Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302-305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopus oocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin's effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.
membrane traffic; SNARE proteins; immunofluorescence; capacitance; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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CYSTIC FIBROSIS IS CAUSED by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which accounts for the cAMP-stimulated Cl conductance at the apical membranes of many secretory and absorptive epithelial cells (22). Biochemical and electrophysiological studies have demonstrated that CFTR Cl channel activity requires protein kinase A (PKA)-mediated phosphorylation of its R-domain (18, 31) and binding and hydrolysis of ATP at one or both nucleotide binding domains (1, 23, 26). In addition to facilitating CFTR Cl channel gating, cAMP/PKA stimulation of CFTR-expressing cells evokes changes in membrane trafficking. cAMP stimulates exocytosis and inhibits endocytosis, and these regulated trafficking events require the expression of wild-type CFTR (5). The close correlation observed between CFTR-dependent increases in Cl current and membrane capacitance suggests that CFTR resides in the membrane vesicles whose trafficking is influenced by cAMP-dependent agonists (25, 30). Accordingly, Cl current stimulation by cAMP/PKA may arise from increases in channel open probability (Po) and/or from increases in the number (N) of active CFTR Cl channels residing in the plasma membrane.
Results of recent studies have implicated a role for membrane traffic regulatory proteins in the control of CFTR Cl currents. Naren et al. (20) showed that syntaxin 1A is expressed in colonic secretory epithelial cells, and they detected an interaction between CFTR and syntaxin 1A using in vitro protein binding assays (21). Coexpression of syntaxin 1A with CFTR in Xenopus oocytes inhibited cAMP-stimulated Cl currents, and this inhibition could be reversed by the syntaxin binding protein, munc-18. They proposed that syntaxin 1A and munc-18 regulate CFTR, and thus Cl secretion, at the apical membranes of secretory epithelial cells.
Using measurements of plasma membrane capacitance and cell surface expression of CFTR, we demonstrate here that syntaxin 1A expression interferes with cAMP/PKA-regulated CFTR trafficking. We used epitope-tagged CFTR to show that cAMP stimulation elicits an increase in plasma membrane CFTR content, which is paralleled by a cAMP-induced increase in plasma membrane capacitance. Syntaxin 1A coexpression selectively inhibited the cAMP-induced increase in membrane capacitance and reduced the amount of CFTR detected in the plasma membrane. This syntaxin-induced reduction in N is consistent with the role of this protein as a component of the membrane fusion machinery.
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MATERIALS AND METHODS |
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Oocyte preparation. Oocyte isolations and RNA injections were performed as described previously (8, 11). Xenopus laevis was obtained from Xenopus 1 (Ann Arbor, MI). Oocytes were isolated surgically and follicular cells removed during a 60- to 90-min collagenase incubation (2 mg/ml, type 2; Worthington). Stage 5-6 oocytes were maintained in a modified Barth's solution overnight before injection (50 nl vol) with cRNA for CFTR, syntaxin 1A, or syntaxin 3. Noninjected oocytes served as controls. Expression was allowed to proceed at 18°C for 1-3 days before current recordings or immunofluorescence measurements were performed.
Electrophysiology. The ND-96 solution
utilized for the current measurements contained (in mM) 96 NaCl, l KCl,
1.8 CaCl2, 1.0 MgCl2, and 5 HEPES. Recordings of
CFTR-dependent Cl current
(ICl) and
membrane capacitance
(Cm) changes
were performed as described in Ref. 30. Briefly, cells were impaled
with two 3 M KCl-filled electrodes connected to a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA) via Ag-AgCl pellet
electrodes and referenced to the bath by an Ag-AgCl pellet. Membrane
potentials were allowed to stabilize before current and capacitance
recordings. The voltage clamp was controlled by an AD/DA interface
board (AXOLAB 1100) using a PC-running software generated in the
laboratory. Membrane capacitance was calculated from the time course of
current decay during a 10-mV hyperpolarizing voltage pulse. The decay
time constant was derived from a single exponential fit of the pulse
currents, and Cm
was calculated on-line as described (30). Steady-state membrane
currents were recorded at 60 mV; these inward currents reflect
Cl flow through CFTR during stimulation by cAMP, as shown in prior
studies (8, 11). Values of
ICl and
Cm were obtained at 2-s intervals; steady-state values are given in the figures.
Cell surface CFTR expression. We used immunofluorescence techniques to monitor the expression of CFTR in the oocyte plasma membrane. Cells were injected with cRNA encoding an epitope-tagged CFTR, which contains the flag epitope (DYKDDDK) inserted into the predicted fourth extracellular loop of CFTR, after amino acid position 901, and designated M2-901/CFTR. Prior studies have shown that this flag-tagged CFTR behaves identically to its wild-type counterpart during macroscopic current stimulation (13), and single channel measurements detect no difference in channel gating or PKA/ATP regulation relative to wild-type CFTR (24).
M2-901/CFTR-expressing oocytes were subjected to a staining protocol to quantitate CFTR protein expression at the cell surface using the monoclonal M2 antibody (Kodak). Oocytes were cooled rapidly to 0-4°C and incubated with M2 antibody overnight at this temperature (1:1,250 dilution). After they were washed with 5% FCS-ND-96, oocytes were incubated at 4°C in fluorescein-conjugated goat anti-mouse IgG (1:100 dilution) and then washed five times as above. This protocol avoids permeabilization or fixation conditions that might expose intracellular flag-CFTR to the M2 antibody. Thus the staining conditions and epitope position were selected to permit detection of M2-901/CFTR only when it is localized in the plasma membrane and the epitope is exposed at the cell surface. For some experiments, CFTR Cl currents were recorded from M2-901/CFTR-expressing oocytes before implementing the staining protocol. This permitted CFTR Cl currents and cell surface expression to be determined in the same cells. In other experiments, oocytes were fixed and permeabilized to assess total flag-CFTR labeling using ice-cold Caltag reagents (proprietary). Cells were immersed in 200 µl medium A for 15 min, washed once in 5% BSA-C (Aurion)/ND-96, and placed in 200 µl medium B for 30 min. After three washes in BSA-C/ND-96, they were subjected to the staining protocol described above.
Individual oocytes were scanned on a Molecular Dynamics multiprobe 2001 laser confocal microscope at ×10 magnification using 10-µm optical sections. Sections were scanned at 512 × 512 pixels (488 nm laser, 510 nm primary beam splitter, 510 nm secondary beam splitter). Control (uninjected) oocytes were scanned to set the fluorescence background; the laser intensity and voltage were set accordingly. Image planes throughout the depth of the specimen were collected. A quantitative measure of protein expression at the surface of the oocyte was derived from a maximal intensity rendered image. The periphery of the image was delineated and the mean pixel intensity calculated.
Reagents. cRNAs were transcribed in vitro using the Message Machine kit (Ambion) from linearized plasmids. Rat syntaxin cDNAs were kindly provided by the laboratory of Dr. Richard Scheller (Stanford University, Stanford, CA). Their expression was confirmed by immunoprecipitation of 35-kDa proteins from oocytes expressing syntaxin 1A or syntaxin 3 (data not shown). Plasmids encoding the control proteins (see Fig. 5) were gifts of the laboratories of Drs. Ernst Wright (University of California, Los Angeles, CA), John Edelman (Vollum Institute), and David Julius (University of California, San Francisco, CA). Botulinum toxin/C1 (BoT/C1) was obtained from Wako Chemical Industries (Japan) and injected in 25-nl PBS. All results are expressed as means ± SE.
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RESULTS |
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cAMP increases plasma membrane CFTR.
Figure 1 shows composite
images of intact, nonpermeabilized oocytes expressing epitope-tagged M2-901/CFTR (Fig. 1, B and
C). A representative image from a
control, noninjected oocyte is displayed in Fig.
1A. CFTR currents and cell surface
fluorescence were determined 2-3 days after cRNA injection. In
noninjected oocytes, background fluorescence was low (Fig.
1A). Oocytes expressing
M2-901/CFTR under nonstimulated conditions showed low levels of
surface staining that were slightly above background, suggesting a low
level of CFTR expression in the plasma membrane in the absence of
agonist stimulation (Fig. 1B).
M2-901/CFTR oocytes stimulated with forskolin/IBMX for 15 min
showed a large and significant increase in surface fluorescence (Fig.
1C). The low fluorescence intensity
detected in the absence of agonist suggests that the staining protocol
does not permit access of the M2 antibody to intracellular epitope. In
assays performed on permeabilized oocytes, the addition of
forskolin/IBMX had no influence on fluorescence intensity using the
same labeling conditions (CFTR basal, 25.1 ± 3.6; CFTR stimulated,
25.6 ± 3.8; n = 6). This result
indicates that recognition of the flag epitope by the M2 antibody does
not require CFTR stimulation.
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Average data from three experiments in nonpermeabilized oocytes are provided in Fig. 1D. Also shown are the results from parallel measurements of steady-state current obtained before and after addition of forskolin/IBMX; these values are consistent with the magnitude of CFTR current stimulation observed in prior studies (8, 11). The immunofluorescence data indicate that the increase in Cl current is paralleled by an increase in plasma membrane CFTR. For oocytes in the stimulation group, the staining protocol was initiated 15 min after addition of forskolin/IBMX, the time when Cl currents rise to their plateau level. Thus detection of the M2 epitope at the cell surface occurs within the time frame required for the cAMP-induced increases in Cl current and membrane capacitance (30). These findings are consistent with the idea that cAMP/PKA stimulation induces the insertion of CFTR-containing vesicles into the plasma membrane.
Syntaxin 1A inhibits cAMP/PKA-induced increases in
plasma membrane capacitance and CFTR. The effects on
ICl and
Cm of syntaxin coexpression with CFTR are illustrated in Fig.
2. When syntaxin 1A was coexpressed with
CFTR, Cl current stimulation by cAMP was inhibited, as reported by
Naren et al. (20). In parallel with the syntaxin-induced reduction in
Cl current, cAMP stimulation of membrane capacitance is also markedly
inhibited. The inhibition of changes in both
ICl
(ICl) and
Cm
(
Cm)
is selective for syntaxin 1A. Coexpression of syntaxin 3 with CFTR did
not affect these parameters significantly, suggesting that these
effects are syntaxin isoform specific. This finding also eliminates
translational competition between the CFTR and syntaxin cRNAs as an
explanation of the inhibitory effect of syntaxin 1A on the
agonist-induced
ICl and
Cm.
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The effect of syntaxin 1A coexpression on CFTR detected at the oocyte
surface during cAMP/PKA stimulation is illustrated in Fig.
3. As in Fig.
1C, these images were collected from
oocytes stimulated with maximally effective concentrations of forskolin and IBMX (see legend to Fig. 3). The noninjected control
oocyte illustrates the background level of cell surface staining in the presence of agonists (Fig. 3A). This
low fluorescence intensity indicates that cAMP stimulation does not
increase background fluorescence in the absence of CFTR expression
(compare with Fig. 1A). During cAMP stimulation, M2-901/CFTR was readily detected in the surface membrane (Fig. 3B), but coexpression
with syntaxin 1A markedly reduced plasma membrane CFTR during cAMP
stimulation (Fig. 3C). This result is consistent with the
syntaxin 1A-induced decrease in cAMP-dependent
Cm, shown in
Fig. 2. As with the membrane capacitance and current responses,
syntaxin 3 coexpression had no effect on cell surface CFTR staining
(data not shown). Quantitation of the results from four experiments
(Fig. 3D) shows that CFTR surface expression correlates with the magnitude of currents recorded from the
same oocytes before antibody labeling. With syntaxin 1A coexpression,
cell surface CFTR fluorescence intensity fell nearly to the level of
the water-injected controls. Thus insertion of CFTR into the plasma
membrane is required for activation of the CFTR Cl currents recorded
from these cells during stimulation, and syntaxin 1A reduces CFTR Cl
currents by decreasing the amount of CFTR in the plasma membrane during
stimulation.
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The effects of BoT/C1 on CFTR
ICl and
Cm with and
without syntaxin 1A coexpression are shown in Fig.
4. We injected BoT/C1 60 min before
recording the current and capacitance responses to forskolin/IBMX.
BoT/C1 acutely reversed the syntaxin 1A inhibition of
ICl and
Cm, presumably
due to cleavage of syntaxin 1A. The toxin did not interfere with the
ICl or
Cm responses
in oocytes injected with CFTR alone. This relatively rapid reversal of
CFTR function argues against an effect of syntaxin on the level of CFTR
protein expression as an explanation for the inhibitory effect on CFTR
current or capacitance. For comparison, the half time for inhibition of
CFTR currents in oocytes by brefeldin A is ~6 h (data not shown).
This finding indicates that the steady-state rates of CFTR production
and degradation in these cells are not sufficiently rapid for protein
synthesis to account for the BoT/C1 reversal of syntaxin's effects on
CFTR.
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Finally, we examined the influence of syntaxin coexpression on the
activities of several other plasma membrane transport or receptor
proteins. These control experiments were performed to examine the
possibility that syntaxin 1A may block the functional expression of
CFTR by generally interfering with the migration of proteins through
the biosynthetic pathway leading to the plasma membrane. cRNA amounts
were selected based on prior reports using these constructs (see
references below), and the currents obtained are similar
in magnitude. Coexpression of syntaxin 1A with the Na-dependent glucose transporter SGLT1 (12), the
ATP-sensitive Ca-activated K channel IK1 (14), or the P2Y2
purinergic receptor (17) had no effect on the currents associated with
activation of these proteins (Fig. 5, see
legend for activation conditions). In addition, syntaxin 1A did not
inhibit plasma membrane expression of the viral hemagglutinin (HA)
protein at the oocyte surface, which was detected by immunofluorescence
using techniques similar to those used for detection of cell surface
M2-901/CFTR [background-subtracted HA fluorescence
intensities: HA alone, 24.1 ± 4.1 (n = 6); HA + syntaxin 1A, 29.6 ± 5.0 (n = 5); cRNAs: 5 ng HA, 10 ng
syntaxin 1A]. These data show that syntaxin 1A does not impair HA
protein expression, even when the cRNA dose ratio is in favor of
syntaxin 1A. In addition, relative protein expression level for hIK1
can be judged from the current, which is comparable with that obtained from CFTR (compare Figs. 2 and 5). Because the single-channel current
amplitude and Po
of hIK1 are greater than those of CFTR (12), a similar macroscopic
current requires the expression of less protein. Despite this, hIK1
currents were not inhibited by syntaxin 1A, even at five times the dose
required for maximal inhibition of CFTR (see legends to Figs. 2 and 5).
Accordingly, syntaxin 1A is not simply disrupting the expression and
targeting of integral plasma membrane proteins, rather its inhibitory
effect was specific for CFTR.
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DISCUSSION |
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In a previous study (30), we showed that cAMP stimulation increased
both Cl current and membrane capacitance in CFTR-expressing Xenopus oocytes. These effects were
reversible with agonist washout and required CFTR expression. Changes
in ICl and
Cm were closely correlated with one another as the agonist dose or CFTR expression level were varied, or when CFTR mutants with different
ICl responses were expressed. The increase in cell surface area detected by
Cm was
confirmed by morphometry of the oocyte surface in the same cells used
for electrical recordings (30). This close correlation between
ICl and
Cm suggests
that CFTR activation by cAMP/PKA elicits reversible stimulation of
membrane vesicle insertion into the plasma membrane, a process that is
implicated in a variety of CFTR-expressing cells (4).
The results obtained from oocytes expressing an epitope-tagged CFTR verify this concept. Incorporation of an extracellular flag epitope permits detection of CFTR protein at the surface of nonpermeabilized cells (13). This approach obviates problems of resolving protein localization in the plasma membrane, since standard immunofluorescence techniques do not have sufficient resolution to distinguish protein in 100-nm submembrane vesicles from that in the plasma membrane. CFTR bearing a flag tag in its fourth extracellular loop was not detected appreciably at the surface of oocytes under basal, nonstimulated conditions, when CFTR Cl currents are near zero (Fig. 1). cAMP stimulation increased plasma membrane CFTR sevenfold within the time frame associated with stimulation of ICl and Cm (~15 min, Figs. 1 and 4). Control experiments indicated that M2 antibody staining of noninjected oocytes was not affected by cAMP stimulation, i.e., the surface labeling signal is CFTR and stimulation dependent. The correlations between cAMP-stimulated CFTR current, capacitance, and cell surface labeling show that CFTR insertion by exocytosis is an important event in the stimulation of plasma membrane Cl conductance of these cells by cAMP.
The oocyte has proven to be a useful model for studies of other
transporter-related membrane-trafficking events. Previous studies
include relocation of -aminobutyric acid transporters to the plasma
membrane in response to protein kinase C activation (7) and regulation
of the cell surface expression of several Na-dependent cotransport
processes by protein kinases A and C (12). In addition, the influence
of mutations in the amiloride-sensitive Na channel (ENaC), which
produce hypertension by prolonging the plasma membrane residency time
of ENaC, was characterized mechanistically using the oocyte expression
system (28).
The biochemical events that control membrane insertion and retrieval processes at the plasma membrane are thought to conform to principles first identified for vesicle transit among compartments of the protein biosynthetic pathway (2, 29). Interactions among proteins that comprise the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein complex control the targeting, docking, fusion, and retrieval of membrane vesicles that contain secretory products or integral membrane proteins, like CFTR, that will become constituents of the plasma membrane. The physiological importance of SNARE proteins is exemplified in the highly regulated process of neurosecretion; our understanding of the actions of these proteins in membrane-trafficking events in epithelial cells is relatively meager.
Our findings implicate a role for syntaxin in regulated CFTR trafficking. Coexpression of CFTR with syntaxin 1A reduced the cAMP-dependent membrane insertion event that is detected as an increase in membrane capacitance (Fig. 2). Syntaxin also blocked the increase in plasma membrane CFTR that occurs during stimulation of its activity by cAMP/PKA (Fig. 3). CFTR was not detected at significant levels in the plasma membrane under basal conditions (Fig. 1), and it was not present appreciably at the cell surface during cAMP stimulation in cells coexpressing syntaxin 1A. These effects were specific for CFTR, as opposed to other integral membrane proteins examined, they were selective for this syntaxin isoform, and they could not be explained by an effect of syntaxin on CFTR synthesis. Taken together, these findings indicate that syntaxin 1A interferes with cAMP/PKA-stimulated trafficking of CFTR to the cell surface.
What is the mechanism of this effect? The results of Naren et al. (20, 21) suggest that CFTR Cl currents are inhibited due to a physical interaction between these proteins. A protein-protein interaction has been observed also between N-type Ca channels and syntaxin 1A (27), which may contribute to syntaxin-associated downregulation of Ca currents (3, 32). Assuming that a physical interaction occurs between syntaxin 1A and CFTR in vivo, expressed syntaxin 1A may prevent CFTR insertion into the plasma membrane by binding with CFTR contained in vesicles that would normally fuse with the plasma membrane. Second, a CFTR-syntaxin 1A complex may be retrieved rapidly from the plasma membrane by endocytosis. Our measurements detect net membrane area (Cm) or steady-state plasma membrane CFTR and do not distinguish whether the effect of syntaxin is on insertion or retrieval of CFTR.
It is possible also that overexpressed syntaxin associates primarily
not with CFTR but with other SNARE components to block the membrane
fusion process. According to this view, syntaxin 1A overexpression
would disrupt the stoichiometric interactions among SNARE proteins that
are required for the plasma membrane fusion of CFTR-containing
vesicles. This effect of expressed syntaxin has been observed
previously. For example, exogenous expression of the Golgi t-SNARE,
syntaxin 5, inhibited protein traffic from endoplasmic reticulum to
Golgi, the step in which this syntaxin isoform functions (9). Likewise,
expression of syntaxin 1A, but not 1B, blocked glucose-stimulated
insulin secretion in pancreatic cells (19). In MDCK cells, syntaxin
3 is apically localized, and its exogenous expression selectively
inhibited apical targeting and apical recycling of the immunoglobulin
receptor (16). These findings suggest that overexpression of a specific
syntaxin isoform disrupts the pathway in which that isoform normally
plays a role in membrane-trafficking events. If this is true, then our
results suggest that endogenous SNARE proteins, including a
Xenopus syntaxin 1A ortholog, are
responsible for cAMP-regulated plasma membrane CFTR insertion in
oocytes. Expression of the core components of the SNARE machinery has
been observed in the cortical granules of sea urchin oocytes (6).
Accordingly, the SNARE fusion machinery would be employed to mediate
the membrane insertion of CFTR, and an interaction of CFTR with SNARE
proteins could explain how the expression of CFTR alone can confer
cAMP-dependent membrane-trafficking events on cells where none was
present before CFTR expression (5, 30).
The requirement for specific SNARE components in this process could also account for the apparent failure of certain cell types to support regulated CFTR-trafficking events (10, 15). The demonstration that syntaxin 1A is expressed in colonic secretory cells (1) is consistent with the concept that this protein, together with other components of the SNARE machinery, mediates the cAMP/PKA-regulated insertion of CFTR into the apical membranes of these cells. Although our findings suggest that the SNARE machinery plays an important role in the regulated trafficking of CFTR at the plasma membrane, they do not rule out a direct regulation of CFTR's gating (Po) that could result from a physical interaction with syntaxin. Accordingly, syntaxin could directly regulate CFTR Cl currents, perhaps in relation to SNARE protein-mediated membrane-trafficking reactions that control the amount of CFTR present in the plasma membrane.
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ACKNOWLEDGEMENTS |
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K. W. Peters and J. Qi contributed equally to this work.
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FOOTNOTES |
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We thank Megan Weiss for technical assistance, Patricia Connelly for help with the manuscript, and the laboratory of Richard Scheller for syntaxin cDNAs.
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50829 and by the Cystic Fibrosis Foundation Grants 97RO and 96PO.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. A. Frizzell, Dept. of Cell Biology and Physiology, S362 BST, 3500 Terrace St., Univ. of Pittsburgh School of Medicine, Pittsburgh, PA 15261 (E-mail: frizzell+{at}pitt.edu).
Received 19 February 1999; accepted in final form 5 April 1999.
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