From the Department of Physiology and Biophysics,
Gregory Fleming James Cystic Fibrosis Research Center, University
of Alabama at Birmingham, Birmingham, Alabama 35294, the
§ Department of Neurobiology, Pharmacology, and Physiology,
University of Chicago, Chicago, Illinois 60637, and the
¶ Department of Biological Sciences, University of Southern
California, HNB 228, Los Angeles, California 90089
Received for publication, November 19, 2002
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ABSTRACT |
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Syntaxin 1A binds to and inhibits epithelial
cystic fibrosis transmembrane conductance regulator (CFTR)
Cl The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is a cyclic
AMP-activated anion channel that mediates salt and fluid transport
across epithelial cells (1). The CFTR protein consists of a symmetric
arrangement of two membrane-spanning regions, two nucleotide binding
domains (nucleotide binding domains 1 and 2), and a central
regulatory domain with multiple phosphorylation sites (2). The
amino-terminal cytoplasmic tail binds to syntaxin 1A, a component of
the membrane traffic machinery, and this interaction is blocked by
Munc-18, a high affinity, syntaxin-binding protein (3). Syntaxin 1A
inhibits CFTR-mediated chloride currents in a variety of cell types and
expression systems. This effect of syntaxin 1A on CFTR channel activity
may be due to the fact that the amino-terminal cytoplasmic tail of
CFTR, to which syntaxin 1A binds, regulates channel gating apparently
by interacting with the regulatory domain and/or nucleotide binding
domain 1 (4).
Syntaxin 1A is a t-SNARE that is highly expressed in neurons and, to a
lesser extent, in a variety of epithelial cells (5). Together with
SNAP-25 (another t-SNARE) and VAMP-2/synaptobrevin (a v-SNARE),
syntaxin 1A assembles into core SNARE complexes that regulate membrane
fusion at the presynaptic membrane in neurons. The ternary SNARE
complex consists of a parallel four-helix bundle containing one
coiled-coil domain each from syntaxin and VAMP-2/synaptobrevin and two
from SNAP-25 (6). The four associating The helical region of syntaxin 1A that participates in SNARE complex
assembly, termed the SNARE motif or the H3 domain (8), is proximal to
the COOH-terminal membrane anchor region and lies downstream of another
helical domain referred to as Habc. The SNARE motif, which forms an
amphipathic helix, is composed of residues that are arranged in a
heptad repeat manner designated a-g. The residues that map
to heptad positions a and d are typically hydrophobic, well conserved across various syntaxin isoforms, and
usually buried in the hydrophobic core of the SNARE complex. Conversely, residues at the b, c, and
f heptad positions are more variable and are exposed on the
surface of the ternary complex. The cytoplasmic domain of syntaxin 1A,
encompassing the Habc and H3 domains, also interacts with Munc-18a with
high affinity, and this interaction prevents syntaxin 1A from
participating in SNARE complex assembly in vitro (9).
In addition to its role in the assembly of SNARE complexes, syntaxin 1A
also has been reported to modulate multiple types of ion channels and
transporters (3, 10-12). For example, syntaxin 1A inhibits the
activities of several types of voltage-gated Ca2+ channels
in a variety of expression systems and cell types. It has been proposed
that the simultaneous association of syntaxin 1A with synaptic vesicle
proteins and voltage-gated Ca2+ channels may help couple
SNARE complex formation/dissociation to the influx of Ca2+
at neurotransmitter release sites (13). With respect to CFTR-syntaxin interactions, syntaxin 1A, but not any other isoform that has been
tested (i.e. syntaxins 2-5), binds to the CFTR
amino-terminal tail (N-tail) and inhibits channel activity
(5).2 In addition, CFTR binds
to the H3 domain of syntaxin 1A, and soluble syntaxin 1A peptides that
lack the transmembrane region but include the H3 region can rescue
CFTR-mediated currents from inhibition by membrane-anchored syntaxin 1A
(3, 5). As argued for the syntaxin 1A-Ca2+ channel
interaction, the interaction between this t-SNARE and CFTR channels may
help synchronize the activity of this channel with protein traffic in
epithelial cells. Thus, the ability of syntaxin 1A to influence the
function of ion channels and to couple their activity to membrane
traffic may be a general phenomenon. However, it is not clear if the
structural basis of the interactions between syntaxin 1A and different
ion channels is similar. In this regard, Bezprozvanny et al.
(14) have reported that the regulation of voltage-gated
Ca2+ channels by syntaxin 1A is disrupted by point
mutations in specific hydrophobic residues in the H3 domain that are
also implicated in SNARE complex stability.
In the present study, we exploited the isoform specificity of the
syntaxin 1A interaction with CFTR to identify unique residues in the H3
domain of syntaxin 1A that participate in CFTR binding and channel
regulation. These residues are hydrophilic and are located in the outer
shell of the SNARE complex structure (i.e. distinct from
those in the hydrophobic layers that stabilize the SNARE complex).
Mutating these residues diminished both the physical and functional
interactions of syntaxin 1A with CFTR but had no effect on the
biochemical association of syntaxin 1A with other SNARE proteins or
with Munc-18. Conversely, the CFTR-syntaxin 1A interaction was not
affected by mutations in specific hydrophobic residues of the H3 domain
that disrupt Ca2+ channel regulation and compromise SNARE
stability. Our results indicate that syntaxin 1A regulates CFTR
channels by a protein-protein interaction that is mechanistically
distinct from how syntaxin 1A participates in SNARE complex assembly or
regulates voltage-gated calcium channels.
Antibodies--
A mouse monoclonal antibody to syntaxin 1A
(Cl 78.3) was obtained from Synaptic Systems (Germany). An
affinity-purified polyclonal antibody raised against human SNAP-23 and
a mouse monoclonal antibody raised against the COOH-terminal tail of
human CFTR have been described earlier (15). A mouse monoclonal
antibody raised against Munc-18a was purchased from Transduction Laboratories.
cDNA Constructs and Purification of GST Fusion
Proteins--
The cDNA encoding human syntaxin 1A was obtained
from a T84 cDNA library (3). Single and double alanine or leucine
substitutions in the H3 domain of syntaxin 1A were generated by PCR
mutagenesis. An appropriate mutagenic oligonucleotide was incorporated
in the PCR reaction using cDNA encoding human syntaxin 1A as
template. The upstream primer contained an EcoRI site, and
the downstream primer included a XhoI site. After digestion
with EcoRI and XhoI, the PCR product was ligated
back into the appropriate sites in the pCDNA3 eukaryotic expression
vector (Invitrogen). The cytosolic region (residues 1-266) of syntaxin
1A was amplified by PCR and subcloned into pGEX bacterial GST fusion
protein expression vector (Amersham Biosciences). All point
mutations were confirmed by DNA sequencing of the entire coding region
of the syntaxin 1A cytosolic domain. Full-length rat syntaxin 1A
mutated at two hydrophobic residues (A240V/V244A) was a gift from I. Bezprozvanny and R. Tsien (14). The cytosolic region (residues 1-266)
of this syntaxin 1A mutant and the full-length coding region were
amplified by PCR and subcloned into pGEX-5X-1 and pCDNA3,
respectively. All GST-syntaxin 1A fusion proteins (GST-syn1A Cell Culture and Lysate Preparation--
BHK cells that were
stably transfected with full-length wild-type human CFTR were a gift
from J. W. Hanrahan and were cultured as described (20).
HT29-Cl19A human colonic epithelial cells expressing native CFTR,
SNAP23, and Munc18 were propagated as described previously (3). Mouse
L-fibroblasts stably transfected with full-length human CFTR and
full-length human syntaxin 1A have been described previously (15).
Cells grown in 10-cm dishes were lysed in situ on ice with
chilled lysis buffer (phosphate-buffered saline containing 1% (v/v)
Triton X-100, 5% (v/v) glycerol, 1 mM dithiothreitol, and
a protease inhibitor mixture of aprotinin, pepstatin A, and leupeptin
at 2 µg/ml each and phenylmethylsulfonyl fluoride at 1 mM). Lysates were centrifuged at 10,000 × g (4 °C) to remove cell debris before being used for
binding assays.
Binding Assays--
The indicated amounts of the GST-syn1A Analysis of SDS-resistant SNARE Complexes--
Binary or ternary
combinations of GST-free wild-type or mutant syntaxin 1A (residues
1-266), SNAP25, and VAMP2/synaptobrevin (residues 1-94) (2 µM each) were mixed overnight (16 h) at 4 °C as
described (19). After the addition of Laemmli buffer, samples were
divided into two aliquots. One aliquot was incubated at 37 °C for 3 min and the other at 100 °C for 3 min. To generate the thermal
melting profiles of the ternary complexes, the samples were divided
into 14 aliquots after adding Laemmli buffer containing 2% SDS and
incubated at temperatures between 25 and 100 °C (in increments of
6 °C) for 3 min in a programmable thermal cycler (MJ Research). The
samples were resolved by SDS-PAGE on 4-20% gradient gels (Bio-Rad)
and immunoblotted for syntaxin 1A. To quantitate the dissociation of
the complexes, the signal intensities of the 100- and 200-kDa complexes
were measured by densitometric scanning for each point on the
temperature axis and normalized to the signal intensity of the
complexes at 25 °C (100%). For SNARE titration experiments, various
concentrations (0.05-1 µM) of recombinant SNAP25 were
added to a binary mixture containing 1 µM each of
recombinant syntaxin 1A (wild-type or mutant) and VAMP2/synaptobrevin
and mixed overnight (16 h) at 4 °C as above. After the addition of
Laemmli buffer, samples were incubated at 37 °C for 3 min and
immunoblotted for syntaxin 1A.
Electrophysiology--
The methods and solutions used for the
Xenopus oocyte expression studies are described in detail
elsewhere (16). The methods for the whole-cell patch clamp experiments
performed on L-fibroblasts stably transfected with CFTR and syntaxin 1A
are similar to those described earlier (15). The pipette solution
contained 140 mM N-methyl-D-glucamine, 40 mM HCl, 100 mM L-glutamic acid, 0.2 mM CaCl2, 2 mM MgCl2, 1 mM
EGTA, 10 mM HEPES, and 5 mM ATP-Mg, pH 7.2. The
bath solution contained 140 mM NMDG, 140 mM
HCl, 2 mM CaCl2, 1 mM
MgCl2, and 10 mM HEPES, pH 7.4. CFTR-mediated
Cl Mutating Syntaxin 1A at Isoform-specific Residues Inhibits CFTR
Binding or Munc-18 Binding--
Our previous findings indicated that
CFTR channels physically and functionally interact with human or mouse
syntaxin 1A but not with syntaxins 2-5 (5). Therefore, we mutated four
residues in the H3 domain of syntaxin 1A that are unique to this
isoform and that are well conserved across species (namely
Ser225, Tyr235, Glu238, and
Val248 (see Fig. 1). These
residues were replaced with alanine singly or in combination. In
addition, we replaced Glu238 with leucine, since human
syntaxins 4 and 5 have leucine at the corresponding position. The
abilities of these syntaxin 1A mutants to bind to CFTR as well as to
SNAP-23 (a t-SNARE) and to Munc-18 were tested using the indicated
GST-syn1A
Since the E238A mutation resulted in the greatest loss of binding to
CFTR, we performed titration experiments over a range of protein
concentrations to provide a semiquantitative index of the binding
strength of this mutant versus wild-type syntaxin 1A. To
detect CFTR binding signals over a broad range of CFTR protein amounts
and syntaxin 1A concentrations, we used lysates made from BHK cells
that express high levels of recombinant CFTR. In the first assay,
recombinant CFTR was pulled down with varying amounts of GST-syn1A
Similar titration experiments were performed to compare the binding of
Y235A, E238A, and wild-type syntaxin 1A to Munc-18 and SNAP-23 using
fixed amounts of GST-syn1A CFTR Binding Is Not Inhibited by Mutating Hydrophobic Residues
Implicated in SNARE Complex Formation and Ca2+ Channel
Regulation (A240V/V244A)--
Since the preceding results indicate
that two hydrophilic residues (Ser225 and
Glu238) of syntaxin 1A are important for its interaction
with CFTR, we next examined the effect of mutating residues buried in
the hydrophobic layers of the SNARE complex on CFTR binding. For this purpose, we selected a syntaxin 1A double mutant, A240V/V244A, in which
the mutated residues map to the a and d heptad
positions predicted to be in the hydrophobic core of the SNARE complex. Another important feature of this double mutant is that it was reported
to be ineffective for regulating N-type Ca2+ channels in
functional assays (14). We compared the CFTR binding of this double
mutant with that exhibited by another double mutant in which the two
hydrophilic residues that were implicated in CFTR binding based on the
preceding data (Figs. 2 and 3A) were converted to alanine
(S225A/E238A). As seen in Fig. 3B (upper panel), the A240V/V244A double mutant is not inhibited for
binding CFTR unlike the S225A/E238A double mutant. Although residues
Ala240 and Val244 are implicated in mediating
SNARE-SNARE interactions, due to their location in the hydrophobic
layers, the double mutation at these positions did not inhibit binding
of syntaxin 1A to SNAP-23 in these pull-down experiments (see Fig. 3,
lower panel). However, in studies described
below, the A240V/V244A double mutation was found to destabilize core
SNARE complexes assembled in vitro.
The E238A Mutation Does Not Inhibit the Formation of SDS-resistant
SNARE Complexes in Vitro--
A hallmark feature of syntaxin 1A is its
ability to assemble into SDS-resistant complexes with SNAP-25 and
VAMP-2 in vitro that mimic the properties of native fusion
complexes isolated from brain vesicles (21). Accordingly we tested the
abilities of the syntaxin 1A mutants to form SDS-resistant complexes
with recombinant SNAP-25 and VAMP-2 proteins. As shown in Fig.
4A, at least three prominent
high molecular weight complexes, presumably representing monomers
(50-70 kDa) and multimers (>100 kDa) of the core SNARE complexes,
could be detected when either wild-type or E238A syntaxin 1A was mixed
with SNAP-25 and VAMP-2. These complexes were resistant to SDS at
37 °C but could be melted by heating to 100 °C. As expected,
SDS-resistant complex formation required all three proteins. To rule
out a more subtle effect of the E238A mutation on SNARE complex
assembly, we also performed a titration experiment in which we
monitored SDS-resistant complex formation induced by mixing increasing
amounts of SNAP-25 with fixed amounts of VAMP-2 and syntaxin 1A
(wild-type or E238A). As seen from Fig. 4B, titrating in
increasing amounts of SNAP-25 resulted in an increased appearance of
SDS-resistant complexes to the same extent for either wild-type or
E238A syntaxin 1A. Thus, a mutation that profoundly affects the binding
of syntaxin 1A to CFTR produced no discernible effect on its ability to
form SDS-resistant SNARE complexes with VAMP-2 and SNAP-25.
Mutating Hydrophobic Residues (A240V/V244A) Destabilizes SNARE
Complexes, but Mutating Hydrophilic Residues That Participate in CFTR
Binding (Ser225 and Glu238) Does Not--
The
steady-state formation of SNARE complexes at 37 °C may not be a
sensitive indicator of subtle effects of syntaxin 1A mutations on the
stability of these complexes. Thus, in order to examine whether point
mutations in syntaxin 1A altered the stability of the ternary
complexes, we compared the thermal melting profiles of SNARE complexes
assembled using wild-type and mutant syntaxin 1A proteins. In complexes
assembled with wild-type syntaxin 1A (Fig.
5A), the major complexes of
sizes 50-70, 100, and 200 kDa began dissociating around 60 °C, and
the larger two complexes could not be detected beyond 67 °C, similar
to previous findings (19). The melting profile for E238A was similar to
that observed for the wild-type protein (Fig. 5B), which was
verified by quantifying the dissociation of SNARE complexes as a
function of increasing temperature (Fig. 5C).
This analysis was next extended to the S225A/E238A and A240V/V244A
double mutants that were also able to assemble SDS-resistant SNARE
complexes with SNAP-25 and VAMP-2 in vitro similar to
wild-type and E238A syntaxin 1A (results not shown). Upon comparing the thermostabilities of the SNARE complexes (Fig. 5D), we
observed a leftward shift in the melting curve for the A240V/V244A
mutant (i.e. the core complexes began dissociating at a
lower temperature when they were assembled with this mutant
(49-55 °C) as compared with wild-type syntaxin 1A (61-67 °C)).
On the other hand, complexes assembled with the S225A/E238A syntaxin 1A
mutant exhibited very similar melting profiles as compared with
wild-type syntaxin 1A. Thus, mutations that inhibit CFTR binding (E238A
and S225A) do not detectably affect SNARE complex formation or
stability in vitro. On the other hand, mutations that do
destabilize SNARE complexes and inhibit Ca2+ channel
regulation (A240V/V244A) do not appreciably affect CFTR binding (Fig.
3B).
Syntaxin 1A Mutants That Are Inhibited for Binding to CFTR Are Also
Inhibited for Regulating CFTR-mediated Cl
In a complementary set of functional experiments, wild-type and mutant
GST-syn1A Munc-18 Is Unable to Rescue CFTR-mediated Currents from Inhibition
by Y235A Syntaxin 1A--
Munc-18 blocks the inhibitory effect of
syntaxin 1A on CFTR-mediated currents, due presumably to its ability to
block the binding of syntaxin 1A to CFTR (4). In the present study, we identified a novel syntaxin 1A mutation (Y235A) that was inhibited for
Munc-18 binding (Fig. 2, A and D). Furthermore,
this mutant was able to interact with CFTR as detected in our in
vitro binding experiments (Fig. 2A) and
electrophysiological assays (Figs. 6A and 7). Accordingly,
we examined the functional effect of this mutation in the context of
the reciprocal regulation of CFTR by syntaxin 1A and Munc-18 by
recording currents in Xenopus oocytes co-expressing the
three proteins (Fig. 8A).
Co-expression of Munc-18 could rescue CFTR currents from inhibition by
wild-type syntaxin 1A in oocytes but not from inhibition by Y235A
syntaxin 1A. The studies were next extended to L-fibroblasts expressing
recombinant CFTR and syntaxin 1A, where CFTR-mediated currents were
stimulated by GST-Munc-18a included in whole-cell patch pipettes. As
reported previously (3), premixing of GST-Munc-18a with the soluble GST-syn1A The Syntaxin 1A-CFTR Interaction Involves Hydrophilic Residues That
Are Not Involved in SNARE-SNARE Interactions--
The goals of this
project were to characterize the molecular basis of the interactions
between syntaxin 1A and CFTR and to compare the nature of these
interactions with that of SNARE-SNARE interactions. We identified two
hydrophilic amino acids (Glu238 and Ser225)
that are specific to the syntaxin 1A isoform and that are necessary for
binding to CFTR and regulating its channel activity. Mutating these
residues (E238A and S225A) disrupted the biochemical association of
syntaxin 1A with CFTR and the ability of syntaxin 1A to modulate CFTR-mediated currents in the oocyte expression system. The mutations did not, however, inhibit syntaxin 1A binding to SNAP-23 or Munc-18; nor did they affect the ability of syntaxin 1A to assemble into SNARE
complexes with SNAP-25 and VAMP-2 or the stability of these complexes.
This is consistent with the positions of residues Glu238
and Ser225 on the outer surface (heptad positions
b and c) of the ternary SNARE complex structure.
Conversely, mutations in neighboring hydrophobic amino acids
(A240V/V244A) that are located in the inner layers (heptad positions
a and d), where they stabilize the core SNARE
complex, did not detectably affect either the binding of syntaxin 1A to
CFTR or its ability to regulate this channel. Thus, the nature of the
syntaxin 1A-CFTR interaction appears to be mechanistically distinct
from syntaxin 1A-SNARE interactions. The former involves hydrophilic
surface residues that are specific to this isoform (Glu238
and Ser225), whereas the latter is mediated by hydrophobic
residues present in other syntaxin isoforms and other SNAREs as well.
This probably explains why CFTR regulation is specific to the syntaxin
1A isoform, whereas SNARE-SNARE interactions are promiscuous at least
in vitro.
The E238A and S225A Mutations Do Not Produce Equivalent Effects
on CFTR Binding--
The results of the titration binding assays
indicated that mutating residue Ser225 alone produced an
intermediate effect on CFTR binding as compared with mutating residue
Glu238 alone or mutating both residues (S225A/E238A).
Conceivably, the negative charge on the Glu238 residue may
render it more important for mediating the CFTR-syntaxin 1A
interaction. In this regard, the binding interaction between CFTR and
syntaxin 1A is
salt-sensitive,3 which
implies that electrostatic interactions contribute to the binding
between these two proteins. In our titration experiments, the CFTR
binding signal for the E238A mutant was considerably lower as compared
with the wild-type protein even at saturating concentrations. An
explanation for this observation could be that CFTR binding at higher
concentrations of syntaxin 1A is limited because of oligomerization of
this t-SNARE. It has been shown that syntaxin 1A oligomerizes at
protein concentrations >2 µM and that the homo-oligomers
are not random aggregates but form parallel helices similar to SNARE
complexes (22). Preliminary gel filtration analysis revealed that there
was an increase in the oligomeric forms of both the wild-type and E238A
syntaxin 1A fusion proteins as the protein concentration was increased from 0.5 to 2 µM. The E238A mutation per se
had no apparent effect on oligomerization as analyzed in this manner
(data not shown). Thus, although one might expect that the E238A mutant
should be able to achieve wild-type levels of CFTR binding at higher
protein concentrations (>2 µM), the decrease in the
concentration of the form competent to bind CFTR might prevent this. It
is also possible that the E238A mutation has an indirect effect on CFTR
binding by altering multimerization (although see above) or by
producing a conformational change in syntaxin 1A, which, in effect,
would shield other residues in the H3 domain that mediate CFTR binding. On the other hand, it is noteworthy that the E238A mutation had a very
specific effect on CFTR binding (namely this mutation did not inhibit
binding to other proteins that interact with the SNARE motif such as
SNAP23 and Munc18). In addition, the E238A mutant, but not the
A240V/V244A mutant, was able to assemble into stable SNARE complexes
like wild-type syntaxin 1A. Thus, although we cannot completely exclude
the possibility that the E238A mutation affects CFTR binding as a
consequence of altering the oligomerization or conformation of syntaxin
1A, it clearly has a very specific effect on the CFTR-syntaxin 1A interaction.
The CFTR-Syntaxin 1A Interaction Appears to Be Different from the
Syntaxin 1A-Ca2+ Channel Interaction--
There is
increasing evidence that syntaxin 1A modulates the activities of
multiple ion channels at the plasma membrane. Such interactions may
link the functions of certain ion channels to the exocytotic machinery.
Catterall and colleagues (23) reported that syntaxin 1A biochemically
interacts with the II/III cytosolic loop ("synprint loop") of the
The strong correlation between the binding of the various syntaxin 1A
mutants to CFTR and their abilities to regulate CFTR channels in
oocytes supports a simple model in which syntaxin 1A regulates CFTR via
a specific protein-protein interaction. Whether this interaction
affects the gating of the CFTR channel, its intracellular traffic, or
both is a matter of some debate (25, 26), but the results of this and
other studies (16) favor the view that syntaxin 1A must bind to CFTR in
order to regulate its function. On this basis, one might conclude that syntaxin 1A by itself is sufficient to modulate CFTR activity in
vivo. However, two observations indicate that there may be more
than one SNARE protein involved in regulating CFTR at least in
mammalian cells. First, we reported recently that CFTR channels preferentially associate with a t-SNARE heterodimer in mammalian cells
that includes both SNAP-23 and syntaxin 1A. These two t-SNAREs appear
to have additive or possibly cooperative effects on CFTR channel
function (15). In support of these findings, the present results
indicate that syntaxin 1A residues that mediate CFTR binding do not
participate in SNARE-SNARE interactions, which, therefore, could allow
syntaxin 1A to simultaneously complex with a t-SNARE partner via
hydrophobic interactions as well as interact with CFTR via hydrophilic
surface residues. Second, in the present study, we observed that the
V248A syntaxin 1A peptide (unlike the wild-type syntaxin 1A peptide)
was unable to rescue CFTR-mediated currents from inhibition by
membrane-anchored syntaxin 1A in mouse L-fibroblasts. This contrasts
with the inhibitory effect of full-length V248A syntaxin 1A on
CFTR-mediated currents in oocytes. Conceivably, the V248A mutation
inhibits the ability of the syntaxin 1A peptide to rescue CFTR channels
from this t-SNARE complex in mammalian cells without directly affecting
the binding of syntaxin 1A to the channel or the ability of the
full-length protein to inhibit CFTR-mediated currents in oocytes. In
support of this hypothesis, the V248A syntaxin 1A peptide could rescue
CFTR-mediated currents from inhibition by full-length wild-type
syntaxin 1A in the oocyte expression system, which, of course, lacks
mammalian SNAP-23 (data not shown). However, more detailed studies will
be required to determine whether the V248A mutation does indeed inhibit
the ability of the syntaxin 1A peptide to disrupt the interactions
between CFTR and the t-SNARE complex in mammalian cells.
The Syntaxin 1A-CFTR Interaction Can Be Uncoupled from the Syntaxin
1A-Munc-18 Interaction--
Munc-18a inhibits the syntaxin 1A-CFTR
interaction due presumably to its ability to block the binding of
syntaxin 1A to the CFTR channel (4). Unlike other proteins that
interact with only the H3 region of syntaxin 1A, Munc-18 requires the
entire NH2-terminal region of syntaxin 1A for high affinity
binding (18). Moreover, Munc-18 is believed to bind to a closed
syntaxin conformation, and mutations that destabilize this closed
conformation compromise binding (9). In the present study, we
identified a novel syntaxin 1A mutant (Y235A) that is inhibited for
binding Munc-18 but is able to physically and functionally interact
with CFTR. The Y235A mutation eliminates the ability of Munc-18 to
neutralize the inhibitory effect of syntaxin 1A on CFTR activity
presumably by disrupting the Munc-18-syntaxin 1A interaction. In the
crystal structure of the syntaxin 1A-Munc-18a complex,
Tyr235 of syntaxin 1A does not directly contact any residue
in Munc-18a; rather, it is flanked on either side by residues that do
directly interact with Munc-18a (27). Presumably, this mutation alters the local conformation of syntaxin 1A to a sufficient degree to inhibit
Munc-18 binding.
In conclusion, our results indicate that syntaxin 1A biochemically and
functionally interacts with the CFTR channel by a mechanism that is
different from how this t-SNARE interacts with other SNAREs or
apparently with voltage-gated calcium channels. The CFTR interaction involves surface-exposed hydrophilic residues that are unique to this
isoform rather than hydrophobic residues that mediate SNARE complex
assembly. The strong correlation between the binding and functional
regulation of CFTR channels by the syntaxin 1A mutants that were tested
favors the hypothesis that this t-SNARE regulates CFTR channels via a
specific protein-protein interaction.
channels and synaptic Ca2+ channels
in addition to participating in SNARE complex assembly and membrane
fusion. We exploited the isoform-specific nature of the interaction
between syntaxin 1A and CFTR to identify residues in the H3 domain of
this SNARE (SNARE motif) that influence CFTR binding and regulation.
Mutating isoform-specific residues that map to the surface of syntaxin
1A in the SNARE complex led to the identification of two sets of
hydrophilic residues that are important for binding to and regulating
CFTR channels or for binding to the syntaxin regulatory protein
Munc-18a. None of these mutations affected syntaxin 1A binding to other
SNAREs or the assembly and stability of SNARE complexes in
vitro. Conversely, the syntaxin 1A-CFTR interaction was
unaffected by mutating hydrophobic residues in the H3 domain that
influence SNARE complex stability and Ca2+ channel
regulation. Thus, CFTR channel regulation by syntaxin 1A involves
hydrophilic interactions that are mechanistically distinct from the
hydrophobic interactions that mediate SNARE complex formation and
Ca2+ channel regulation by this t-SNARE.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices contain hydrophobic
residues that are grouped into "layers" that zipper together the
four-helix bundle, resulting directly or indirectly in bilayer mixing.
This paradigm applies to nonneuronal as well as to neuronal fusion
events; in the former case, other SNARE isoforms are involved. The
interactions among v-SNAREs and t-SNAREs are typically nonselective, at
least in vitro (7), presumably because hydrophobic residues
that are common to multiple isoforms mediate these interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C;
C
refers to deletion of the COOH-terminal membrane domain) and GST alone
were expressed and purified from bacterial cells and dialyzed against
phosphate-buffered saline as described (16). GST fusion constructs for
mouse SNAP25 (residues 1-206) and rat VAMP2/synaptobrevin (residues
1-94) were gifts of Drs. J. Pevsner and M. Bennett, respectively, and
have been described previously (17, 18). In experiments where GST-free proteins were used, fusion proteins were cleaved with thrombin as
described (19). Protein concentrations were estimated by standard
micro-BCA protein assay (Pierce) or by Coomassie Blue staining of
protein bands after SDS-PAGE using bovine serum albumin as a standard.
C
proteins were added to clarified cell lysates (1-ml final reaction
volume) and incubated overnight (12-16 h) at 4 °C on a rotator. For
titration binding experiments, the lysates were diluted in lysis buffer
before adding fusion protein. The complexes were captured by mixing
with excess glutathione-Sepharose for 2 h, and the beads were
washed in several volumes of lysis buffer minus the protease
inhibitors. The bound proteins were eluted with Laemmli buffer and
separated by SDS-PAGE on 4-20% gradient gels (Bio-Rad) followed by
immunoblotting on polyvinylidene difluoride membranes (PerkinElmer Life
Sciences) using conditions described (5). CFTR
immunoprecipitations were carried out to monitor CFTR protein levels in
the lysates as described previously (15). The immunoreactive protein
bands were visualized by enhanced chemiluminescence (Pierce). Signals
were quantified by densitometric scanning followed by analysis with
Scion Image (release beta 4.0.2) (Scion Corp.).
current was induced within 10-15 min of breaking into
the cell by perfusion with a mixture containing 10 µM
forskolin, 1 mM isobutylmethylxanthine, and 400 µM cpt-cAMP. cAMP-activated currents were recorded at +110 mV from a holding potential of
40 mV. All experiments were performed at 22-24 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C proteins to pull down native proteins from HT29-Cl19A
epithelial cell lysates (Fig. 2A). Introducing the alanine
substitutions at residues Ser225 and Glu238
(singly or in combination) as well as the leucine substitution at
Glu238 of syntaxin 1A resulted in a substantial loss of
binding to CFTR as compared with the wild-type protein
(upper panel). The E238A and E238L mutations had
a more pronounced effect on CFTR binding than the S225A mutation (see
also Fig. 3A). Conversely,
mutating Tyr235 or Val248 did not appreciably
reduce syntaxin 1A binding to CFTR. Of all the syntaxin 1A mutants that
were tested, only the tyrosine mutant (Y235A) showed a marked decrease
in its binding to Munc-18 (middle panel).
Notably, none of the syntaxin 1A mutants that showed reduced binding to
CFTR or to Munc-18 were affected for binding to SNAP-23 (bottom panel). GST alone was used as a negative
control in all binding experiments and did not bind to any of the
target proteins.
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Fig. 1.
Domain organization (bottom) and sequence
alignment of the SNARE motif of human syntaxin isoforms (top) and of
the corresponding regions in syntaxin 1A of human
(hum), mouse (mou), and
Drosophila (drome)
(middle). Heptad positions a-g are
indicated below the sequences. Hydrophobic residues at the
a and d heptad positions are boxed.
Residues unique to the syntaxin 1A isoform are in boldface
type, and the corresponding residues in all isoforms are
shaded.
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Fig. 2.
Screening of syntaxin 1A mutants for CFTR,
Munc-18, and SNAP-23 binding. A, GST alone or GST
fusion proteins of the cytosolic domain of the wild-type
(WT) or indicated syntaxin 1A mutants (0.5 µM
each) were used to pull down native CFTR, Munc-18, and SNAP-23 from
HT29-Cl19A cell lysates. Band C represents
mature, fully glycosylated CFTR. Since CFTR is less abundant than
SNAP-23 and Munc-18 in these cells, a more concentrated lysate (5×)
was used for the CFTR pull-down. This experiment was repeated 10 times
with similar results. B, recombinant CFTR was pulled down
from BHK cell lysates with the indicated amounts of wild-type or E238A
GST-syn1A C proteins. The graph shows the CFTR binding
signals for each fusion protein as a function of protein concentration.
Data points represent means ± S.E. calculated from three separate
experiments. All binding signals were normalized to the CFTR binding
signal at 2 µM wild-type GST-syn1A
C. C,
recombinant CFTR and native SNAP-23 were pulled down from BHK cell
lysates (at the indicated dilutions) with the indicated GST-syn1A
C
proteins (0.25 µM). Control pull-downs with GST and
GST-syn3
C (0.25 µM each) were performed using
undiluted lysates. D, native Munc-18 and SNAP-23 were pulled
down from HT29-Cl19A lysates with the indicated GST-syn1A
C proteins
(0.5 µM).
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Fig. 3.
CFTR binding is inhibited by mutating
hydrophilic residues but not hydrophobic residues of syntaxin 1A.
A, recombinant CFTR was pulled down from BHK cell lysates
with the indicated GST-syn1A C proteins (0.25 µM). CFTR
was immunoprecipitated from undiluted lysate using a CFTR antibody (2 µg) (IP). Note that mutating residue Ser225
alone produced an intermediate effect on CFTR binding as compared with
mutating E238A alone or mutating both residues. B,
recombinant CFTR and native SNAP-23 were pulled down from BHK cell
lysates with indicated GST-syn1A
C proteins or GST alone (0.5 µM each). Mutating the hydrophobic residues at positions
240 and 244 had no effect on CFTR binding.
C
(wild-type or E238A) added to a fixed volume of BHK cell lysate. The
amount of CFTR that bound to the E238A mutant was considerably lower at
all concentrations tested (Fig. 2B). Binding appeared to
saturate between 1 and 2 µM for both the wild-type and
mutant syntaxin 1A fusion proteins (see "Discussion" for
explanation). In a complementary assay, various dilutions of BHK cell
lysates containing recombinant CFTR and native SNAP-23 were incubated
with a fixed concentration of wild-type or mutant GST-syn1A
C. As
seen from the upper panel of Fig. 2C,
the CFTR binding signal was substantially lower for the E238A mutant as compared with wild-type protein at all lysate dilutions. However, the
SNAP-23 binding signals were comparable for both syntaxin 1A fusion
constructs at all lysate dilutions (lower panel).
GST and GST-syn3
C were used as controls, and, as expected,
GST-syn3
C could bind SNAP-23 but not CFTR, whereas GST alone could
bind neither. In summary, the data obtained from multiple binding
assays performed using either native or recombinant CFTR indicate that the E238A syntaxin 1A mutant is specifically inhibited for CFTR binding.
C fusion proteins and varying dilutions of
HT29-Cl19A epithelial cell lysates. As shown in the upper
panel of Fig. 2D, the Y235A mutant showed
considerably reduced Munc-18 binding as compared with wild-type or
E238A syntaxin 1A even at the highest concentration of lysate. All
three syntaxin 1A fusion proteins showed comparable binding to SNAP-23.
In summary, we have generated point mutations in the H3 domain of
syntaxin 1A that differentially inhibit binding to CFTR (S225A and
E238A,L) and to Munc-18 (Y235A). Both types of mutations map to
residues that are not implicated directly in SNARE complex formation
(i.e. heptad positions b, c, and
f), and, as expected, neither inhibited SNAP-23 binding.
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Fig. 4.
The E238A mutation does not inhibit the
formation of SDS-resistant SNARE complexes. A, binary
or ternary complexes of GST-free, wild-type (WT), or E238A
syntaxin 1A, SNAP-25, and VAMP-2 were incubated with Laemmli buffer
(2% SDS) at the indicated temperatures and immunoblotted with syntaxin
1A antibody. SNAP-25 was used in this assay, because formation of
SDS-resistant SNARE complexes was not detected when SNAP-23 replaced
SNAP-25 (28) (results not shown). Bands represent monomers (50-70 kDa)
and multimers (>100 kDa) of core SNARE complexes. The lowest band at
~25 kDa represents cross-reactivity of the syntaxin 1A antibody with
SNAP-25. B, soluble SNAP-25 was titrated into a binary
mixture (1 µM each) of soluble VAMP-2 and wild-type or
E238A syntaxin 1A, and the resulting complexes were analyzed by
immunoblotting for syntaxin 1A.
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Fig. 5.
Thermostabilities of SNARE complexes
assembled with wild-type and mutant syntaxin 1A. SNARE complexes
assembled using wild-type (A) or E238A syntaxin 1A
(B) were incubated with Laemmli buffer (2% SDS) at the
indicated temperatures and immunoblotted for syntaxin 1A. C
and D, the signal intensities of the 100- and 200-kDa bands
were plotted as a function of the incubation temperature for SNARE
complexes that were assembled using the wild-type (WT) or
E238A, S225A/E238A, or A240V/V244A mutant, respectively. The
intensities of the two bands were summed for each experiment and then
averaged over three separate experiments. The data points represent
means ± S.E. calculated from three experiments. An
asterisk indicates p < 0.05 compared with
wild-type (unpaired t test).
Currents--
We next tested the abilities of these syntaxin 1A
mutants to regulate CFTR-mediated chloride currents in order to
determine (i) whether syntaxin 1A mutations that disrupt CFTR binding
also inhibit CFTR channel regulation by this t-SNARE and (ii) whether syntaxin 1A mutations that destabilize SNARE complexes and inhibit regulation of voltage-gated Ca2+ channels (A240V/V244A) are
disrupted for CFTR regulation. CFTR-mediated currents were measured in
Xenopus oocytes co-expressing CFTR and full-length syntaxin
1A proteins by two-electrode voltage clamp analysis. As documented in
Fig. 6A, CFTR-mediated
currents in oocytes were inhibited in a dose-dependent
manner when these oocytes were co-injected with increasing amounts of
wild-type syntaxin 1A cRNA, as previously observed (3). Similar degrees
of inhibition were observed when CFTR was co-expressed with the V248A
and Y235A syntaxin 1A mutants that displayed wild-type levels of
binding to CFTR (Fig. 2A). The A240V/V244A double mutant
also inhibited CFTR-mediated currents like wild-type syntaxin 1A. On
the other hand, the S225A mutation, which moderately compromised CFTR
binding, and the E238A and S225A/E238A mutations, which severely
inhibited CFTR binding (Fig. 3A), were moderately and
severely inhibited for regulating CFTR currents. The attenuation of
CFTR-mediated currents by wild-type or mutant syntaxin 1A could be
reversed in all cases by first microinjecting into the oocytes
botulinum neurotoxin C1 (BoNT/C1), a protease that cleaves syntaxin 1A
(Fig. 6B). In order to verify that the relative
ineffectiveness of certain syntaxin 1A mutants to inhibit CFTR currents
was not due to poor protein expression, we immunoblotted oocyte
membranes to confirm the expression of the various syntaxin 1A
constructs (Fig. 6C). In combination with our binding data,
the results of Fig. 6 indicate that (i) there is a strong correlation
between CFTR binding strength and CFTR channel regulation for the
various syntaxin 1A mutants that were tested and (ii) a syntaxin 1A
mutation (A240V/V244A) that destabilizes SNARE complexes and that
disrupts Ca2+ channel regulation in oocytes has no
discernible effect on CFTR regulation in this expression system.
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Fig. 6.
Mutations in syntaxin 1A that disrupt CFTR
binding also disrupt regulation of CFTR-mediated currents in
oocytes. A, peak cAMP-activated CFTR-mediated currents
were recorded in oocytes injected with cRNAs for wild-type CFTR (1 ng)
and the indicated amounts of wild-type or mutant full-length syntaxin
1A. For each data point, n = 8-19 oocytes.
B, effect of BoNT/C1 (1 ng/oocyte) on CFTR-mediated currents
in oocytes co-expressing wild-type or mutant syntaxin 1A (5 ng).
cAMP-activated currents were recorded before and after BoNT/C1
injection in the same oocytes (n = 5-7 oocytes per
data point). All values in A and B are normalized
to the peak currents recorded in oocytes expressing CFTR alone. Data
points represent means ± S.E. Statistical comparisons between two
groups were performed using Student's t test. The
asterisks indicate p < 0.05 relative to
untreated samples. C, immunoblot analysis of oocyte
membranes (10-12 oocytes/lane) to confirm the expression of
recombinant wild-type or mutant syntaxin 1A. Control represents
uninjected oocytes. Total membrane fraction was purified from oocytes
and analyzed by immunoblotting (29).
C proteins were screened for their abilities to stimulate
CFTR-mediated Cl
currents in L-fibroblasts that were
stably transfected with recombinant CFTR and full-length wild-type
syntaxin 1A. Previously, we had shown that a GST fusion protein
containing the soluble cytosolic domain of syntaxin 1A (GST-syn1A
C),
when introduced into these cells via whole cell patch pipettes, could
stimulate CFTR-mediated currents presumably by competing with the
membrane-anchored syntaxin 1A for CFTR binding (15). This was also true
for GST-syn1A
C containing the Y235A or A240/V244A mutations, both of
which bind CFTR (Fig. 7). Conversely, the
E238A mutant and to a lesser extent the S225A mutant (both of which
showed reduced binding to CFTR) were compromised in their abilities to
stimulate currents in this functional assay. Unexpectedly, the V248A
mutant fusion protein, which does bind to CFTR, was also inactive when
tested in this assay, despite the fact that the full-length V248A
syntaxin 1A did inhibit CFTR-mediated currents in the oocyte expression
system (Fig. 6). At present, we have no explanation for this disparity other than the possibility that the peptide rescue experiments may not
provide a direct measure of the strength of the CFTR-syntaxin 1A
interaction. Given that this assay may require that the GST-syn1A
C peptide disrupt CFTR interactions with multiple SNARE proteins (see
"Discussion"), the binding assays and the oocyte expression experiments may provide more direct measures of the strength of the
interaction between CFTR and syntaxin 1A.
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Fig. 7.
Mutant syntaxin 1A fusion proteins that are
inhibited for binding to CFTR are also inhibited for stimulating
CFTR-mediated Cl currents in
L-fibroblasts. Whole cell current measurements were performed as
described under "Experimental Procedures." cAMP-induced increase in
current density recorded at 110 mV is shown for cells in the absence
(control) or presence of the indicated GST-syn1A
C peptides (0.35 µM) in the pipette solution. The number of cells per data
point is shown in parentheses. Data are mean ± S.E.
The asterisks indicate p < 0.05 relative to
control determined by unpaired t test.
C peptide neutralized each other's stimulatory effect on
CFTR currents, most likely by blocking the binding of this soluble
syntaxin 1A peptide to CFTR. However, such a neutralizing effect was
not observed when GST-Munc-18a was premixed with the mutant
Y235A peptide. Thus, the regulatory effect of syntaxin 1A on
CFTR was physically and functionally uncoupled from its interaction with Munc-18a by mutating residue
Tyr235.
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Fig. 8.
Munc-18 is unable to rescue CFTR-mediated
currents from inhibition by Y235A syntaxin 1A. A,
co-expression of Munc-18a blocks the inhibition of CFTR-mediated
currents by full-length wild-type (WT) syntaxin 1A but not
by Y235A syntaxin 1A in oocytes (n = 4-8 oocytes).
Comparisons across multiple groups were performed using one-way
analysis of variance followed by Tukey's honestly significant
difference post hoc tests. The
asterisks indicate p < 0.05 in comparison
with CFTR alone (*) or in comparison with CFTR + wild-type syntaxin 1A
(**). B, comparison of cAMP-activated current densities in
L-cells in the absence or presence of 0.35 µM
GST-syn1A C (wild-type or Y235A) with or without 0.35 µM GST-Munc-18a in the patch pipette. The
numbers in parentheses indicate the
number of cells per data point.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1B subunit of the N-type Ca2+ channel.
However, functional studies using the oocyte expression system revealed
that mutating hydrophobic residues Ala240 and
Val244 of syntaxin 1A, located in the SNARE-stabilizing
layers, resulted in appreciable loss of Ca2+ channel
regulation but did not disrupt binding to the synprint motif.
Furthermore, deletions within the II/III loop region of
1B that eliminated the synprint site did not altogether
abolish calcium channel modulation by syntaxin (14). This suggests that the binding and modulatory functions of syntaxin 1A can be disconnected in the case of the Ca2+ channel and/or that syntaxin 1A
binds to multiple regions of this channel. In this respect, the
regulation of CFTR by syntaxin 1A appears to be different. Point
mutations in syntaxin 1A that disrupted CFTR binding also disrupted
CFTR regulation. Also, the A240V/V244A double mutation did not affect
the regulation of CFTR by syntaxin 1A but did destabilize core SNARE
complexes, consistent with the predicted roles of these two hydrophobic
residues. The latter results are in agreement with the findings of a
related study in which mutations of the corresponding residues of
Drosophila syntaxin 1 (A243V/V247A) severely compromised
SNARE complex stability and disrupted synaptic transmission in flies
(24). Thus, residues in the SNARE motif that facilitate the inhibition
of N-type Ca2+ channels also govern SNARE complex formation
and stability. In contrast, our results obtained with the S225A/E238A
double mutant indicate that these hydrophilic residues are not involved
in forming or stabilizing SNARE complexes, although mutating these
residues had a pronounced effect on CFTR binding and regulation. This
is the first report of mutations in the SNARE motif of syntaxin 1A that
affect ion channel regulation without affecting SNARE complex assembly
or stability.
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ACKNOWLEDGEMENTS |
---|
We thank Shui He for making the syntaxin 1A mutants and Yongming Wang and Katie Davis for technical support. We also thank I. Bezprozvanny, R. Tsien, M. Bennett, and J. Pevsner for providing cDNA constructs and mutants.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL58341.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis
Research Center, University of Alabama at Birmingham, 1918 University
Blvd., MCLM 985, Birmingham, AL 35294. Tel.: 205-934-3122; Fax:
205-934-5787; E-mail: kirk@physiology.uab.edu.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M211790200
2 A. P. Naren and K. L. Kirk, unpublished observations.
3 A. P. Naren and K. L. Kirk, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; SNARE, soluble NSF attachment protein receptor; GST, glutathione S-transferase; BHK, baby hamster kidney; cpt-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate.
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