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Botulinum neurotoxin (BoNT), an exotoxin produced by
the bacterium Clostridium botulinum, is the etiological agent
in botulism poisoning. Although there are 7 distinct
serotypes (designated A-G), they have the same general
molecular properties. Each serotype of BoNT consists of a
~100 kDa heavy chain (HC) and a ~50 kDa light chain (LC),
linked by a single disulfide bond and non-covalent
forces[1,2]. A 4-step mechanism consisting of binding, internalization,
translocation and cleaving the soluble NSF accessory
protein receptor (SNARE) protein is currently the accepted
explanation for BoNT intoxication[3,4]. It is believed that BoNT
enters cells via receptor-mediated
endocytosis[4]. Exposure of the BoNT holotoxin to the acidic pH of the endosomes
induces a conformational change of the disulfide-linked
dichain toxin, allowing the LC protease to pass across the
membrane through the putative HC channel and into the
cytosol[5]. When the LC of the toxin reaches the cytosol, it
acts as a zinc-dependent endoprotease to cleave
polypeptides that are essential for exocytosis, which results in
synaptic transmission blockade. It has been known for some
time that BoNT is capable of forming ion channels in
artificial bilayers[6-9] and PC12 cell
membrane[10]. A recent study demonstrated that toxin translocation and channel
formation were correlated[11]. The finding suggested to us that the
BoNT channel represented a potential target for
intervention to attenuate BoNT neurotoxicity.
Toosendanin (TSN,
C30H38O11, FW=574), a triterpenoid
derivative[12,13], is extracted from the bark of
Melia toosendan Sieb et Zucc. It is used in Chinese traditional medicine as an
ascarifuge[14]. Despite having some actions in common with
BoNT, TSN has been shown to have a markedly
antibotu-lismic effect both in vivo and in
vitro. For example, TSN can prevent death in animals (mice, rats and monkeys) treated
with several times the normal lethal dose of
BoNT[15,16]. The time to paralysis of the neuromuscular junction after
treatment with BoNT is lengthened by several times when the
preparations were preincubated with
TSN[16,17]. Our recent study showed that TSN treatment made synaptosomes
resistant to BoNT/A-mediated proteolytic cleavage on their
SNAP-25[18]. The protective effect did not result from
inhibiting the endopeptidase activity of the toxin, but from
interference with the approach of the LC to the substrate.
The purpose of this study was to investigate whether
TSN affects BoNT/A-induced channels, and hence protects
SNAP-25 from cleavage. NGF-differentiated PC12 cells were
used in this study. The cells become very sensitive to BoNT
after differentiation[19].
Materials and methods
Cell culture PC12 cells were obtained from the Type
Culture Collection of the Chinese Academy of Sciences and
were serially passaged in Dulbecco¡¯s modified Eagle¡¯s
medium (DMEM, Gibco, NY, USA) supplemented with 5% fetal
calf serum (HyClone, UT, USA). The cells were plated at low
density in culture medium supplemented with 100 ng/mL
nerve growth factor (NGF, 2.5S) in 35-mm plastic dishes
(Corning, NY, USA). After a minimum of 4 d, the cells were
used for electrophysiological recordings.
Solutions and chemicals The standard bath solution
used for the inside-out patch recording contained 200
mmol/L CsCl and 1 mmol/L dithiothreitol, and was buffered to pH
7.0 with 5 mmol/L Na·MOPS
(3-(N-morpholino)propane-sulfonate). The solution to fill the pipettes contained 200
mmol/L CsCl and was buffered to pH 5.3 with 5 mmol/L
Na·MES (2-(N-morpholino)ethanesulfonate). All solutions
were filtered at 0.45 mm before use. Unless otherwise
specified, all chemicals were from Sigma (St Louis, MO, USA).
The 500 kDa BoNT/A was from Wako (Japan). The toosendanin used in this work was a sample recrystallized in
ethanol with a purity of >98%[20]. Toosendanin was added
to the standard bath or pipette solution at a final
concentration of 35 µmol/L. Previous studies have demonstrated that
at a concentration of 17 µmol/L, TSN abolishes the action of
BoNT/A to cleave its substrate[18]. Here, a higher
concentration was used.
Electrophysiological recordings Patch pipettes were
pulled from 1.5 mm (outer diameter) capillary glass (type 95,
Shanghai Institute of Physiology, Shanghai, China) using a
Narishige PP-83 electrode puller (Narishige,
Japan). The tip of each pipette was coated with
N-trimethylsilydiethylamine and fire-polished on a microforge (FP-1, Shanghai Institute
for Biological Sciences, CAS). Tip resistance was 4-8
MW when filled with pipette solution. Single channel currents
were recorded in the inside-out
configuration[21] using a patch-clamp amplifier (Axopatch 200A, Axon Instruments,
Foster City, CA, USA) at room temperature (22-25
°C). After the pipettes were removed from the cell somas, the isolated
membranes were exposed to symmetrical salt solutions with
a pH gradient that was acidic on the pipette, or extracellular,
membrane surface. The previously intracellular side was
exposed to a bath solution at neutral pH.
Data analysis Membrane current was filtered at 1 kHz
and digitized at 20 kHz. Single channel analysis was
performed using the pClamp 6.0.4 program (Axon
Instruments). The membrane voltages are presented as the voltages at the
pipette referenced to the grounded bath and represent the
potentials on the side of the membrane where the toxin was
applied. As a preliminary criterion for selecting
BoNT-induced ion channels, membrane patches showing significant
channel activity immediately following excision were
discarded, because these were most likely endogenous ion
channels. In fact, most of the patches of membrane were
initially electrically silent. To generate toxin-induced ion
channels in these patches, 20 µg/mL of 500 kDa BoNT/A
was added to the pipette filling solution described earlier
before formation of the patch. As an index of channel
activity we used NPo; that is, the product of the number of
channels present in the patch membrane (N) and the probability
that a particular channel is open (Po).
All data are expressed as mean±SEM. Statistical
analysis was performed with Student¡¯s t-test, and
P<0.05 was considered significant.
Results
BoNT/A formed ion channels in PC12 cell
membrane No ion channel activity was detected in 8 patches examined
in the absence of BoNT/A in the pipette over 20
min. With the toxin, spontaneous ion channel openings due to
BoNT/A were observed in 27 of 31 patches examined after a delay of
2-9 min following first contact with the membrane patch
(average time 3.5±1.9 min). Typical membrane ion channels
formed by BoNT/A are shown in Figure 1A. Once these
toxin-induced ion channels appeared they remained active
until the gigohm patch seal decayed. During this period, the
apparent conductance of individual channels increased with
time after their first appearance. As shown in Figure 2A, for
membrane patches exposed to BoNT/A, conductance through the toxin-induced channel became larger over
time. The conductance of toxin-induced channels was essentially
independent of membrane potential, as shown in the
current-voltage plot in Figure 1D. In addition, channel activity
and time-averaged current through the membrane patch
increased with time (Figure 2B). In part, this represented an
obvious increase in the number of individual active
BoNT-induced channels in the membrane.
TSN interfered with the formation of the BoNT/A
channels To test whether TSN affects the formation of the
toxin-induced channel, two groups of experiments were carried
out. In the first group of experiments, PC12 cells were
preincubated with TSN for more than 30 min prior to the
formation of the gigohm patch seal. In control experiments,
no ion channel opening was observed without BoNT/A in
the recording pipette (n=6). After BoNT/A was added into
the pipette solution, spontaneous ion channel openings due
to BoNT/A were observed in 16 of 36 patches examined after a delay of 8.0±2.5 min (Figure 1B). When these values are
compared with those obtained without TSN, the probability
of toxin-induced channel appearance was significantly
decreased (P<0.05) and the delay before channel appearance
was prolonged in the presence of TSN. In addition, the
conductance of the toxin-induced channels observed under
these conditions (Figure 1B, 2A) was smaller than that
investigated without TSN. Under these conditions, 20 min
after the channels appeared, the conductance was 56±9 pS
(n=7), whereas without TSN the conductance was 120±11 pS
(n=10). As seen in Figure 2B, after preincubation with TSN,
the activities (NPo) of the channels that appeared were also
reduced. Moreover, even in the membrane patches in which
toxin-induced channels could form, not more than one
active channel was observed in each patch examined. Under
these conditions, the shape of the current-voltage curve was
not changed (Figure 1D).
In the second group of experiments, TSN was added in
the pipette solution in company with BoNT/A.
In the control experiments with TSN only in the patch pipette, no ion
channel opening was observed in 6 cells over 20
min. When BoNT/A was added accompanied by TSN, ion channel
openings due to BoNT/A were observed in 14 of 31 patches
examined after a delay of 5.5±1.2 min (Figure
1C). The channel formation rate was also substantially
reduced. The conductance and activity of the toxin-induced channels under these
conditions (Figure 2A, B) were similar to those observed in
the first group of experiments, which were both smaller than
those without TSN. These results indicate that TSN
interfered with the pore-forming activity of BoNT/A.
TSN affected the BoNT/A-induced channels To
investigate whether TSN blocks the toxin-induced channels, TSN
was applied to the PC12 cells shortly after the ion channels
appeared. As shown in Table 1, 10 min after perfusing the
inside-out patch with the TSN-containing solution, neither
the open probability (Po) nor the mean open time
(mo) of the channels was affected
(n=5). The conductance through the channels was significantly increased
(P<0.05). However, the increased value was smaller than that obtained without TSN
(Table 1). The results indicate that TSN delays the increase
in the conductance of the BoNT/A-induced channels.
Discussion
Unlike many ligand or voltage-gated ion channels, the
channels formed by BoNT are not easily identified by the
action of well-identified inhibitors or
agonists. BoNT-induced ion channels are usually identified by using the
correlation between the presence or absence of the active toxin
and the presence or absence of channel activity.
In the
present study, in the absence of BoNT/A, no channel
activity was observed in 8 initially silent membrane patches
examined. However, when the active toxin was present, ion
channels formed in 27 of 31 patches with a delay similar to
that found in a previous study[10].
The probability that the channels are not due to BoNT/A is very
low. The properties of the channels we observed were similar to those
previously reported[10]. The pH conditions in the present study,
low pH on the side of toxin application and high pH on the
membrane surface away from the toxin, were selected to
optimize the formation of ion channels. These have the same
orientation and magnitude of the pH gradients in
endocytotic vesicles, which are thought to play a role in the
translocation of toxin from an extracellular compartment to an
intracellular site of action[22,23].
It is known that pore formation is involved in BoNT
translocation[11]. In the present study we observed that TSN
interfered with the formation of BoNT/A-induced ion
channels. After the cells were preincubated with TSN, ion
channels were observed in only 16 of 36 patches
examined. When TSN was added into the pipette solution, ion
channels were observed in only 14 of 31 patches
examined. Under both of these conditions, the probability that a
toxin-induced channel would appear was reduced by approximately
50%. Moreover, the conductance and activity of the
channels that appeared were reduced by more than
50%. The reduction in conductance in part represented a decrease in
the size of the toxin-induced pore. As we know, the size of
the HC-formed pore is an essential factor for BoNT
translocation. It seems likely that the LC is unable to pass
through the reduced pore formed in the TSN-treated cells
because of its large dimensions. Therefore, the LC of the
toxin can not approach its substrate protein in the
TSN-treated cells, a fact that has already been demonstrated in
our previous study[18].
During the binding, internalization and translocation of
the toxin, interaction and recognition between toxin and
biomembrane must be involved. In a previous study, we
demonstrated that TSN inhibited delayed rectifier
K+ channels, irrespective of intracellular application or
extracellular addition[20]. TSN can modulate the channels on both
sides of the membrane, meaning that it is membrane
permeant. Moreover, TSN can modulate various kinds of
K+ chan-nels[24-26] and L-type Ca2+
channels [27], whose common denominator appears to be that they are imbedded into, and
span the membrane¡¯s bilayer. Therefore we hypothesized
that TSN might derange the membrane environment after
entering the membrane bilayer, and hence prevent BoNT from
interacting with the membrane. Similarly, the change in the bilayer microenvironment caused by TSN affects ion
channel activity, and results in inhibition of
K+ currents and an increase of
Ca2+ influx.
Together, the data obtained in the present study indicate
that the antibotulismic effect of TSN might be achieved by
interfering with translocation of the toxin.
Acknowledgement
The authors acknowledge the help provided by
Wen-ping WANG in carrying out the experiments.
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