments carried out in this study sought to resolve two related issues: (a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Using a combination of membrane capacitance measurements coupled with FM1-43 fluorescence we demonstrate that cAMP-dependent changes in membrane turnover that accompany CFTR activation are cell specific and can be revealed in some cell types only when dynamin-dependent endocytosis is inhibited. In addition, cAMP-dependent changes in membrane turnover are independent of the disruption of syntaxin 1A-CFTR interactions.
We have previously shown that syntaxin 1A and CFTR physically interact and
that this interaction results in a negative modulation of cAMP-mediated CFTR
function (Naren et al., 1997;
Naren et al., 1998
;
Naren et al., 1999
;
Naren et al., 2000
).
Furthermore, three different reagents rescue CFTR from inhibition by
plasma-membrane-anchored syntaxin 1A, namely, botulinum toxin C1, the
syntaxin-binding protein Munc 18 and the cytosolic domain of syntaxin 1A
(Naren et al., 1997
). The
interaction between CFTR and membrane-anchored syntaxin 1A is dynamic
(reversible) and is dependent upon an intact actin cytoskeleton. Data in this
investigation demonstrate that while changes in membrane turnover do not
appear to be dependent upon the presence or absence of a direct interaction
between membrane-anchored syntaxin 1A and CFTR, the interaction does result in
a decrease in single channel open state probability. Disruption of the
t-SNARE-channel interaction by the cytosolic domain of syntaxin 1A results in
a significant increase in CFTR open-state probability in excised patches.
These results indicate that binding of full length syntaxin 1A to CFTR
inhibits PKA-dependent activation through direct protein-protein interaction.
We speculate that second messenger pathways that regulate the physical
interaction of these two proteins via alterations in the actin cytoskeleton or
CFTR-syntaxin-Munc-18 interactions will fine tune channel activity through
subtle changes in membrane-resident channel gating and not alterations in
membrane turnover.
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Materials and Methods |
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Electrophysiology
Whole-cell recordings were obtained from 16HBE14o- human airway epithelial
and from HT29-CL19A human colonic epithelial cells using methods described
previously (Hamill et al.,
1981) and solutions published previously
(Naren et al., 1997
). The cAMP
cocktail used to elicit CFTR activation in the whole-cell experiments
contained 1 mM IBMX, 10 µM Forskolin and 400 µM cpt-cAMP. Peptides were
added to the pipette solution at their specified concentrations.
Single-channel recordings were obtained from 16HBE14o- cells using the
inside-out configuration. The extracellular (pipette) solution contained 140
mM NMDG-Cl, 2 mM MgCl2, 10 mM HEPES and 200 µM DIDS (to
pharmacologically block non-CFTR anion channels) titrated to a pH of 7.4 with
NMDG. The intracellular (bath) solution contained 140 mM NMDG-Cl, 10 mM EGTA,
2 mM MgCl2, 8 mM Tris and 5 mM Mg-ATP adjusted to a pH of 7.4. Once
the inside-out configuration was achieved, 75 U/ml of PKA catalytic subunit
(Promega, Madison, WI) was added to activate CFTR. Subsequently, either 350 nM
GST-Syn1A
C or 350 nM GST-Syn1A
H3 were added to the bath. In this
manner, the same inside-out patch served as its own internal control.
Single-channel currents were filtered at 1 kHz and sampled at 2 kHz. All
experiments were conducted at room temperature (22-24°C) using an EPC-9
patch clamp amplifier (HEKA Electronik GmbH, Lambrecht, Germany) and using the
Pulse V 8.31 acquisition program (HEKA Electronik GmbH, Lambrecht, Germany).
Data analysis was performed using Tac V4.1.1 (Bruxton Corp., Seattle, WA,
USA). Statistical significance of results was determined using the Student's
t-test.
Simultaneous recording of membrane capacitance, conductance, and
fluorescence
The EPC-9 includes a built-in data acquisition interface (ITC-16,
Instrutech, Port Washington, NY, USA). The software package controlled the
stimulus and data acquisition for the software lock-in amplifier in the `sine
+ dc' mode as described (Gillis,
2000). The temporal resolution of the capacitance data was 40
mseconds per point using a 1 kHz, 20 mV sine wave. The holding potential in
the capacitance experiments was -10 mV. The fluorescence intensity of FM1-43
excited at 470 nm was simultaneously measured with a photomultiplier system as
described (Katnik and Nelson,
1993
) using a Leitz inverted DM-IRB microscope. The
photo-multiplier output was collected at 510 nm and averaged online. These
data were stored together with the Cm and Gm
measurements. Pipette and bath solutions were as described above. Peptides
were added to the pipette solutions as in whole-cell experiments. All
experiments were conducted at room temperature (22-24°C). Statistical
significance of results was determined using the Student's
t-test.
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Results |
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|
Rescue of CFTR from inhibition by membrane-anchored syntaxin 1A by the cytosolic domain of syntaxin 1A was dependent on the actin cytoskeleton. If the 16HBE14o- cells were treated with cytochalasin D (0.5 µg/ml), which disrupts the actin cytoskeleton, cAMP-dependent current activation was not significantly different from that observed under control conditions (Fig. 2). However, the syntaxin 1A cytosolic domain failed to rescue CFTR from membrane-anchored syntaxin 1A inhibition in the cytochalasin-treated cells. Thus, the presence of an intact cytoskeleton appears to maintain the reversibility of the dynamic interaction between CFTR and membrane-anchored syntaxin 1A. This result suggested to us that CFTR current augmentation could involve a trafficking event mediated via cytoskeletal interactions.
|
Regulation of CFTR by syntaxin 1A and PKA does not involve membrane
turnover
Reasoning that cytoskeleton-dependent vesicle insertion or inhibition of
membrane endocytosis could account for the soluble syntaxin 1A rescue of CFTR
inhibition by membrane anchored syntaxin 1A, we measured membrane turnover,
ICFTR activation and the regulation of these parameters by the
cytoplasmic domain of syntaxin 1A in the 16HBE14o- cells. Membrane capacitance
(Cm) is proportional to cell surface area, and the measurement of
changes in Cm can give insight into the change in surface area
resulting from exocytosis and endocytosis
(Neher, 1988;
Neher and Marty, 1982
). Time
resolved changes in membrane capacitance provide a direct and quantitative
record of the time course of plasma membrane turnover in response to stimuli.
Conductance (Gm) was markedly augmented by dissociation of the
syntaxin 1A-CFTR interaction by either GST-Syn1A
C
(Fig. 3A and B) or GST-Munc18
(Fig. 3B). As expected,
addition of 350 nM GST-Syn1A
H3 to the pipette solution did not result
in an increase in Gm. Under all conditions tested, the capacitance
of the 16HBE14o- cells remained unchanged. Thus, neither cAMP activation alone
nor cAMP activation in combination with GST-Syn1A
C or GST-Munc18 had
any detectable effect on Cm in human airway cells.
|
Studies of capacitance are limited by the fact that they measure net
changes in cell surface area owing to simultaneous exocytosis and endocytosis.
Examination of each process independently could lend valuable insight into the
underlying membrane trafficking events. Exocytosis can be examined
independently of endocytosis using the fluorescent styryl dye FM1-43, since it
labels but does not permeate cell membranes. Fluorescence intensity of cells
in the presence of FM1-43 remains unchanged during the process of endocytosis.
As vesicles fuse with the plasma membrane, however, the newly exposed surface
is labeled by the FM1-43 and fluorescence intensity increases. Thus,
exocytosis can be measured without contamination from endocytotic processes
(Smith and Betz, 1996). As
summarized in Fig. 3B, we
detected no changes in FM1-43 fluorescence upon activation of CFTR in the
16HBE14o- cells with or without GST-Syn1A
C, GST-Munc18 or
GST-Syn1A
H3 (fluorescent intensity only increased 1.1 to 1.3% under the
various conditions where n=4-10).
Dynamin mediates different types of endocytosis in various cell types
(Hinshaw, 2000). Disruption of
its function with anti-pan-dynamin IgG (
-Dyn) inhibits rapid
endocytosis in chromaffin cells (Artalejo
et al., 1995
; Elhamdani et al.,
2000
). Therefore, we carried out a set of experiments to determine
if we could detect effects of syntaxin-1A-CFTR disruptive reagents on
Cm or Gm by blocking endocytosis with
-Dyn.
Disruption of the syntaxin-1A-CFTR interaction with either GST-Syn1A
C
or GST-Munc18 did not change capacitance in 16HBE14o- human airway epithelial
cells even in the presence of
-Dyn, suggesting again that membrane
recycling is not involved in the augmentation of ICFTR by the
cytosolic domain of syntaxin 1A (Fig.
3). Capacitance changes were also not apparent in the presence of
GST-Syn1A
H3 plus
-Dyn. Thus, using two different assays, we
could not detect an increase in exocytosis during the time course of the
current augmentation response, whether or not the interaction between syntaxin
1A with CFTR was disrupted.
The possibility that SNARE-dependent membrane turnover contributes to the
regulation of ICFTR in other epithelial cell types lead us to
examine the t-SNARE-channel interaction in a cell line of colonic origin.
Support for these experiments came from reports that cAMP induces the
insertion of heterologously expressed CFTR into the plasma membranes of
Xenopus oocytes in the absence of syntaxin 1A
(Peters et al., 1999). As it
had been previously shown that cAMP stimulates apical protein secretion in
CFTR-expressing HT29-CL19A human colonic epithelial cells
(Jilling and Kirk, 1996
), we
assayed Gm, Cm and FM1-43 exocytosis in these cells in
order to determine if membrane trafficking contributes to CFTR regulation by
syntaxin 1A in cells of gastrointestinal origin. Results of these experiments
are given in Fig. 4. We failed
to observe an increase in Cm in response to cAMP in the presence or
absence of GST-Syn1A
C or GST-Munc18. However, there was a significant
increase in the FM1-43 fluorescence following the addition of the cAMP
cocktail, indicating that the simultaneity of the processes of exocytosis and
endocytosis might result in an undetectable change in membrane capacitance.
The cAMP-induced change in Cm in the HT29-CL19A cells was not
detectable unless dynamin-dependent endocytosis was inhibited. The addition of
-Dyn to the pipette in the presence of the cytosolic domain of syntaxin
1A resulted in a large increase in Cm, conductance and
fluorescence. This increase in Gm was, however, not significantly
different from that observed with GST-syn 1A
C alone, indicating that
inhibition of membrane retrieval did not contribute to Gm
augmentation by either GST-Syn1A
C or GST-Munc18
(Fig. 4B). Moreover, the
increase in Gm induced by cAMP in the presence of
-Dyn was
essentially complete before the increase in Cm was detectable,
indicating that inhibition of membrane turnover was not causally linked to the
conductance increase as seen in Fig.
5. There were no measurable changes in either capacitance or
conductance induced by
-Dyn alone in the absence of activation with
cAMP, as summarized in Fig.
6.
|
|
|
Syntaxin 1A decreases the open probability of CFTR
In order to examine the hypothesis that syntaxin 1A regulates CFTR by
direct protein-protein interactions rather than through a modulation of
membrane trafficking, we performed single channel studies using excised
inside-out patches obtained from 16HBE14o- cells the results of which are
illustrated in Fig. 7. The
extracellular (pipette) solution contained 200 µM DIDS to pharmacologically
block non-CFTR anion channels. In these experiments, the reversal potential
for Cl- was zero, and Cl- was the only permeant ion in
both bath and pipette solutions (see Methods). Once the inside-out
configuration was achieved, 75 U/ml of PKA catalytic subunit was added to the
bath solution to activate CFTR. Patches were exposed to the catalytic subunit
only if background Cl- currents were not observed during a 300
second baseline recording period. A channel with the characteristics of CFTR
was associated with the addition of the catalytic subunit to the bath as seen
in Fig. 7A. Channel activity
was observed following exposure of the excised patch to the catalytic subunit
of PKA and was determined to be CFTR on the basis of the following criteria:
(1) current activation was observed in the presence of 200 µM DIDS to the
pipette (extracellular solution), (2) currents had a linear I-V with a single
channel conductance of 6 pS as expected for CFTR and (3) currents were not
observed in the absence of the catalytic subunit. Patches with an open-state
probability of greater than approximately 0.10 following PKA activation were
discarded. Low open-state probability patches were subsequently exposed to
either 350 nM GST-Syn1AC or 350 nM GST-Syn1A
H3 added to the
bath. Selection of patches with initial low levels of current activation
allowed for an accurate quantitation of CFTR rescue from inhibition by
membrane-anchored sytaxin 1A via the soluble reagents. We observed increases
in channel activity in 5 of the 9 patches exposed to GST-Syn1A
C as seen
in Fig. 7A. The apparent level
of patch variability was probably due to cytoskeletal disruption during patch
excision that prevented the rescue of CFTR from inhibition by the cytosolic
domain of syntaxin 1A in the whole cell experiments
(Fig. 2). However, even if the
cytoskeleton were intact, such variability in response to the soluble syntaxin
1A peptide would not be unexpected as the syntaxin 1A-CFTR interaction is
reversible and likely to be dynamic such that not all channels would be
expected to be bound to syntaxin 1A at a given time.
|
Analysis of open times (o) for a representative patch
revealed no change prior to or after disruption of the syntaxin-1A-CFTR
interaction (Fig. 7C). Closed
time analysis revealed that disrupting syntaxin 1A from CFTR resulted in a
decreased closed time (
c) consistent with an increase in the
likelihood of opening events. Open probability (Po) increased
threefold (similar to the increase seen in whole-cell current measurements)
after addition of GST-Syn1A
C from 0.06±0.03 to 0.19±0.04
(Fig. 7D). Channel activity,
both prior to and after interruption of the syntaxin-1A-CFTR interaction, was
stable for the duration of each 300 second segment of recording. The changes
in Po after addition of GST-Syn1A
C were immediate. To
examine the specificity of the response, we tested GST-Syn1A
H3 for its
effects on CFTR activity in excised membrane patches. Po was
unaffected by the soluble peptide lacking the CFTR binding domain as
summarized in Fig. 7D.
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Discussion |
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In agreement with studies in Calu-3 cells that are derived from human
airway submucosal glands and express high levels of CFTR
(Shen et al., 1994), we found
that ICFTR activation in human airway epithelia did not involve
membrane trafficking (Chen et al.,
2001
; Loffing et al.,
1998
). Rather, the increase in Cm occurred
independently of the increase in current. This is not necessarily incongruous
with studies that demonstrate that exocytosis of CFTR-containing vesicles are
involved in current activation (Lehrich et
al., 1998
; Takahashi et al.,
1996
). As has been suggested, the mechanism by which cAMP
stimulates CFTR activation may be variously regulated in different cell lines
and, furthermore, may be dependent on the state of tissue differentiation
(Guggino, 1998
;
Moyer et al., 1998
).
Endocytosis is the cellular process that serves the dual function of
maintaining cell surface area constant while retrieving vesicular components
for recycling. The severing activity that releases endocytotic vesicles from
the plasma membrane is controlled by the dynamin family of proteins, which
show high levels of GTPase activity. Dynamin is found in cells in both a
soluble and membrane-associated form and is thought to oligomerize around the
neck of retracting vesicular structures
(Henley et al., 1999). Rapid
endocytosis in neurons requires GTP hydrolysis, and the critical G protein
regulating this process appears to be dynamin
(Artalejo et al., 1995
;
Artalejo et al., 1997
). The
increase in exocytosis induced by cAMP and unmasked by
-Dyn that we
observed for the HT-29 cells is normally balanced by a compensatory increase
in endocytosis in a manner analogous to that in kidney epithelial cells
expressing aquaporin 2 water channels
(Katsura et al., 1995
).
Consistent with this interpretation, we were unable to observe an effect of
-Dyn on Cm unless exocytosis was stimulated by cAMP
(Fig. 4B). There were no
measurable changes in either capacitance or conductance in the presence of
-Dyn in the absence of activation with cAMP
(Fig. 6). These results
indicated that membrane-anchored syntaxin 1A inhibits CFTR via direct
interactions rather than by modulation of membrane insertion in both human
colonic and airway epithelial cells. In addition, cAMP activates two distinct
processes in colonic epithelial cells: one involving activation of CFTR and
the other involving membrane trafficking.
Understanding the complexities of CFTR regulation is an important
prerequisite in the design of novel therapeutic strategies targeting cystic
fibrosis and perhaps secretory diarrhea. We have previously shown that
syntaxin 1A binds specifically and stoichiometrically to the N-terminal tail
of CFTR (Naren et al., 1998).
The N-terminal tail of CFTR modulates PKA-dependent channel gating, possibly
by interacting with the cytoplasmic regulatory (R) domain
(Naren et al., 1999
). We
hypothesize that membrane-bound syntaxin 1A sterically prevents the N-terminus
from interacting with its R domain, thus resulting in a decrease in
Po. The present findings demonstrate that CFTR ion channel function
can be stimulated directly by reagents that disrupt the association of
syntaxin 1A with its N-terminal tail. While syntaxin 1A may also influence the
intracellular traffic of CFTR in some systems (e.g. Xenopus oocytes)
(Peters et al., 1999
), this
does not appear to be the case in epithelial cells. We show, in this study,
that CFTR is modulated directly by SNARE interactions. A possible
physiological role of such SNAREion-channel interactions may well link
the activities of certain ion channels and transporters to membrane traffic
events such as exocytosis in tissues for which such coordination is
functionally advantageous. Given the growing body of evidence regarding direct
SNARE-ion transport interactions, we speculate that SNAREs play roles both in
modulation of ion channels and in trafficking of proteins and vesicles.
In summary, our data demonstrate that cAMP augments membrane turnover in a cell-specific manner. The cAMP-induced changes in membrane turnover, when observed, are not correlated with cAMP-induced changes in CFTR activation. In fact, the conductance change appears to be complete before the capacitance change is initiated. Finally, dissociation of syntaxin 1A from CFTR does not alter the cAMP-induced enhancement of membrane trafficking that we observed for the colonic secretory epithelial cells. Thus, in chloride transporting epithelia, there is a clear separation of the modulatory from the trafficking or anchoring functions of the syntaxins, and this is likely to be a generalized phenomenon for all membrane ion channels that interact with the ubiquitous SNARE proteins.
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Acknowledgments |
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![]() |
Footnotes |
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