Centre for Neurobiochemistry, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, UK
* Author for correspondence (e-mail: o.dolly{at}ic.ac.uk )
Accepted 15 April 2002
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Summary |
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Key words: Secretion, Large dense-core granules, SNAP-25, Synaptobrevin, Clostridial neurotoxins
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Introduction |
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In neurones and neuroendocrine cells, the release of neurotransmitters or
hormones is tightly linked to depolarisation-induced Ca2+ influx.
It has been suggested that Ca2+-triggered SNARE complex formation
mediates vesicle fusion in PC-12 cells, but complexes also form during a
MgATP-dependent priming step without fusion occurring
(Chen et al., 1999). Moreover,
Ca2+ triggers secretion from semi-intact synaptosomes but has no
effect on the amounts of SNAREs co-immunoprecipitated together
(Leveque et al., 2000
); also,
neither formation nor dissociation of SNARE complexes is concomitant with the
fusion of sea-urchin egg cortical vesicles
(Coorssen et al., 1998
;
Tahara et al., 1998
).
Elevations of intracellular Ca2+ concentration
([Ca2+]i) may be communicated to the SNAREs by
synaptotagmin I [itself a SNARE (Schiavo
et al., 1995
)], which changes conformation upon binding the cation
(Davletov and Sudhof, 1994
),
but the mechanism of such signal transduction remains speculative. In vitro,
synaptotagmin I associates with syntaxin in a Ca2+-dependent manner
(Li et al., 1995b
;
Shao et al., 1997
) and, also,
binds to SNAP-25, but this does not require Ca2+, the interaction
being enhanced only weakly (Li et al.,
1995b
; Schiavo et al.,
1997
). In contrast, Ca2+ triggers dissociation of
synaptotagmin I from SNARE complexes in permeabilised nerve endings
(Leveque et al., 2000
;
Mehta et al., 1996
). In most
cases, the Ca2+ sensitivity of the aforementioned reactions is
incompatible with the Ca2+ dependency of regulated exocytosis from
neuroendocrine cells; such a discrepancy has given rise to conjecture that the
latter may utilise a different Ca2+ sensor from that employed in
nerve terminals (Bennett, 1997
;
Burgoyne and Morgan, 1995
;
Gerona et al., 2000
;
Li et al., 1995b
). Indeed, a
recent study of chromaffin cells from synaptotagmin-I-deficient mice showed
that this protein is not essential for Ca2+-triggered exocytosis
but is required for an extremely fast phase, known as the exocytotic burst
(Voets et al., 2001
). Also,
following analyses of the ternary SNARE complex structure, determined by X-ray
crystallography, it has been suggested that it may bind Ca2+
directly (Fasshauer et al.,
1998b
; Sutton et al.,
1998
). Furthermore, the C-terminus of SNAP-25 has been implicated
in Ca2+ sensing because the blockade of neuroexocytosis induced by
BoNT/A can be alleviated by stimulation protocols that increase
[Ca2+]i (Sellin,
1987
; Simpson,
1989
; Dolly et al.,
1994
). In view of these conflicting data, further investigations
are needed to clarify the Ca2+ dependencies of SNAREs and
synaptotagmin I in neuroendocrine cells and their relationship to
Ca2+-elicited exocytosis.
In the present study, an accepted assay of changes in protein structure -
acquisition of resistance to protease digestion
(Davletov and Sudhof, 1994) -
was exploited to demonstrate alterations in the SNAREs in response to
Ca2+. For the first time, it is shown that equivalent
Ca2+ concentrations trigger both vesicle fusion and alterations in
the structure of synaptotagmin I and the other SNAREs. During
Ca2+-triggered exocytosis, the proteins became less susceptible to
trypsin or proteinase K via a mechanism that is not related to the formation
of SDS-resistant SNARE complexes. Removal by BoNTs of part of the cytoplasmic
domain of Sbr (which prevents ternary SNARE complex formation) or the nine
C-terminal residues of SNAP-25 did not prevent transmission of the
Ca2+ signal to the SNAREs, although BoNT/A reduced the extent to
which changes occurred.
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Materials and Methods |
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Chromaffin cell preparation and culture
Bovine adrenal chromaffin cells were prepared and maintained as monolayer
cultures as described previously (Foran et
al., 1996; Lawrence et al.,
1996
; Lawrence et al.,
1994
). The cells were used for experiments between three and 10
days after isolation. When required, they were pre-intoxicated with BoNT/A or
B using a protocol that facilitates their uptake (see
Lawrence et al., 1996
), then
returned to the standard culture medium and maintained for 24-72 hours before
further manipulations.
Stimulation and assay of catecholamine release
Immediately prior to experiments, which were all performed at room
temperature (22°C), cells were rinsed with a HEPES-buffered saline
solution (HBS; 145 mM NaCl, 4.8 mM KCl, 1.2 mM NaH2PO4,
20 mM HEPES, pH 7.4) before permeabilisation by exposure to 20 µM digitonin
in KGEP [139 mM potassium glutamate, 5 mM EGTA, 20 mM
piperazine-N,N'-bis-(2-ethanesulfonic acid), pH 6.5]. Aliquots of
CaCl2 were added to the KGEP to produce the desired concentrations
of buffered, free Ca2+ and, where indicated in the figures, 2 mM
ATP and 4 mM MgCl2 were also included. After 15 minutes of
stimulation, an aliquot was removed and the amount of catecholamines released
from the cells assayed, as described elsewhere
(Lawrence et al., 1996
). Mean
values (±s.d.) were determined from four wells of cells. Control
untreated cells were solubilised with 1% Triton X-100 in HBS, and aliquots
were assayed to determine the total cell content of catecholamines; release
values were expressed as a percentage of the latter. In an exceptional set of
experiments, a 30 minute delay was included, between cell permeabilisation
with digitonin and the addition of Ca2+, so that the effect of
run-down (detailed later) could be examined. In experiments using either
trypsin or proteinase K (see figure legends for details), it was added
directly to the digitonin-containing KGEP from a 10 mg/ml stock in the same
buffer.
Enrichment, SDS-PAGE and western blotting of membrane proteins
A membrane-enriched fraction was prepared as detailed previously
(Foran et al., 1996;
Lawrence et al., 1996
),
dissolved in 50 mM Tris.HCl, pH 5.8 containing 1% SDS plus 10 mM
ß-mercaptoethanol (without or with boiling; see Figure legends for
details) and PAGE performed using the NuPAGE system (Novex, San Diego, USA).
Proteins were transferred from the gels onto the PVDF membrane, and western
blotting was performed using standard protocols
(Lawrence et al., 1996
);
binding of primary antibodies was detected using horseradish-peroxidase- or
alkaline-phosphatase-conjugated secondary antibodies and ECL or colorimetric
development. Signals on western blots were quantified using a flatbed scanner
linked to a PC running NIH Image software. The data presented in each of the
figures are from a single experiment representative of results obtained
consistently and on at least three separate occasions.
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Results |
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Ca2+ also makes the other SNAREs less susceptible to
trypsinisation
The same assay of protection against protease digestion was then applied to
determine whether Ca2+ induces changes in other SNAREs. In the
absence of Ca2+, syntaxin, SNAP-25 and Sbr all proved to be
susceptible to trypsin; increasing amounts of each protein were retained upon
the incremental addition of Ca2+, peaking at 20 µM
(Fig. 1C). Note that
Ca2+ maintains each SNARE protein intact because, with the
occasional exception of a minor proteolytic fragment of SNAP-25, no other
signals were observed (not shown). The Ca2+ dose dependency for
acquisition of resistance to trypsin was ascertained for each of the SNAREs
(Fig. 1E), as described for
synaptotagmin I. Importantly, these proved to be equivalent to that for both
the Ca2+ sensitivity of synaptotagmin I protection and
exocytosis.
MgATP enhances secretion but does not alter SNARE susceptibility to
trypsin
Inclusion of 2 mM MgATP increased the amount of hormone released at each
Ca2+ concentration (Fig.
1A). In the presence of the nucleotide, the level of exocytosis
clearly peaked at 20 µM Ca2+ and is significantly lower at 100
µM Ca2+. By contrast, 20 and 100 µM Ca2+ elicit
similar amounts of MgATP-independent secretion. These findings accord with a
previous study on the Ca2+ dependency of both MgATP-independent and
-requiring stages of exocytosis from chromaffin cells
(Bittner and Holz, 1992); in
the presence of MgATP, secretion peaks at
20 µM Ca2+
because higher concentrations inhibit priming. MgATP failed to alter
significantly the Ca2+ dependence for SNAP-25, Sbr and syntaxin
acquiring resistance to trypsin (Fig.
1C). Despite its enhancement of secretion, the amount of SNAREs
surviving tryptic digestion was only marginally augmented by the nucleotide;
in fact, the Ca2+-induced protection of synaptotagmin I against
proteolysis was attenuated by MgATP. For the other SNAREs, there was a good
agreement overall between extent of protection and the level of
Ca2+-induced secretion; at 100 µM Ca2+ there were
some anomalous points, the reason for which is not clear.
Ca2+ fails to induce SNARE resistance to trypsin in cells
that are rendered incompetent for exocytosis owing to run-down
To gain further evidence that the Ca2+-induced changes in the
SNAREs are caused by their involvement in the exocytotic process, the
experimental conditions were manipulated such that the cells could be exposed
to the cation without catecholamine secretion being elicited. Delaying the
addition of Ca2+ to permeabilised cells by 15 minutes following
their permeabilisation abolishes their Ca2+-triggered secretory
response (Hay and Martin,
1992
; Holz et al.,
1989
; Lawrence et al.,
1994
). This phenomenon, termed `run-down', is caused by the loss
of proteins and metabolites (e.g. MgATP) required for exocytotic reactions
(priming and fusion), and it can be attenuated if the latter factors are added
exogenously (Hay and Martin,
1992
; Holz et al.,
1989
; Sarafian et al.,
1987
). Thus, susceptibility to trypsin was compared in cells
treated with or without 30 minutes of run-down before application of the
cation. As expected, Ca2+ elicited only a minimal level of
exocytosis after run-down, in contrast to the robust secretion seen when it
was co-applied with digitonin (Fig.
2A). As noted previously, samples that had exhibited exocytosis
showed protection of synaptotagmin I against tryptic proteolysis, whereas
virtually no full-length synaptotagmin I was detected in the cells that had
been subjected to run-down, despite extensive development of the western blot
(Fig. 2B). Likewise, the
Ca2+-induced acquisition of resistance to trypsin by syntaxin,
SNAP-25 and Sbr was severely attenuated when the secretory response was
diminished by run-down (Fig.
2B). Thus, it appears that the trypsin resistance of each of the
SNAREs can be induced only in cells that are competent for regulated
exocytosis (but see below).
|
Ca2+ induces SNARE resistance to proteinase K
The induction by Ca2+ of protease-resistant SNAREs was confirmed
using proteinase K, which targets distinct peptide bonds from those broken by
trypsin. The latter cleaves between the carboxylic side of the basic amino
acids lysine and arginine and any residue, whereas proteinase K cuts bonds on
the carboxylic side of aliphatic, aromatic or hydrophobic amino acids linked
with any other residue. In general, proteinase K cleaves each SNARE slightly
closer to their coiled-coil domains than trypsin when pre-formed complexes are
exposed to either protease in vitro, thereby, producing a slightly smaller
`minimal core SNARE complex' (Fasshauer et
al., 1998a). In permeabilised chromaffin cells, increasing
[Ca2+] induced incremental levels of resistance to proteinase K in
all of the four SNAREs (Fig.
2C). As noted for trypsin, major signals were observed for
full-length SNAREs, with truncated forms being less abundant, presumably
because of rapid proteolysis or severance from the membrane.
BoNT/A reduces the amounts of Ca2+-induced SNAP-25, Sbr
and syntaxin resistant to trypsin, but their concentration dependence remains
unaltered
BoNTs cause neuromuscular paralysis via the blockade of acetylcholine
release from motor nerve terminals (Dolly et al., 2001). BoNT/A differs from
the other BoNTs in that its effect can be overcome transiently by
high-frequency nerve stimulation, the addition of agents that facilitate
Ca2+ entry into the presynaptic neurone (e.g. 4-aminopyridines) or
by increasing the extracellular Ca2+ concentration
(Simpson, 1989). Likewise,
blockade by BoNT/A of evoked transmitter release from brain isolated nerve
terminals can be reversed by Ca2+-specific ionophores that render
the presynaptic membrane permeable to the cation
(Dolly et al., 1994
). Thus, it
has been proposed that BoNT/A inhibits evoked exocytosis by lowering the
Ca2+ sensitivity of the membrane fusion apparatus
(Sellin, 1987
). Notably, the
removal of nine amino acids from the C-terminus of SNAP-25 does not stop this
protein participating in SNARE complexes in vitro or in vivo, although their
stability (i.e. resistance to SDS denaturation) is reduced
(Hayashi et al., 1995
;
Pellegrini et al., 1995
;
Lawrence and Dolly, 2002
). If
formation of these complexes drives membrane fusion, and BoNT/A does not block
this, it might be that the toxin inhibits exocytosis by preventing
transmission of the Ca2+ signal to the SNAREs. To test this
hypothesis, chromaffin cells were exposed to BoNT/A using a protocol that
results in internalisation of the toxin, with consequent cleavage of virtually
all the cells' complement of SNAP-25 and near-complete blockade of
catecholamine exocytosis in response to depolarising stimuli such as nicotine,
2 mM Ba2+ or 55 mM K+
(Lawrence et al., 1996
).
Nevertheless, when these cells are permeabilised, a fraction of the secretory
response can be elicited by the addition of Ca2+
(Lawrence et al., 1996
). To
ascertain whether the Ca2+ sensitivity of the exocytotic trigger
had been altered by BoNT/A, pre-poisoned cells were permeabilised and exposed
to the cation at various concentrations, as described previously for
toxin-free cells (see Fig. 1).
Notably, the BoNT/A-poisoned cells secreted much less catecholamine than the
toxin-free controls (Fig. 3A),
but the Ca2+-concentration dependence for the residual secretion
remained unaltered. Membranes were prepared from these BoNT/A-poisoned and
control cells and boiled before being subjected to analysis for each SNARE by
western blotting (Fig. 3B,C).
BoNT/A cleaved SNAP-25, revealed by the exclusive disappearance of the signal
for IgG reactive with its C-terminal residues
(Fig. 3B), with no change in
the other SNAREs as expected from the toxin's absolute specificity. Next, the
influence of toxin treatment on the susceptibilities of the SNAREs to trypsin
was investigated. The levels protected against trypsinisation were attenuated
for SNAP-25, syntaxin and Sbr, but not synaptotagmin, following treatment with
BoNT/A (Fig. 3C; note that
equivalent amounts of protein from BoNT/A-treated and control cells were used
and the samples analysed together by identical procedures). This experiment
was repeated several times with no consistent BoNT/A-induced change in the
amounts of trypsin-resistant synaptotagmin being observed. Notably, despite a
reduction in the amount of SNAREs (except synaptotagmin) being protected, the
[Ca2+] dependencies of their protection were unchanged
(Fig. 3D-G); the reasons for
the somewhat anomalous values observed at 100 µM Ca2+ are
unclear.
|
Ca2+ protects SNARE monomers against trypsinisation
A plausible explanation for the SNAREs' acquisition of resistance to
trypsin is that during the triggering of exocytosis by the cation, monomeric
SNAREs associate to form a heterotrimeric complex. Several in vitro
experimental findings lend support to this hypothesis. (i) Recombinant
syntaxin, SNAP-25 and Sbr spontaneously form a complex when mixed
(Hayashi et al., 1994); (ii)
the SNARE complex protects its individual components against cleavage by
trypsin or BoNTs (Fasshauer et al.,
1998a
; Hayashi et al.,
1994
; Poirier et al.,
1998
); (iii) homotypic yeast vacuole fusion only proceeds between
vesicles proffering `monomeric' (i.e. can be solubilised by SDS without
boiling) SNAREs rather than cis complexes of SNAREs
(Ungermann et al., 1998
); and
(iv) in BoNT/E-poisoned PC-12 cells, a SNAP-25 C-terminal peptide that
restored Ca2+-triggered exocytosis was incorporated into a SNARE
complex in the cell membrane (Chen et al.,
1999
).
To determine whether SDS-resistant complex formation underlies the acquisition by SNAREs of resistance to trypsin, cells were exposed to various concentrations of Ca2+; some samples were subsequently treated with 100 µg/ml trypsin where indicated, then membranes were prepared, solubilised in SDS sample buffer and divided into two. One half of each sample was boiled, the other not; both were subjected to SDS-PAGE followed by western blotting for the SNAREs. Notably, boiling of non-trypsin-treated samples clearly increased the intensity of signals for each of the SNAREs at the expected position for their respective monomers (Fig. 4A). Thus, it appears that a sizeable fraction of each protein is resistant to solubilisation by SDS at ambient temperatures. Nevertheless, the majority of the trypsin-resistant SNAREs in cells exposed to the protease were not in complexes that are stable in 1% SDS sample buffer containing ß-mercaptoethanol, because boiling of the samples resulted in only a slight increase in band intensity for each of them (Fig. 4B). These data imply that the Ca2+-induced protection against trypsin observed for the SNAREs is not, or only partly, caused by their increased incorporation into SDS-resistant complexes. In contrast to control samples, after BoNT/A intoxication, minimal levels of trypsin-resistant SNAREs were observed in non-boiled samples (Fig. 4B), but some was recovered after boiling, suggesting that only SNAREs in complexes were protected under these conditions. This would explain why much lower amounts of the SNAREs are protected in the BoNT/A-poisoned cells.
|
Synaptotagmin I, SNAP-25 and syntaxin respond to Ca2+ in
the absence of Sbr
BoNT/B specifically proteolyses Sbr, removing a large portion of its
cytoplasmic domain from the membrane-associated moiety. As with BoNT/A,
chromaffin cells can be extensively poisoned with BoNT/B such that >90% of
the cells' Sbr is degraded (Fig.
5A) and evoked transmitter release almost abolished
(Foran et al., 1995;
Lawrence et al., 1996
). In
contrast to BoNT/A, little recovery of Ca2+-triggered exocytosis
was obtained after permeabilisation of chromaffin cells intoxicated with
BoNT/B (Fig. 5B), as expected
(Lawrence et al., 1996
). In
the BoNT/B-treated chromaffin cells lacking most of the intact Sbr, it might
seem reasonable to postulate that ternary SNARE complexes could not form and,
therefore, SNAP-25 and syntaxin should not be protected against
trypsinisation. However, western analysis of the membranes from cells
pre-incubated with BoNT/B that had been permeabilised and exposed to trypsin
in the presence of incremental [Ca2+] revealed that, as well as
synaptotagmin, SNAP-25 and syntaxin remained responsive to the cation and
still became resistant to trypsin (Fig.
5C). In general, the Ca2+ sensitivity and amounts of
synaptotagmin, syntaxin and SNAP-25 protected against trypsin were similar for
toxin-free and BoNT/B-poisoned cells (Fig.
5C-F), although at lower [Ca2+] slightly more
trypsin-resistant SNAREs were observed in BoNT-poisoned cells than in control
cells. Of course, only a faint immuno-signal for trypsin-resistant Sbr was
observed in the BoNT/B-poisoned cells (Fig.
5C, single asterisk), but extended photographic exposure (double
asterisk) showed that Ca2+ protected this residual level against
proteolysis.
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Discussion |
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The SNARE complex itself has been proposed as a candidate Ca2+
sensor (Fasshauer et al.,
1998b; Sutton et al.,
1998
), but Ca2+ binding at physiologically relevant
concentrations remains to be shown. Furthermore, the structure solved for the
cis SNARE complex is likely to be an end product of fusion
(Chen et al., 1999
) and,
therefore, could not sense Ca2+ before fusion. Also, the bulk of
the catecholamine released from permeabilised cells exposed to >10 µM
Ca2+ derives from undocked granules distal to the plasmalemma,
which have to be mobilised to reach the cell membrane before fusing; these
granules are enriched for synaptotagmin and Sbr but not SNAP-25 nor syntaxin,
thus, SNARE complexes are unlikely to be involved in their
Ca2+-triggered mobilisation.
SDS-resistant complex formation is not the major means by which
SNAREs acquire resistance to trypsin
In non-toxin-treated cells, the majority of the SNAREs protected by
Ca2+ against trypsin were solubilised by SDS as monomers without
boiling, indicating that they were not in SDS-resistant complexes. Although
the increased trypsin resistance could be induced within each protein
individually, their identical Ca2+ sensitivity suggests that they
acquire this property simultaneously. The data do not preclude a role for
SDS-resistant SNARE complex formation during exocytosis, as proposed by Chen
et al. (Chen et al., 1999), but
shows that Ca2+ induces other persistent changes in the SNAREs.
Unfortunately, it is impractical to use milder detergents to preserve and
assay weak complexes formed in situ because they do not prevent the generation
of artefactual SNARE interactions during sample preparation for PAGE
(Otto et al., 1997
).
Priming is essential for synaptotagmin and the other SNAREs to remain
responsive to Ca2+
In cells that were rendered incompetent for exocytosis owing to `run-down',
synaptotagmin and the other SNAREs did not acquire increased resistance to
trypsin in the presence of Ca2+
(Fig. 2). The diminution in
secretory activity is caused by loss from the cells of proteins and
metabolites (e.g. MgATP) that are essential for priming reactions that precede
Ca2+-elicited fusion (Hay and
Martin, 1992; Holz et al.,
1989
; Sarafian et al.,
1987
). Thus, it appears that the role of priming is to maintain
synaptotagmin and the other SNAREs in a Ca2+-responsive state,
whereas fusion entails Ca2+ induction of conformational changes in
these `primed' SNAREs. Importantly, these data clearly indicate that
acquisition of trypsin resistance is gained via synaptotagmin and SNARE
function and is not some artefact of the assay.
Distinct effects of Clostridial toxins provide clues to the
mechanism of Ca2+ signal transduction to the SNAREs
As noted above, in BoNT/B-poisoned cells, the Ca2+ signal can
still be communicated to SNAP-25 and syntaxin despite their lack of Sbr; this
supports the hypothesis that SDS-resistant ternary complex formation is not
the only way in which the SNAREs can become less susceptible to trypsin.
Binary SNAP-25-syntaxin complexes may be induced by Ca2+, either to
form or to adopt a novel `activated' conformation
(Lawrence and Dolly, 2002)
before association with Sbr. The data does not rule out Ca2+ signal
transmission to SNAP-25-syntaxin already associated with Sbr in toxin-free
cells, but mitigates against hypotheses that propose an Sbr-SNAP-25
interaction preceding the binding of syntaxin
(Chen et al., 2001
). In cells
exposed to BoNT/A, SNAP-25 is C-terminally truncated, and the amounts of
SNAP-25, syntaxin and Sbr that resist trypsin degradation are severely
reduced; by contrast, the abundance of trypsin-resistant synaptotagmin I was
not lowered. Thus, the toxin does not perturb Ca2+ sensing by the
latter, but attenuates transmission of the Ca2+ signal to the other
SNAREs. The data dispel the popular hypothesis that BoNT/A simply lowers the
Ca2+ affinity of the exocytotic apparatus
[(Gerona et al., 2000
;
Sellin, 1987
;
Simpson, 1989
) and see above].
The toxin reduces the amount of each SNARE being protected against trypsin,
but the Ca2+ sensitivity for this and (most importantly) exocytosis
is not shifted. Moreover, the above-noted hypothesis cannot explain the lack
of response from BoNT/A-treated intact chromaffin cells to depolarising
stimuli such as elevated [K+] or nicotine
(Lawrence et al., 1996
),
because these treatments raise the free [Ca2+] at sub-plasmalemmal
exocytotic release sites to >100 µM
(Burgoyne, 1991
). Rather, the
severe inhibition of responses to the latter could be a consequence of the
transient Ca2+ signal they induce [<1 minute
(Burgoyne, 1991
)]; in this
context, it is noteworthy that the stoichiometry of SNAP-25-syntaxin in
complexes, which may be vital for the fast exocytotic response, is altered by
BoNT/A (Lawrence and Dolly,
2002
). In permeabilised cells, the Ca2+ stimulus is
maintained for an extended period (15 minutes), and this may be why exocytosis
can still proceed, albeit at an attenuated rate, after inhibition with BoNT/A;
the toxin selectively inhibits fast phases of exocytosis more strongly than
slow responses (Xu et al.,
1998
).
Finally, a sequence for putative reactions that occur in response to Ca2+ can be proposed from the differing effects of BoNT/A and B on acquisition of resistance to trypsin. Firstly, synaptotagmin may be the mediator for recruitment of undocked granules to release sites. Upon docking, the C-terminus of SNAP-25 is implicated in Ca2+ sensing by Sbr and syntaxin, as BoNT/A reduces the amounts of all three proteins,
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Acknowledgments |
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