From the Laboratory of Developmental Neurobiology, NICHD, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, December 6, 2000, and in revised form, January 8, 2001
Botulinum neurotoxin serotype A (BoNT/A) is
distinguished from BoNT/E by longer duration of paralysis and greater
potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137-142).
This single quality of toxin activity can account for the differences
observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the
synaptic protein SNAP-25 whereas BoNT/A removes only nine residues
creating a 197-amino acid fragment (P197) that is 95% the
length of SNAP-25. We show that inhibition of neurotransmitter release
by BoNT/E is equivalent to the damage caused to SNAP-25. However,
synaptic blockade by BoNT/A is greater than the extent of SNAP-25
proteolysis. These findings can be explained if P197 produces an
inhibitory effect on neurotransmitter release. A mathematical model of
the experimentally determined relationship between SNAP-25 damage and
blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca2+. In this case, vesicular
release is restored by increasing intracellular Ca2+. These
data demonstrate that P197 competes with intact SNAP-25, but is unable
to initiate normal synaptic vesicle fusion in physiological concentrations of Ca2+.
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INTRODUCTION |
Intoxication with botulinum neurotoxin
(BoNT)1 in vivo
leads to flaccid paralysis by blockade of acetylcholine release at the neuromuscular junction (1-3). The proteolytic activity of the toxin responsible for causing paralysis resides within the 50-kDa light
chain domain (4, 5) and is directed against three synaptic proteins:
synaptosomal-associated protein of 25-kDa (SNAP-25), vesicle-associated
membrane protein (VAMP), or syntaxin (6-11). BoNT/A and -E both cleave
SNAP-25 but at distinct sites (12). Interestingly, paralysis from
BoNT/A lasts for many months whereas blockade caused by BoNT/E lasts
for relatively brief periods (13, 14). Two hypotheses were proposed to
account for this difference: (a) BoNT/A remains
catalytically active for a longer interval than BoNT/E or,
alternatively (b) the catalytic activity of both toxins is
very short-term but the major SNAP-25 fragment generated by BoNT/A
(P197) persists in nerve terminals and interferes with neurotransmitter
release (15). The first hypothesis has been substantiated by direct
demonstration of BoNT/A persistence in primary mouse spinal cord
cultures, electroporated chromaffin cells, and mammalian neuromuscular
junction preparations in vivo (16-18). Evidence for the
second hypothesis is indirect. Overexpressed P197 in an insulinoma
Hit-T15 cell line inhibits insulin secretion (19). BoNT/A action at the
neuromuscular junction produces a strong immunogenic signal for P197
within nerve terminals, indicating that P197 resides for some time in
the proper intracellular regions where it could exert an inhibitory
effect on neurotransmission (20, 21). However, there has been no
quantitative analysis to determine whether the concentrations of P197
generated by toxin activity are able to inhibit neurotransmitter release.
BoNT/A and -E are unique among the BoNTs in that, under certain
experimental conditions, elevated Ca2+ can partially
relieve a toxin-induced blockade (22-24). The effect of
Ca2+ on reversing BoNT/A-induced paralysis has been
reported for muscle preparations (25, 26), hippocampal brain slices
(27, 28), synaptosomes (29), and chromaffin cells (31). The molecular relationship between SNAP-25 and Ca2+ is not well
understood, however. The altered Ca2+ sensitivity observed
in PC12 cells for either BoNT/A or -E (30) is consistent with earlier
observations using neuromuscular or chromaffin cell preparations prior
to the discovery of SNAP-25 as the toxin substrate (32-34).
Furthermore, electrophysiological results indicate that the C terminus
of SNAP-25, which is directly affected by BoNT/A and -E, contributes to
the initiation of two distinct Ca2+-sensitive steps in the
neurotransmitter release process (31).
SNAP-25 and P197 each bind to VAMP and syntaxin although the
P197-containing complex is less stable to detergent solubilization than
a complex containing SNAP-25 (35-37). Examining the possible inhibitory role of P197 within the framework of the SNARE hypothesis (38, 39) suggests that BoNT/A damage compromises the ability of the
complex to facilitate vesicle fusion. Calcium induces a conformational
transition of the trimeric SNARE complex from a cis
(nonsecretory) to a trans orientation which triggers vesicle fusion and neurotransmitter release (40, 41). Given these two effects,
SNAP-25 cleavage and reduced Ca2+ sensitivity, it is
possible that BoNT/A action on SNAP-25 alters other interactions which
affect neurotransmitter release. To this end, it was demonstrated that
Ca2+ stimulates binding between SNAP-25 and P197 with
synaptotagmin (30). Hence, there may be multiple roles for SNAP-25 and
Ca2+ in the release process (34, 41-43).
Calcium ionophores have been used successfully to discriminate between
the effects of voltage-gated Ca2+ channels and direct
effects of elevated intracellular Ca2+ on BoNT/A action
(27, 28, 44, 45). This approach has been extended to the present study
to investigate the requirement for intracellular Ca2+ in
overcoming the inhibitory action of BoNT/A in mouse spinal cord
cultures. The findings demonstrate that a mathematical correlation exists between the generation of P197 by BoNT/A and inhibition of
Ca2+-dependent neurotransmitter release,
indicating that the amount of P197 attained within neurons will block exocytosis.
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EXPERIMENTAL PROCEDURES |
Materials--
Monoclonal antibody to SNAP-25 (SMI-81) was
obtained from Sternberger Monoclonals, Inc. (Lutherville, MD).
Thapsigargin, ionomycin, monoclonal antibody to syntaxin, and alkaline
phosphatase-labeled anti-mouse antibody were purchased from
Sigma-Aldrich Chemical Co. All electrophoresis reagents were from
Bio-Rad. Polyvinylidene difluoride membrane was obtained from Bio-Rad.
Preparations of BoNT/A and -E toxin complex were from Wako Chemicals
Inc. (Richmond, VA) with reported activities of 2.0 × 107 LD50 and 1.0 × 107
LD50 per mg of protein, respectively. BoNT/E (1 mg/ml) was
activated (nicked) by incubating for 30 min at 37 °C with 0.3 mg/ml
trypsin (type XI, bovine pancreas) in 30 mM HEPES, pH 6.75. Trypsin was subsequently inhibited by addition of 0.5 mg/ml trypsin
inhibitor (type I-S, soybean) and incubation for 15 min at 20 °C
(16). Toxins were aliquoted and stored at
20 °C; each experiment
utilized a new aliquot of toxin to ensure uniform activity. Molar
concentrations stated in the text were based upon masses of 500 and 300 kDa for BoNT/A and -E, respectively.
Spinal Cord Cultures--
Timed pregnant C57BL/6NCR mice were
obtained from the Frederick Cancer Research and Development Center,
Frederick, MD. Spinal cord cell cultures were prepared as described
(46, 47). Briefly, spinal cords were removed from fetal mice at
gestation day 13, dissociated with trypsin, and plated on
VitrogenTM 100-coated dishes (Collagen Corp., Palo Alto,
CA) at a density of 106 cells/dish. Cultures were
maintained for 3 weeks at 37 °C in an atmosphere of 90% air,
10% CO2 before addition of toxins. Growth medium consisted
of minimum essential medium (formula 82-0234AJ; Life Technologies,
Inc., Bethesda, MD) supplemented with 5% heat-inactivated horse serum
and a mixture of complex factors (48). Cultures were incubated with
BoNT diluted with growth medium for times indicated in figure legends.
BoNT-containing medium was removed by aspiration; cells were rinsed
with toxin-free medium and prepared for Western blot analysis or
neurotransmitter release.
Protein and Western Blot Analysis--
Protein was prepared by
dissolving cells in 1% SDS with 1 mM EDTA and 1 mM EGTA. The slurry was transferred to 1.5-ml
microcentrifuge tubes, incubated in a 95 °C water bath for 5 min to inactivate proteases, and then stored at
20 °C. Immediately
prior to use samples were thawed, mixed with equal volumes of
Tris-Tricine sample buffer (Bio-Rad), heated at 95 °C for 4 min, and
then separated by SDS-polyacrylamide gel electrophoresis. Equal
quantities of protein were loaded onto 16.5% acrylamide gels prepared
by the method of Laemmli (49). Proteins were separated using 0.1 M Tris-Tricine, pH 8.3 (50), and then transferred to
polyvinylidene difluoride membrane with a buffer containing 192 mM glycine, 25 mM Tris, pH 8.3, and 12%
methanol. Membrane development was performed as previously described
(16).
Neurotransmitter Release and
Quantitation--
Potassium-stimulated
Ca2+-dependent neurotransmitter release was
determined as previously described (51). Cultures were labeled with
[3H]glycine for 0.5 h at 35 °C and then washed
with a series of low K+-containing buffers. Unless
otherwise indicated, glycine release was stimulated by addition of 56 mM K+ and 2 mM Ca2+ to
cultures; stimulation medium was collected after 5 min at 35 °C.
Calcium-dependent release was determined by subtracting baseline radioactivity secreted from cultures in the absence of Ca2+, and expressed as a percentage of the total cellular radioactivity.
Regression Analysis and Curve Fitting--
Scanned images of
Western blots were produced and edited utilizing NIH Image
(National Institutes of Health, Bethesda, MD). Where indicated, images
were digitally analyzed using IPLab Gel software (Scanalytics, Inc.,
Fairfax, VA). Nonlinear regression analysis of data was performed with
Sigma Plot (SPSS Science, Inc., Chicago, IL). Error bars represent
standard deviations of triplicate determinations unless otherwise indicated.
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RESULTS |
Concentration-dependent Effects of BoNT/E on SNAP-25
Cleavage and Neurotransmitter Release--
BoNT/E treatment of spinal
cord cell cultures results in cleavage of SNAP-25 in a
concentration-dependent manner (Fig.
1A). Under the conditions of
these experiments, the IC50 for BoNT/E cleavage of SNAP-25
is ~50 pM. Measurement of synaptic function indicates
that K+-evoked glycine release is inhibited to the same
extent as SNAP-25 cleavage (Fig. 1B). In control cultures,
the extent of glycine release is related to Ca2+
concentrations in the external medium. Cultures depolarized in the
presence of 0.15, 0.25, 2, and 10 mM Ca2+
release 4.3, 15.7, 26.5, and 25.8% of the total cellular radiolabel. Maximal release is achieved with 2 mM Ca2+
(Fig. 3). Varying external Ca2+ concentration over this
range did not alter significantly the IC50 for the BoNT/E
block of glycine release which varies from 49 to 55 pM for
the conditions tested (Fig. 1B).

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Fig. 1.
Effects of BoNT/E on neurotransmitter release
and SNAP-25 cleavage. A, Western blot showing cleavage
of SNAP-25 at indicated concentrations of BoNT/E. Spinal cord cultures
were incubated in toxin for 24 h at 37 °C. Cells were
solubilized in sample buffer and 7 µg of total cellular protein was
used for Western blotting with SNAP-25 and syntaxin antibodies.
Cleavage of SNAP-25 increased in direct proportion to the toxin
concentration. B, potassium-evoked glycine release was
measured in the presence of 0.15 ( ), 0.25 ( ), 2.0 ( ), or 10 mM ( ) Ca2+ in the depolarizing medium.
SNAP-25 cleavage as determined by densitometric analysis of Western
blots similar to that of A is also plotted ( ).
IC50 values for SNAP-25 proteolysis and glycine release
were determined by regression analysis using the equation:
Rres = Rmax/(1 + [toxin]/IC50)n. Rres
represents the release determined at each dose of BoNT/E, [toxin].
Rmax is the maximal signal detected at zero
toxin concentration. IC50 values ranged from 49 to 55 pM with a Hill coefficient (n) of 1.1 ± 0.1. C, model of the relationship of BoNT/E, SNAP-25, and
K+-evoked release. In this depiction, SNAP-25 is cleaved by
BoNT/E. Each incremental decrease in SNAP-25 results in an equivalent
decrease in neurotransmitter release. A graphed representation of this
modeled relationship generated the same overlapping pattern as the
experimental data.
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The model in Fig. 1C illustrates the effect of BoNT/E on the
release process. In this model, SNAP-25 is shown to interact with
elements (X) required for neurotransmitter release with an affinity represented by Kd. Potassium depolarization stimulates release in a Ca2+-dependent manner.
Release is directly proportional to the amount of SNAP-25 present. As
SNAP-25 is cleaved by increasing concentrations of BoNT/E,
neurotransmitter release decreases to a similar extent. BoNT/E-cleaved
SNAP-25 (P180) has been shown to interact with VAMP and syntaxin
in vitro (36, 37). However, our data indicate that in this
intact neuronal system, P180 does not interfere with K+-evoked release, possibly because P180 interacts with
X very weakly relative to SNAP-25. Furthermore, increasing
extracellular Ca2+ above physiological concentrations does
not overcome the block (Fig. 3), similar to findings with neuromuscular
preparations (14).
Concentration-dependent Effects of BoNT/A on SNAP-25
Cleavage and Neurotransmitter Release--
Unlike BoNT/E,
dose-response data with BoNT/A produce nonoverlapping curves for
SNAP-25 proteolysis and blockade of neurotransmitter release (Fig.
2B). SNAP-25 cleavage occurs
with an approximate IC50 of 3 pM and glycine
release is inhibited with IC50 values decreasing from 0.3 to 0.1 pM as Ca2+ concentrations decrease from
2 to 0.15 mM in the release medium (Fig. 2B).
Increasing Ca2+ to 8 or 10 mM yielded results
identical to that obtained with 2 mM Ca2+ (data
not shown). At Ca2+ concentrations lower than 2 mM, a greater degree of inhibition occurs relative to
SNAP-25 cleavage (Fig. 2B). At all Ca2+
concentrations tested, the slopes for neurotransmitter release were the
same. Comparing these slopes with the slope for SNAP-25 yields a ratio
of ~2:1.

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Fig. 2.
BoNT/A effects on neurotransmitter release
and SNAP-25 cleavage. A, Western blot showing cleavage
of SNAP-25 at the indicated concentrations of BoNT/A. Cultures were
treated as described in the legend to Fig. 1. B,
potassium-evoked glycine release was measured with 0.15 ( ), 0.25 ( ), or 2.0 mM ( ) Ca2+. Level of SNAP-25
cleavage as determined by densitometric analysis of Western blots
similar to that of A is also plotted ( ). Error
bars represent standard deviations of at least three
determinations. C, modeling SNAP-25 and P197 effects on
neurotransmitter release. In this depiction, it is assumed that P197
interacts with the same cellular components as SNAP-25 and that this
interaction yields an inhibitory complex, P197-X, that
supports transmitter release only when internal Ca2+
exceeds normal activity-dependent concentrations
(Ca2+'). D, graphed representation of modeled
relationship. When interactions between SNAP-25 or P197 with
X are predicted to have the same binding affinity
(Kd = K ), the above graph is generated
for neurotransmitter release ( ) relative to SNAP-25 ( ). Slopes
from the model curves demonstrate a 2:1 relationship similar to the
experimental findings.
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Fig. 2C depicts possible interactions between SNAP-25 and
P197 and their combined effects on the release process. In this model
P197 binds to elements X much the same as SNAP-25. Because elevated Ca2+ has been shown previously to reverse BoNT/A
inhibition in brain slices (27), PC12 cells (29), and neuromuscular
preparations (52), we hypothesize that neurotransmitter release through
the P197-X pathway would require Ca2+ in excess
of that achieved during normal neuronal stimulation. Elevated
Ca2+ above normal levels is depicted with the term
Ca2+'. This model allows us to predict the relationship
between SNAP-25 cleavage and block of transmitter release. Assuming
(a) that P197 interacts with elements X with the
same or nearly the same affinity as SNAP-25, and (b) that at
any given dose of BoNT/A, the molar sum of the remaining SNAP-25 and
P197 will equal the amount of SNAP-25 in the absence of toxin, the
profile in Fig. 2D is generated. According to this model,
the ratio of the slope for neurotransmitter release relative to SNAP-25
is exactly 2:1. The data for neurotransmitter release measured with 2 mM Ca2+ (Fig. 2B) overlap the
predicted result.
Extracellular Ca2+ Effects on Overcoming BoNT
Intoxication--
Because at lower Ca2+ concentrations,
BoNT/A-exposed cultures show a greater block in transmitter release
(Fig. 2), we examined the effects of extracellular Ca2+
concentration on glycine release following exposure to a single, high
dose of BoNT/A or -E. Both BoNTs at 1 nM cleave all
detectable SNAP-25 after 24 h (Figs. 1A and
2A). [3H]Glycine release from control cultures
(no toxin exposure) is Ca2+ dependent with an
EC50 value for Ca2+ of 0.21 mM and
a Hill coefficient of about 2 (Fig. 3).
Glycine release from BoNT/A-treated cultures is similarly
Ca2+ dependent with an EC50 value of 1.0 ± 0.1 mM and a plateau value that is 23% of control
release (Fig. 3). Maximum release for both control and BoNT/A-treated
cultures is attained when Ca2+ exceeds 2 mM
(Fig. 3) and the slope of release is unaffected by toxin with an
estimated Hill coefficient of 2.2 ± 0.3 (53, 54). Glycine release
from BoNT/E-treated cultures is not detected at any concentration of
Ca2+ tested (Fig. 3). This indicates that the rise of
intracellular Ca2+ resulting from Ca2+-channel
activation can restore some function to BoNT/A- but not to
BoNT/E-treated neurons, consistent with results from Figs. 1 and 2.

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Fig. 3.
Calcium titration and K+-evoked
glycine release. Spinal cord cultures were incubated in growth
medium containing either BoNT/A or -E at 1 nM for 24 h
at 37 °C, after which Ca2+-dependent release
of glycine was assayed. Cultures not treated with toxin ( )
demonstrated a standard titration curve relative to Ca2+
concentration with an EC50 value for Ca2+ of
0.21 ± 0.01 mM and a Hill coefficient of 1.9 ± 0.2. Maximal glycine release for each experiment ranged from 18 to 27%
of the total cellular radioactivity and was set to 100%.
BoNT/A-treated cultures ( ) demonstrated Ca2+ dependence
with an EC50 value of 1.0 ± 0.1 mM and
maximal release when Ca2+ exceeded 2 mM. The
plateau value for this release was 23.2 ± 2.2% of the maximal
release elicited from control cultures. BoNT/E-treated cultures ( )
demonstrated no Ca2+ dependent release when measured in the
presence of 1-8 mM Ca2+. Results were compiled
from five (control) and four (BoNT/A-treated) experiments. Each symbol
represents the average of duplicate determinations.
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Intracellular Ca2+ Effects on Overcoming BoNT/A
Intoxication--
To examine the effects of increased intracellular
Ca2+ above levels attained from Ca2+-channel
activation, glycine release was evoked by 56 mM
K+ in the presence and absence of 2 mM
Ca2+ and 5 µM ionomycin, a
Ca2+-specific ionophore. Ionomycin partially restores
glycine release after exposure to BoNT/A (Fig.
4). Treatment with BoNT/A reduces glycine
release to ~23% of control release. Addition of ionomycin in
combination with Ca2+ reduces the effect of BoNT/A, raising
glycine release to 60% of control values. To determine the source of
Ca2+ that contributes to this reversal, glycine release was
assayed with ionomycin either in the absence of extracellular
Ca2+, or after having treated cultures with thapsigargin to
deplete intracellular Ca2+ stores (55, 56). The absence of
any release with zero external Ca2+ with ionomycin and the
failure of thapsigargin to affect the ionomycin-dependent
release provide evidence that internal Ca2+ stores do not
contribute significantly to ionomycin's reversal of the BoNT/A-induced
block.

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Fig. 4.
The ability of ionomycin to reverse the
BoNT/A block. Spinal cord cultures were treated with 2 nM BoNT/A for 29 h at 37 °C. Upon removal of the
toxin some cultures were treated with 15 µM thapsigargin
in minimal essential medium for 2.5 h. Glycine release was
determined in the presence of 2 mM Ca2+ with
release from control cultures (18.6% of total cellular radiolabel) set
at 100% (first column). BoNT/A treatment reduced glycine
release to 25% of controls (second column). Ionomycin at 5 µM added to release buffer containing 56 mM
K+ and 2 mM Ca2+ did not enhance
the total release from controls (data not shown) but increased release
from BoNT/A-treated cultures to 60% (third column).
Depletion of intracellular stores of Ca2+ with thapsigargin
did not alter substantially the effect of ionomycin, indicating that
ionomycin-induced recovery of release was independent of internal
Ca2+ stores (fifth column). In contrast,
reversal of the BoNT/A block did not occur in zero Ca2+
(fourth column), indicating a requirement for
Ca2+ in the extracellular medium.
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To expand upon this finding, cultures were treated with 1 nM BoNT/A or BoNT/E for 24 h and neurotransmitter
release was measured in the presence of 10 µM ionomycin
in 56 mM K+ with various external
Ca2+ concentrations. Ionomycin produces a significant
reversal of the BoNT/A block at all Ca2+ concentrations
tested with complete recovery occurring near 2 mM
Ca2+. Unlike K+/Ca2+ stimulation
alone, stimulation in the presence of ionomycin partially alleviates
BoNT/E-induced block with maximum glycine release at 53% of control
cultures (Fig. 5). Within these
experimental conditions, BoNT/A- and/E-treated neurons are about 4- and
7-fold less sensitive to Ca2+, respectively, than untreated
neurons. Ionomycin has two measurable effects on reversing BoNT/E
action: neurons release more glycine and demonstrate higher sensitivity
to Ca2+. In the case of BoNT/A, ionomycin stimulates
release of glycine to control values. However, ionomycin does not alter
the EC50 value for Ca2+ which is 0.8 ± 0.1 mM (Fig. 5) compared with 1.0 ± 0.1 mM obtained for the partial release achieved in the absence
of ionomycin (Fig. 3). Ionomycin at the tested concentration does not
affect cultures that were not treated with toxin; neither the
Ca2+ sensitivity nor the extent of glycine release was
altered (Fig. 5).

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Fig. 5.
Relative sensitivity of neurotransmitter
release to Ca2+ in the presence of ionomycin following
exposure to BoNT. Cultures were treated with 1 nM
BoNT/A ( ) or/E ( ) for 24 h. Potassium-stimulated
Ca2+-dependent release of
[3H]glycine was measured in the presence of 10 µM ionomycin and various concentrations of
Ca2+ in release medium. Data are normalized to release
obtained with 2 mM Ca2+ from toxin-free
controls, which ranged from 19.1 to 23.4% of the total cellular
radioactivity for each experiment. EC50 values for
Ca2+ were obtained by regression fit of the equation
R(release) = (Rmax × [Ca2+])/(EC50 + [Ca2+]) to the
data. R(release) represents the fractional
glycine release measured at any given concentration of
Ca2+. Ionomycin did not alter the apparent EC50
value for control cultures ( ) which is 0.20 ± 0.01 mM. EC50 values for BoNT/A- and E-treated
cultures expressed relative to Rmax from
controls are 0.8 ± 0.1 mM and 1.4 ± 0.2 mM, respectively. Error bars represent S.D.
values of three separate determinations.
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DISCUSSION |
In this study, we examined the relationship between blockade of
neurotransmitter release and cleavage of SNAP-25 caused by BoNT/A and
-E to aid in understanding how BoNT/A seems to derive greater potency
than BoNT/E (14, 22, 26, 32). Both BoNTs induce a
concentration-dependent block in synaptic vesicle
exocytosis and proteolysis of SNAP-25. However, it appears likely that
in addition to disabling SNAP-25, BoNT/A action has a secondary
inhibitory effect. The nonoverlapping pattern of SNAP-25 cleavage and
block of K+-evoked transmitter release in this latter case
points to the formation of an inhibitory complex containing a cleavage
fragment produced by BoNT/A. Most simply, BoNT/A action on SNAP-25
produces a dual effect: a reduction in functional SNAP-25 and
production of a fragment that antagonizes synaptic vesicle fusion.
The identity of this fragment could be either the 197-amino acid
fragment, P197, or the smaller 9-residue fragment cleaved from the C
terminus. C-terminal peptides of SNAP-25 disrupt synaptic vesicle
trafficking in permeabilized chromaffin cells (57, 58). Peptides with
sequences corresponding to the C-terminal 26 and 20 amino acids
inhibited neurosecretion with IC50 values of ~0.25 and 20 µM, respectively (58, 59). A 12-residue peptide of the C
terminus failed to inhibit release to any significant extent (60). The
correlation of shorter peptide length with lower potency suggests that
the 9-residue cleavage fragment generated by BoNT/A does not interfere
with neurotransmitter release.
An inhibitory role of P197 was demonstrated by transfection of P197
into an Hit-T15 cell line and subsequent inhibition of insulin
secretion (19). Additionally, P197 has been shown to interact directly
with the SNARE proteins VAMP and syntaxin in a manner similar to
SNAP-25 (35, 37). Based on the estimated half-life for BoNT/A being 3 days (61), detection of P197 in intoxicated motor nerve
terminals in vivo generated the hypothesis that P197 was
degraded slowly relative to SNAP-25 and could, therefore, interfere
with the normal vesicular pathway for neurotransmitter release for
weeks or months (20, 21). Two studies since then have provided direct
evidence that BoNT/A catalytic activity persists within neurons with a
half-life far greater than 3 days (16, 18). Information on the
degradation rate of P197, although indirect at present, suggests that
it occurs at a rate similar to SNAP-25 (16). The prolonged duration of
the catalytic action of BoNT/A does not, however, invalidate the role
of SNAP-25 cleavage products in the extent of paralysis. For example,
the persistence of BoNT/A activity would provide a constant production
of P197 to account for its continued presence in intoxicated nerve
terminals (20, 21). Furthermore, because nerve stimulation accelerates
the onset of paralysis (23, 62, 63), toxin probably accumulates within
nerve terminals where production of P197 would occur in the proximity
of vesicular activity. Additive effects on muscle twitch from the
production of P197 and loss of SNAP-25 could also explain why muscle
paralysis occurs prior to detectable loss of SNAP-25 by
immunohistochemical methods (64).
The model presented in Fig. 2C predicts that cleavage of
x% of the SNAP-25 population would result in a
2x% block of neurotransmitter release. This is based on
simple competition of P197 and SNAP-25 for components of the fusion
complex and the relationship is depicted graphically in Fig.
2D. Otto et al. (72) reported that
cleavage of approximately half of SNAP-25 in synaptosomal preparations caused nearly a complete block of
Ca2+-dependent glutamate secretion, in
agreement with our model and experimental observations. In contrast to
the sigmoidal curve observed for BoNT/E (Fig. 1B), the
linear relationship of SNAP-25 cleavage relative to BoNT/A
concentration may reflect the preferential anchoring of BoNT/A (20) but
not BoNT/E within nerve terminals. SNAP-25 cleavage, therefore, may
represent both BoNT/A concentration and movement of SNAP-25 within
proximity of the toxin, for example, SNAP-25 must be transported to the
toxin. The model does not require that SNAP-25-X is
necessarily composed of VAMP and syntaxin; however, the current SNARE
hypothesis (38, 39) would support the involvement of these or other
proteins (30, 65, 66).
BoNT/E-treated spinal cord cultures developed a block in
neurotransmitter release no greater than the extent of SNAP-25
cleavage. The direct relationship between SNAP-25 cleavage and
neurotransmitter blockade indicates that, in intact neurons, SNAP-25
fragments created by BoNT/E do not interfere with the release process.
Neurotransmitter release from BoNT/E-treated spinal cord neurons is
approximately 7-fold less sensitive to Ca2+ than release
from untreated neurons when ionomycin is used, although in the absence
of ionomycin release does not occur. The ionomycin/Ca2+
combination could not produce a full recovery, however. Similar observations were obtained with other neuronal preparations (28, 45)
and are consistent with observations in vivo that the
minimal amount of the K+ channel blocker
3,4-diaminopyridine that stimulates a full recovery from BoNT/A does
not elicit recovery from BoNT/E (14).
Unlike BoNT/E-treated neurons, glycine release from BoNT/A-treated
neurons could be elicited without ionomycin. This release was
Ca2+-dependent with an EC50 value
for external Ca2+ of 1.0 mM, approximately
5-fold higher than controls (0.21 mM). At the lower
concentrations of Ca2+ tested, BoNT/A completely blocked
K+-stimulated release and at higher Ca2+
concentrations, maximal release was ~23% of the release from control
cultures. The effect of Ca2+ on the release process is
consistent with studies using intact chromaffin cells and hippocampal
neurons (31, 53, 67, 68); i.e. a pool of neurotransmitter
can be released after complete cleavage of SNAP-25 although the
concentration of Ca2+ required for this to occur is higher
than physiological Ca2+ levels. Release that occurs despite
treatment with BoNT/A may reflect the weaker ability of P197 relative
to SNAP-25 to facilitate Ca2+ requiring step(s) given the
extent of Ca2+ influx that occurs under normal
physiological conditions.
To further investigate the possible P197-release pathway (Fig.
2C), we employed ionomycin to determine whether elevated
cytosolic Ca2+ would fully reverse the effects of BoNT/A
(24, 29, 69). Ionomycin improved the efficiency of vesicular release in
BoNT/A-treated neurons. The apparent EC50 for
Ca2+ in the presence and absence of ionomycin was
statistically unaffected, 0.8 ± 0.1 mM compared with
1.0 ± 0.1 mM, respectively. Lack of an effect on
Ca2+ sensitivity and promotion of a full recovery of
transmitter release suggests that ionomycin facilitates the
hypothesized P197 release process by raising Ca2+ above
physiologically attainable levels. An alternative explanation is that
the action of an undetectable population of uncleaved SNAP-25 could
account for the observed synaptic release. If this were the case,
however, and SNAP-25 were solely responsible, then BoNT/A treatment
would reduce the plateau values for transmitter release but the
observed EC50 value for Ca2+ would approximate
the value for neurons not treated with toxin, i.e. 0.2 mM.
The lower Ca2+ sensitivity following BoNT/A exposure may be
due to disruption of several fusion processes. P197, for example, may
hinder vesicle trafficking in nerve terminals. BoNT/A and tetanus
neurotoxin cause significant accumulation of synaptic vesicles at
active zones (63, 70). These vesicles, unable to fuse with the
presynaptic membrane, might produce a physical obstruction for the
recruitment of new synaptic vesicles to dock and fuse upon
Ca2+-dependent stimulation (31, 63, 71). Hence,
we show that an intact neuronal system treated with BoNT/A requires
more Ca2+ to attain maximal release. This effect, in part,
may be due to changes in the dynamics of synaptic vesicle movement in
and near release sites caused by one or more Ca2+-related
changes from SNAP-25 cleavage (30).
Primary spinal cord cultures were used to provide the first proof that
catalytic activity of BoNT/A but not BoNT/E ensues for many months
following intoxication (16). This finding is consistent with in
vivo observations that BoNT/A but not BoNT/E causes long-term
paralysis (13, 18). In the present work, Ca2+ stimulates
release of neurotransmitter with a Hill coefficient of approximately 2 in the absence of toxin, which is consistent with electrophysiological
estimates (54, 67). Calcium stimulates release following exposure to
BoNT/A and to a lesser extent, BoNT/E, each observation being similar
to in vivo experiments with local injections of either toxin
into the extensor digitorum longus muscle of rats (14, 22, 23). Our
data demonstrate that the amount of P197 generated by
BoNT/A is sufficient to block neuronal activity in a
concentration-dependent manner. Furthermore, the ability of
P197 to inhibit release is based upon its ability to compete against uncleaved SNAP-25 in the release mechanism. In support
of this latter assertion, electroporation of anti-toxin antibodies into
BoNT/A-treated, chromaffin cells accelerates the recovery of secretion
by nearly 10-fold (17), indicating that inactivation of BoNT/A allows
newly synthesized SNAP-25 to displace P197. This demonstrates that the
effect of P197 is brief without BoNT/A activity indicating P197 itself
has a brief lifetime relative to BoNT/A. Integrating these observations
into a unified model we suggest that paralysis produced by BoNT/A,
(a) is due to persistent catalytic activity of this toxin
within nerve terminals and (b) is caused by loss of
functional SNAP-25 and concomitant generation of P197 that
competes with the remaining intact SNAP-25 to exacerbate the block.
We are grateful to Drs. Robert Sheridan,
Michael Adler, Sharad Deshpande, and George Oyler for comments during
the preparation of this manuscript. We also thank Dr. Robert Phair for
assistance in interpreting the models depicted in the current work; J. Caitlin Sticco for help in preparing and analyzing Western blot data
and, most certainly, we thank Karen Bateman for preparation and
maintenance of the spinal cord cell cultures and help with preparing
the present paper.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010992200
The abbreviations used are:
BoNT, botulinum neurotoxin;
SNAP-25, synaptosomal-associated protein of 25 kDa;
P197, BoNT/A-truncated SNAP-25;
P180, BoNT/E-truncated SNAP-25;
VAMP, vesicle-associated membrane protein;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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