Elsevier Editorial System(tm) for Toxicon
Manuscript Number: TOXCON-D-06-00201R1
Title: A novel depolarising activity of scorpion venom fraction M1 due to activation of skeletal muscle
Article Type: Short Communication
Keywords: Scorpion venom; Skeletal muscle; Nicotinic acetylcholine receptor agonists; Excitation-
Contraction Coupling; Calcium transient; Confocal microscopy
Corresponding Author: Dr. Christian COGNARD,
Corresponding Author's Institution: CNRS/Université de Poitiers
First Author: Amani CHEIKH, Student
Order of Authors: Amani CHEIKH, Student; Rym BENKHALIFA, PhD; Daniel POTREAU, PhD; Guy
RAYMOND, PhD; Mohamed EL AYEB, Professor; Christian COGNARD
Manuscript Region of Origin:
Université de Poitiers - UFR Sciences Fondamentales et Appliquées
Dr. Christian COGNARD
Directeur de Recherche au CNRS
UMR Université de Poitiers/CNRS n°6187
IPBC Institut de Physiologie et de Biologie Cellulaires
Pôle Biologie Santé
40 avenue du Recteur Pineau
F-86022 Poitiers Cedex
Physiologie et de
To Dr A. Harvey
Editor of Toxicon
Poitiers on September 7th, 2006
Dear Dr Alan Harvey,
Find a revised version of the paper entitled "A novel depolarising activity of scorpion venom fraction
M1 due to activation of skeletal muscle nicotinic receptors" by Amani Cheikh, Rym Benkhalifa, Daniel
Potreau, Guy Raymond, Mohamed El Ayeb & Christian Cognard, to be submitted for publication in Toxicon.
This revised version corresponds to the manuscript MS 06-224 formerly MS 06-201. As proposed by the
reviewer #9 we reformat it to make it compatible with the size and recommendations of a short
communication: in particular 5 double-spaced pages main text, no more than 2 figures and a shorter
abstract (75 words).
To reach this goal the main changes are the following:
- figure 1 was kept unchanged
- figure 5 was kept (becoming figure 2) and the panel C of the former figure 3 was added as panel A of the
new figure 2
- former figures 2 and 3 (effects of blockers of targets other than nAchRs) and figure 4 (effects of M1 on
membrane potential) were deleted
More space was saved at the level of the text content itself, particularly by following the
recommendations of reviewer #9:
- the myotubes culture method was deleted and a reference to a previous paper describing it was added
- the electrophysiological technical details were deleted, as the data obtained with the patch-clamp
technique were only reported but not illustrated, and the technique is became classical.
- the effects of blockers of the ECC steps (other than nAchRs) was reported only through short sentences
and not illustrated
- some old citations were deleted (specific point 1 of reviewer #9) particularly those related to classical
membrane depolarising effects due to nAchRs operation as well as those related to the well-known ECC
steps in skeletal muscle.
Find below our comments about some specific points raised by reviewer #9:
- specific point 2: generally speaking: yes. Control experiments have been performed and they showed the
repeated action of M1 even with long duration intervals provided that technical parameters remained stable
for 1 hour or more.
Email: firstname.lastname@example.org / Tél. 33 5 49454064 / Fax 33 5 49454014
- specific point 3: α-bungarotoxin was used as it is the classical blocker of the nicotinic type of AchRs in
skeletal muscle tissue. A few control experiments were performed with d-tubocurarin with quite similar
results but no extensive investigation was performed with other antagonists of nAchRs.
- specific point 4: high concentrations of TTX were used mainly because some type of sodium channels
exist which are highly resistant (high KD for TTX) to TTX. In case of α-bungarotoxin, such high
concentrations has been used in a previous work on the same preparation (Cognard et al 1993, Cell calcium
14: 333-348) and we kept the concentration unchanged. Nevertheless we agree that nM concentrations
ranges can working well.
- specific point 5: sorry, we are not certain to well understand the question. In our opinion even in
myotubes where nAchRs are not concentrated in a neuromuscular junction but scattered in patches, action
potentials are necessary for spreading the membrane depolarisation into the vicinity of transverse tubules
(TTs). Consequently TTX always blocks the effect of Ach or M1. The situation could be different in myoblasts
or in early developed myotubes (see our work of 1993 cited above) where TTs development is reduced.
- specific point 6: We agree. Such lack of additivity was clearly explained in the text, we hope, by the all-
or-none behaviour of the APs generation.
- specific point 7: We agree. Same explanation than for point 6. Experiments on the same cell has shown
that the obtained responses with 1 and then 10 µg/mL had essentially a 'all-or-none' nature.
Dr Alan Harvey, we thank you again for reconsidering our paper and hope that the new shorter version will
be in good order for publication in Toxicon.
Very truly yours,
Dr Christian COGNARD
Email: email@example.com / Tél. 33 5 49454064 / Fax 33 5 49454014
A novel depolarising activity of scorpion venom fraction M1 due to activation of
skeletal muscle nicotinic receptors
?Amani Cheikh, 1,?
?, Rym Benkhalifa, 2Daniel Potreau, 2Guy Raymond, 1Mohamed El Ayeb
& 2, §Christian Cognard
1 Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, BP 74-1002, Tunis, Tunisia and 2 Institut de Physiologie et
de Biologie Cellulaires, UMR 6187, 40 avenue du Recteur Pineau, 86022 Poitiers Cedex, France
? authors who have contributed equally to this work.
§Corresponding author: Christian COGNARD,
Phone: 33 5 49454064
Fax: 33 5 49454014
A depolarising activity following interaction with nicotinic receptors (nAchRs) in skeletal muscle
cells, was observed for the first time in the non toxic venom fraction (M1) of the yellow scorpion
Buthus occitanus tunetanus (Bot). The effects of M1 fraction were tested on cultured rat myotubes by
recording changes in [Ca2+]i. When applied, M1 (10 µg/mL) induced a transient increase of [Ca2+]i
which could be blocked by a prior application of α-Bungarotoxin.
Scorpion venom, Skeletal muscle, Nicotinic acetylcholine receptor agonists, Excitation-Contraction
Coupling, Calcium transient, Confocal microscopy.
Many studies have shown that crude venoms or isolated toxins can affect in different ways the
structure and function of muscle. Some of them contain active post-synaptic neurotoxins, which were
isolated from the venoms of elapid and hydrophid snakes (Endo and Tamiya, 1987 for review). These
toxins bind with high affinity to certain nicotinic acetylcholine receptors in skeletal muscle and
nervous tissue, where they competitively antagonize the acetylcholine action (Stroud et al., 1990 for
review). In our study we have shown for the first time, a calcium concentration increase effect of a
Bot (Buthus occitanus tunetanus) scorpion venom non-toxic fraction (Drira-Chaabane et al., 1996),
called M1, which corresponds to the first one obtained by sephadex gel filtration and we found that
this effect could be prevented by using α-Bungarotoxin (α-BgTx) which competitively blocks the
nAchRs in a relatively irreversible manner (Berg et al., 1972; Young et al., 2003).
Adult Buthus occitanus tunetanus scorpions were collected in Beni Khedache (South of
Tunisia) and kept in veterinary laboratory. The venom was obtained by inducing the animals to sting
a parafilm membrane on a small Petri dish. The venom was immediately freeze-dried and stored at –
80°C. The collected venom was filtrated using a sephadex G-50 column, 26 x 100 cm, 30 mL/hr/cm2
(Sigma) equilibrated and eluted at 15°C with 10 mM ammonium acetate buffer, pH 4.7 (Miranda et
al., 1970). The non-toxic fraction, M1 (Drira-Chaabane et al., 1996), was freeze-dried and stored at -
20°C. All animal handling and procedures strictly conformed to The Guide for The Care and Use of
Laboratory Animals (National Institutes of Health, publication number 85-23, 1996). Primary
cultures of rat skeletal muscles were prepared according to the procedure described elsewhere
(Cognard et al., 1993). Calcium homeostasis in rat myotubes was studied using an inverted
microscope (Olympus IX 70, Japan). The culture dish (with glass bottom) was used as the
experimental chamber. Myotubes were imaged through a 60X water-immersion 1.2 NA objective
(Olympus) with a confocal laser-scanning unit (Bio-rad MRC 1024) equipped with a 15 mW Ar/Kr
gas laser. The myotubes were incubated for 20 min in the control solution (in mM: 130 NaCl, 5.4
KCl, 2.5 CaCl2, 0.8 MgCl2, 10 HEPES, 5.6 glucose; 300 mOsm, pH 7.4) containing Fluo4-AM (3
µM) (Sigma) which was then de-esterified by the esterases in the cytoplasm, and then, rinsed with the
same control solution. Fluorescence signal collection was performed through the control software
Lasersharp 3.2 (Bio-rad). Fluorescent images (256*256) were acquired every 300 ms by means of
Ar/Kr laser tuned at 488 nm (excitation) through a dichroic mirror and a band pass filter centred at
522 nm (emission). Myotubes were exposed to control or drugs containing media by means of a
home-made micro-superfusion system driven by gravity. Fluorescence images were recorded and
Region of interest (Roi) were selected inside (Roi1 or/and Roi2) and/or outside (Bg) myotubes (Fig.
1A and C for examples) in which the pixels fluorescence mean value (in arbitrary unit) was
computed and plotted against the time as traces (Fig.1B, D and E).
Application of 10 µg/mL M1 fraction by superfusion in the culture dish induced a rapid
increase in the fluo-4 fluorescence as viewed in Fig. 1A. A second M1 application was able to induce
a similar transient fluorescence increase as evidenced by the trace of Fig. 1B corresponding to Roi1.
Depending on the duration of the M1 superfusion (compare Fig1 D and E), the shape of the
fluorescence transient could vary, and it can be noticed that the response amplitude was not held all
along the drug application (see Fig. 1D). Such changes in fluorescence intensity (FI) corresponded to
changes in free calcium concentration in response to M1 applications and varied from a basal value
of 31 ± 10 a.u. to a peak value of 132 ± 14 a.u. (n = 8) during a superfusion of 10µg/mL M1 and
from 36 ± 4 to 133 ± 19 a.u. (n=7) during a 30 µg/mL M1 superfusion. The M1 effect did not follows
a clear dose/response relation since the 30 µM concentration used did not lead to a significant greater
responses. In addition, a high concentration of M1 could induce sometimes myotube damages (see in
Fig. 1C, the 120 s image) with detachments from the dish bottom or even sometimes with the
impossibility to get two consecutive responses (Fig. 1E). This, and the fact that not all of the
myotubes responded (8/23 at 10 µg/mL and 7/18 at 30 µg/mL), led us to test the responsiveness to
M1 of each myotube prior to any inhibitor application. In addition the 10 µg/mL concentration was
used throughout the remaining part of this study, a dose at which the effect was reversible,
reproducible and safe for myotubes.
The next set of experiments aimed to identify mechanism/protein(s) triggering the increase of
[Ca2+]i during M1 application, that is, the target of the M1 fraction. For this purpose several inhibitors
have been separately used to address the different steps of the excitation-contraction coupling (ECC)
mechanism. In skeletal muscle in vivo, ECC is a cascade of events in which calcium elevation results
from the sequential activation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular
junction, tetrodotoxin-sensitive voltage-operated Na+ channels at the sarcolemmal membrane,
dihydropyridine receptors (DHPRs) in the triadic part of T-tubules and, finally, ryanodine receptors
(RyRs) at the sarcoplasmic reticulum (SR) membrane (see for review Melzer et al., 1995). The
termination of such a sequence is a massive calcium release from SR leading to a transient elevation
of calcium concentration in the myoplasm and to contraction. The following inhibitors were
respectively used: α-bungarotoxin, tetrodotoxin, nifedipine, ryanodine. All these inhibitors appeared
to be able to prevent M1-induced calcium transients (data not shown) but as the nicotinic cholinergic
receptor is the first element in the ECC sequence, M1 effect is thought to arise through an action on
nAchRs which depolarize myotube membrane, trigger Action Potentials (APs), activate DHPRs and,
through a change in conformation of RyRs, release calcium ions from SR. In vivo, activation of
AchRs is operated by the neurotransmitter Acetylcholine (Ach). This activation can be antagonized
by molecules such as α-BgTx, a long snake neurotoxin from the Southeast Asian banded krait,
Bungarus multicinctus. In the example of Fig. 2A, after a first test application of M1, myotubes
incubation for about 3 min in presence of 10 µM α-BgTx completely prevented calcium transient
induction by a second M1 application. The proposed hypothesis of an action of M1 on nAchRs was
then addressed through additional tests. It is well established that activation of nAchRs by Ach
results in membrane depolarisation due to the highly permeable open receptor-channel for sodium
and potassium ions. To test the direct activation of nAchRs by M1 fraction, its effects were briefly
investigated and compared (data not shown) to the one of Ach on the membrane potential of
myotubes, measured with the current clamp mode of the whole cell patch clamp technique. In these
conditions M1 (10 µg/mL) application induced a membrane depolarisation to around 0 mV (-1.5 ±
1.0 mV; n = 4). This effect was quite similar to the one observed during application of 10 µM Ach. In
addition, this effect is completely and irreversibly abolished after a previous application of 10 µM α-
BgTx. This control result shows that M1 could interact with nAchRs in a way very similar to the
Ach one. Fig. 2B shows that α-BgTx (10 µM) inhibited the M1 stimulatory effect as well as the one
of Ach (10µM). The inhibiting action of α-BgTx was irreversible as long as its superfusion time was
sufficient (around 2 min in the present conditions) as evidenced in the experiment of Fig. 2C. Finally,
Ach and M1 were applied together. This type of investigation, illustrated in Fig. 2D, provided us with
two data. First the amplitude of response induced by Ach and the one induced by M1 are very close:
the fluorescence intensity was increased to 127 ± 20 a.u. (n = 3) (from a resting value of 24 ± 2 a.u.)
with Ach (10µM), and to 115 ± 23 a.u. (n = 5) (from a resting value of 28 ± 2) with M1 (10µg/mL).
The difference was not significant (p = 0.52). Second, the simultaneous application of Ach and M1
did not show an additive effect and we can properly hypothesize that both drugs interact with the
same binding site on nAchR.
Although a clear conclusion can be drawn from these data, that is the presence in M1 of a
compound which mimics acetylcholine action, some aspects are worth thinking about.
The first question is that these effects could be simply explained by the presence of
acetylcholine in the M1 fraction. But, as reported by our group (see Cheikh et al., 2006), the M1
fraction preparation method ruled out this eventuality. The second question raised by the present
experiments is related to the fact that only a part of the myotubes responded to M1. This could
probably be explained by the particular localization of nAChRs in cultured aneural skeletal cells. On
aneurally cultured rat myotubes, approximately only 10% of the AchRs are aggregated in clusters
while the reminder is uniformly distributed over the entire membrane (see Axelrod et al., 1976).
Therefore some differences in the distribution of AchRs, as well as in the development, from one
myotube to another, could be sufficient to preclude the cell membrane potential to reach AP
generation threshold that, in turn, led to the subsequent blocking of ECC sequence. Another
observation was the adverse effect sometimes observed during high concentrations (30 µg/mL)
application of M1 with myotubes going off the dish bottom. The explanation of this effect could
reside in the nature of M1. M1 is a fraction of a scorpion venom extract and it likely contains a bunch
of other compounds in addition to the main active molecule. Some of them could have deleterious
effects on the membrane and/or cell integrity.
Showing the presence of a depolarising activity in scorpion venom acting via interaction with
nAchRs could be of great interest as, in the perspective of nAchR-based therapeutics improvement,
many efforts are directed toward the identification and characterization of novel, potent nAchR
ligands. Selective neuronal nAchR agonists may provide therapeutic utility in the treatment of some
neurological diseases (Holladay et al., 1997; Lin et al., 1998) such as Alzheimer (Narahashi et al.,
2003). Until now, it has been reported a positive effect of some venoms on intracellular calcium
levels, such as, trachynilysin from fish venom (Colasante et al., 1996; Meunier et al., 2000; Sauviat
et al., 2000) or epibatidine from frog skin (Spande et al., 1992; Bonhaus et al., 1995; Prince and Sine,
1998). As far as we know, snake venoms contain depolarising proteins of nerve and muscle cells (see
Harvey et al., 1983; Dufton and Hider, 1988; Chen and Harvey, 1993) and several nAchR antagonists
had been identified in snake venom (Fulpius et al., 1972; Maelicke et al., 1977), but yet now, no
scorpion venom molecule appeared to be active on nAchR in skeletal muscle.
As above-mentioned, M1 is an extract and identification and peptides purification from M1
will be now the key step in the further elucidation of M1 properties. This will be of great interest as
nAChR agonists are considered as potential analgesics and therapeutics for the treatment of various
neurological and mental disorders related to a decrease in cholinergic function (Holladay et al., 1997;
Tonder and Olesen, 2001).
This work was supported in part by grants from the Comité Mixte Tuniso-Français de
Coopération (CMCU), the Association Française contre les Myopathies (AFM), the Centre National
de la Recherche Scientifique (CNRS) and the Université de Poitiers. We thank Professor Koussay
DELLAGI, head of the Institut Pasteur de Tunis, for constant support. We also thank Dr Bruno
CONSTANTIN for his helpful discussions, Anne CANTEREAU, Nejla SOUDANI, Françoise
MAZIN and Chantal JOUGLA for their technical help.
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Legends of figures:
Figure 1. The non toxic fraction (M1) of Buthus occitanus tunetanus venom induces [Ca2+]i
transient increase in cultured rat myotubes
In A, calcium transients were visualized by laser scanning confocal microscopy in myotubes, loaded
with the calcium probe fluo-4, during two successive 10 µg/mL M1 applications. The M1 effect
appeared to be reproducible. Acquisition frequency for images was 1 every 300 ms. Time mentioned
at bottom of the images corresponds to the beginning time (after the start of the whole experiment) of
each image acquisition presented in the panel. On the left, an inset shows the colours scale used to
code the fluorescence amplitude with the high ones upwards. In B, traces correspond to the mean of
fluorescence, in arbitrary units (a.u.), calculated from pixels belonging to the regions of interest
(Roi1, Roi2 and Bg) selected in myotubes during M1 (10 µg/mL) applications. Bg: a Roi not
localised in myotubes, was chosen as background reference. The superfusion periods are indicated as
black lines above the traces. Not all the myotubes were able to respond to M1 (see Roi2). In C, a
similar experiment to the one in A but with one application of M1 at 30 µg/mL. Myotube damage
could be noted at this concentration (see last image). In D and E, two other examples of responses
induced by superfusion of 30 µg/mL of M1. Note the difference of duration in function of the
superfusion time. In E, at this concentration, a second M1 application did not induce another
From such recorded data statistical analysis was performed using Prism software (v3 or v4;
GraphPad Software, San Diego, CA, USA) and the numerical data were expressed as mean ± SEM.
Differences were tested using an unpaired two-tailed t-test, assuming that the population follows a
Gaussian distribution. Differences were assumed to be significant when p < 0.05.
Figure 2. M1 fraction acts in a Ach similar way and its effect is blocked by α α α α-Bungarotoxin
In A, after a first application of 10µg/mL M1 inducing a transient calcium increase, superfusion of α-
Bungarotoxin prevent the effect of a second application of M1. In B, After control stimulations by
Ach (10 µM) or M1 (10 µg/mL) applications, Ach, as well as M1, had no effect during nicotinic
cholinergic receptors blockade by α-Bungarotoxin (10 µM). In C, a representative trace obtained
during an experiment similar to the one in B but M1 was applied before Ach, and the effect of the
two drugs was also tested after washout. This experiment confirmed the irreversible blocking effect
of α-BgTx on Ach and M1 effect as well. In D, Changes in [Ca2+]i were recorded in response to
successive applications of ACh, M1 and both (M1 + ACh) in the aim to know whether their effect
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