Inhibition of ACh release at an Aplysia synapse by neurotoxic phospholipases A2: specific ...

12 Pages·2005·2.11 MB·English

4124 Journal ofPhysiology (1995),489.1, pp.29-40 29
Inhibition of ACh release at an Aplysia synapse by
neurotoxic phospholipases A2: specific receptors
and mechanisms of action
Philippe Fossier, Gerard Lambeau*, Michel Lazdunski* and Gerard Bauxt
Laboratoire de Neurobiologie Cellulaire et Mole'culaire, CNRS, F-91198 Gifsur-Yvette
cedex and *Institut de Pharmacologie Moleculaire et Cellulaire, UPR 411 CNRS,
660 route des Lucioles, Sophia Antipolis, F-06560 Valbonne, France
1. Monochain (OS2) and multichain (taipoxin) neurotoxic phospholipases A2 (PLA2), purified
from taipan snake venom, both inhibited ACh release at a concentration of 20 nm (90%
inhibitionin 2 h)atanidentifiedsynapsefrombuccalganglionofAplysia californica.
2. The Na+ current was unchanged upon application of either OS2 or taipoxin. Conversely,
presynaptic K+currents (IAand IK)wereincreasedbytaipoxinbutnotbyO82. Inaddition,
OS2 induced a significant decrease of the presynaptic Ca2+ current (30%) while taipoxin
increased thislattercurrentby 20-30%.
3. Bee venom PLA2, another monochain neurotoxic PLA2, also inhibited ACh release while
non-toxic enzymatically active PLA2s like OS1 (also purified from taipan snake venom) or
porcine pancreatic PLA2 elicited a much weaker inhibition of ACh release, suggesting a
specific actionofneurotoxic PLA2s versus non-toxicPLA2s onAChrelease.
4. Using iodinated OS2, specific high affinity binding sites with molecular masses of 140 and
18 kDa have been identified on Aplysia ganglia. The maximal binding capacities were 55
and 300-400 fmol (mg protein)-' for membrane preparations from whole and buccal
ganglia, respectively. These binding sites are of high affinity for neurotoxic PLA2s (Kd
values, 100-800 pM) and of very low affinity for non-toxic PLA2s (Kd values in the
micromolar range), thus indicating that these binding sites are presumably involved in the
blockade ofAChreleasebyneurotoxic PLA2s.
Snake venoms contain various phospholipases (PLA2s) (for venom (Fohlman, Eaker, Karlsson & Thesleff, 1976).
recent reviews see Kini & Evans, 1989; Harris, 1991; Taipoxin from Oxyuranus scutellatus scutellatus snake
Hawgood & Bon, 1991). The ancestral function of these venom is one of the most toxic PLA2s and is composed of
venom PLA2 enzymes is probably digestive. However, a three PLA2 subunits (Fohlman et al. 1976). The third class
small group ofsnake venom PLA2s has evolved into potent of PLA2 neurotoxins includes /3-bungarotoxins. This type
neurotoxins that block neuromuscular transmission by of toxin is composed of a subunit with PLA2 activity
modifying transmitter release. Three classes of PLA2 covalently linked by a single disulphide bridge with
neurotoxins have been distinguished on the basis of their another small subunit homologous with dendrotoxins,
quaternary structure (Hawgood & Bon, 1991). The first which are inhibitors of certain classes of K+ channel
class comprises single chain polypeptides of 13-15 kDa. It (reviewedinRehm, 1991; Hawgood&Bon, 1991).
includes notexin, agkistrodotoxin, ammodytoxin,
Electrophysiological studies carried out at mouse nerve
pseudexin and the bee venom PLA2 as well as 082, a very
terminals have previously suggested that the three
potent PLA2 neurotoxin purified from the taipan snake
different classes of PLA2 neurotoxins act by blocking
venom (Lambeau, Barhanin, Schweitz, Qar & Lazdunski,
presynaptic K+channels(Dreyer&Penner, 1987; Rowan &
1989). The second class includes multichain neurotoxic
Harvey, 1988). /J-Bungarotoxin blocks a subtype of
PLA2s which are made of several non-covalently linked
voltage-gated K+ channels by binding directly onto the
polypeptide subunits with at least one with PLA2 activity.
channel via its small subunit that is homologous with
Members of this class are crotoxin, textilotoxin and the
dendrotoxins (Rehm & Betz, 1982; Othman, Spokes &
very neurotoxic taipoxin also purified from the taipan
tTowhomcorrespondenceshouldbeaddressed.
30 P Fossier, C. Lambeau, Il. Lazdunski and G. Baux J. Physiol.489.1
Dolly, 1982; Petersen, Penner, Pierau &Dryer, 1986; Rehm OS1, OS2 and taipoxin from Oxyuranus scutellatus scutellatus
& Lazdunski, 1988; Rehm, 1991; Guillemnare et al. 1992). venom and the bee venom PLA2 wrere purified as described
Other classes of toxic PLA2s, including taipoxin, crotoxin, previously (Lambeau et al. 1989). Porcine pancreatic PLA2 and
notexin and OS2, do not inhibit the binding of suberic acid bis-N-hydroxysuccinimide ester (DSS) were from
BoehringerMannheim andSigma, respectively.
fl-bungarotoxin toitsspecific K+ channel-binding site, thus
suggesting that they may act by binding to different Taipoxin and OS2 were applied at concentrations ranging between
targets. The existence of specific toxin-binding sites for 0 1 and 200 nim. Most experiments, however, were performed at
20 nAi, as this concentration of drug gave clear and consistent
other monochain and multichain PLA2 neurotoxins
results.
including taipoxin and O82 have been identified in ratbrain
(Lambeau et al. 1989) and skeletal muscle (Lambeau, Study ofAChrelease
Schmnid-Alliana, Lazdunski & Barhanin, 1990) using The postsynaptic neurone was voltage clamped at -80 mV with a
iodinated 082 as a ligand. /-Bungarotoxin exhibits a veryT double-electrode voltage-clamp apparatus in such a way that the
low affinity for these binding sites, indicating that the postsynaptic responses due to the opening of ACh-gated Cl-
channels were recorded as currents. Low-resistance KCl-filled
targets of /3-bungarotoxin are different from those of
microelectrodes were used to obtain an optimal voltage clamping
monochain and multichain PLA2s (Lamnbeau et al. 1989,
of these large postsynaptic neurones (200,um in diameter). This
1990). Moreover, the binding sites identified in brain were
poses the problem of loading the postsynaptic neurone with Cl-
found to be involved in the neurotoxicity of various ions leaking from the electrodes and inducing a shift in the Cl-
monochain and multichain venom PLA2s (Lambeau et at. equilibrium potential that would increase the size of the
1989). postsynaptic responses. To account for this problem, the reversal
potential of the response was regularly reassessed during the
In order to better understand the effects ofmonochain and
course of the experiments by measuring the membrane potential
multichain PLA2 neurotoxins on synaptic efficacy, xve have at which the postsynaptic response had zero amplitude. The
applied OS2 and taipoxin, which are monochain and postsynaptic current was then expressed as an apparent
multichain neurotoxic PLA2s, respectively, purified from conductance by dividing the recorded value of current by the
the taipan snake venom, on a well-identified cholinergic driving force, V- Veq, wNhere Vis the holding potential of the
neuroneuronal synapse ofAplysia californica, at which it is postsynaptic neurone (-80 mV) and Veq the reversal potential for
Cl- ions. ACh release was evoked either by a presynaptic action
possible not only to quantify ACh release (Baux, Fossier &
potential orbyadirectdepolarization ofthe presynapticterminal.
Tauc, 1990) but also to record the presynaptic K+ and Ca2+
This latter method was used in order to study transmitter release
currents (Baux, Fossier, Trudeau & Tauc, 1993; Fossier,
independently of spike conduction. For this purpose, in the
Baux & Tauc, 1994) and to discriminate the pre- and presence ofTTX toblockspike generation in all afferent neurones
postsynaptic effects of a drug. Another important to the studied postsynaptic neurone, we cdelivered a 3s square
advantage ofthis synaptic preparation is the possibility of voltage command (from -50 to +10 mV) to the test presynaptic
carrying out experiments lasting several hours, allowing neurone voltage clamped at -50 mV. Thisprecise control freesthe
the testingofthe long term effectsofadrug. The datathus presynaptic membrane potential from any presynaptic ionic
obtained indicate that both taipoxin and O82 decrease ACh current involved in the polarization of the presynaptic neurone
and thuspermitsacontrolled longdurationcurrentresponseinthe
release but in different ways. Moreover, binding studies
voltage-clamped postsynaptic neurone tobe induced. Fluctuations
with iodinated O82 show the presence of high affinity
appeared at the top of this long duration-induced postsynaptic
binding sites in Aplysia ganglions, which are probably current (LDIPSC), which can be analysed statistically to calculate
related totheaboveeffectsonsynaptic transmission. the mean amplitude and decay time of evoked miniature
postsynaptic currents wNhich sum to build up the LDIPSC. A
detailed explanation ofthis method hasalready appeared (Baux et
METHODS
al. 1990). The number ofevoked miniature postsynaptic currents
Biological preparation andsolutions comnposing theLDIPSCcorrespondsexactly tothe numberofACh
Experiments wvere performed at a constant temperature of 22°C quanta released by the presynaptic depolarization. By using this
on neurones ofthe buccal ganglion ofAplysia californiica (Marinus method we can obtain a precise quantification of transmitter
Inc., Long Beach, CA, USA). After removal of the connectixe release.
tissue sheath, the cell bodies of the pre- (B4 or B.) and MeasurementofCa2+andK+currents
postsynaptic (B3 or B6) neurones, which possess well-identified
To record the inward Ca current in the presynapticneurone, the
cholinergic synapses (Baux et al. 1990), were penetrated by two inward Na+ current was blocked by bath application of TTX
microelectiodes filled wvith 3 -m KCM. Following this procedure, thle (10-4 I). The late outward K+ curIent (IK) was blocked by
resting potential ranged between -50 and -60 mX. Experiments
extracellular application of 50 mm tetraethylammonium (TEA)
were carried out in a 1 ml experimental chamber filled with bromide. The early outward K+ current (IA) was eliminated by
artificial sea wvater (ASWA) comprising (mi): NaCl, 460; KCl, 10; bathapplication of4mM 4-aminopyridine (4-AP). Toincrease the
CaCl2, 11; A1gCl2, 25; MgSO4, 28; Tris-HCl buffer, 10; pH 7 8. In Ca2P gradient, the external Ca2P concentration wA-as raised from 11
some experiments in which it was necessary to block Na+ currents to55mM. Ca2Pcurrentswereelicitedbydelivering25 msduration
inordertoprevent action potentialgeneration, tetrodotoxin (TTX) steps from -50 mV to test potentials ranging from -40
wasadded toafinal concentration of 100 # to +50 mV. All currents were corrected for leakage. The early (IA)
J. Physiol.489.1 Effects of neurotoxic PLA2s at an Aplysia synapse 31
and delayed rectifier (IK) K+ currents were measured through the Bindingstudies
same range ofpresynaptic depolarizations as for the Ca2+ current, 15I-S2derivative wasobtainedasdescribed previously (Lamheau
butlengthened to80 ms. Theneuroneswere bathed by the normal et al. 1989). The specific radioactivity obtained was routinely
saline medium (ASWA). To isolate IK' the presynaptic neurone was 2500-3500c.p.m.(fmol toxin)-'. All binding experiments were
voltage clamped at -30 mV, a potential at which IA inactivates performed at 20°C in 1 ml of a buffer consisting of 20 mm
completely in ouIr prepaIation. Then, considering that at -30 mV Tris-HCl (pH 7 4), 140mM NaCl, 1 mM CaCl2 and 0-1% bovine
'K was poorly inactivated, it was possible to measure a pure IA serum albumin. Membranes were incubated with the radiolabelled
current by subtracting the current trace recorded in the neurone ligand in the presence or absence of unlabelled competitor for
voltage clamped at -30 mV from that recorded in the neurone 90 min before filtration (Lambeau et al. 1989). Dilution of
voltage clampedat-50 mVforthesame levelofdepolarization. unlabelledtoxinswasdoneinincubationbuffer.
Membranepreparations Cross-linkingexperiments
Dissected ganglia fiom Aplysia californica were rinsed and Membranes (200jtg ml-l) were incubated in 1 ml of a solution
resuspended in ten volumes of an ice-cold homogenization buffer comprising 20 mM Hepes-NNaGH (pH 7 4), 140 mm. NaCl, 0O1 mm
consisting of 40 mim Tris-HCl (pH 7 4), 0-32 M sucrose, 2 mAi CaCl2 and 0-1% bovine serum albumin with 300 pM 125i-os82 in
EDTA and 05 mM phenylmethylsulphonyl fluoride. The mixture the absence or presence of 100 nm unlabelled competitor. After
wasthen homogenized usingapolytron (Kinematica)and apotter 60 min at 20°C, incubation mixturesweere centrifuged at 12000 g
(Heidholf) homogenizer (20 strokes at 800 r.p.m.). The resulting for 10 min and resuspended in 1 ml of a solution comprising
homogenate was centrifuged at 1000 g for 5 min; the pellet was 20 mM Hepes-NaOH (pH 8 2), 140 mM NaCl and 1 mM CaCl2.
discarded and the supernatant was filtered through a nylon gauze Following addition of 150/AM DSS (freshly dissolved in Me2SO at
and then centrifuged at 100000 gfor 40 min. The final pellet was 15 mM)for5 minat 20°C, thereaction wasstopped byadditionof
then resuspended in the homogenization buffer and stored in 100 1l 1 MTris(pH 80)andcentrifugation at 12000 gfor 10 min.
aliquots at -70°C until used. All the preparations were carried The resulting pellets were solubilized with a SDS sample buffer
out at 4°C and the protein concentrations were determined (Laemmli, 1970) under reducing conditions (4%
according to Bradford (1976) after digestion ofthe membranes in 3-mercaptoethanol) and analysed by SDS-polyacrylamide gel
0O1 NNaOHusingbovineserumalbumin asastandard.
Taipoxin
500 70
"%LN
AT:
400 [ 0o oo8 ,R
IC
C-li w 0 0 .
I 300
0
0cn. OM
a)
--
.9
a. 200 1
co O vitb
cn 35 E
0
100
0°m
POO
0 L
-60 0 60 120 180
Time (min)
200 nS
a b C
20 mV
\ 40 ms
Figure 1. ActionoftaipoxinonAChrelease
The graph represents the evolution of the amplitude of the postsynaptic response (0) and of the
presynaptic action potential (S) before and after taipoxin application (at time 0, arrow). Bottom
recordings: the uppertracerepresentsthepostsynaptic responseevoked byapresynapticaction potential
(lower trace), before (a), 55 min (b) and 115 min (c) after bath application of taipoxin (20 nm). The
recordings a, band care indicated on the graph. After 2 h application oftaipoxin, the amplitude ofthe
postsynapticresponsewasreduced by90%and that ofthespikeby morethan 20%.
32 P Fossier, G. Lambeau, M. Lazdunski and G. Baux J Physiol.489.1
electrophoresis (4-14% gel) (Laemmli, 1970). Gels were stained from that observed with OS2 (Fig. 2). The amplitude ofthe
with Coomassie Brilliant Blue, dried and autoradiographed at spike was always decreased more by taipoxin (more than
-70°C using Amersham MP film and intensifying screen (Du 20% after 2 h) than by OS2 (between 5and 10% after 2 h).
PontCronexHi-plus).
In both cases the presynaptic neurone was maintained at
-50 mV. Differences in the modification of presynaptic
RESULTS action potentials following taipoxin and OS2 application
Effects oftaipoxin and OS2on synaptic transmission pointed to different mechanisms of action to reduce the
release of ACh. Therefore, an analysis ofthe effects ofthe
Theapplication of20 nM taipoxin (Fig. 1)orOS2(Fig. 2)on
toxinonpresynapticioniccurrentswascarriedout.
the identified cholinergic synapse ofthe buccal ganglion of
Aplysia californica led to a progressive decrease in the Effects oftaipoxin and OS2onpresynaptic K+
amplitude of the postsynaptic response evoked by a currents
presynaptic action potential. After application of the The decrease in presynaptic action potential amplitude in
toxins for 2 h, the amplitude of the postsynaptic response the presence oftaipoxin could be due either to a reduction
wasonly 10%ofthe control. The effectstarted 10-20 min of the presynaptic Na+ inward current responsible for the
after toxin application. This latency might be due to the depolarization or toan increasein presynaptic K+ currents
difficulty of the bath-applied toxins in reaching the inducing a more rapid repolarization and then a reduction
synaptic areas situated deep in the neuropile and/or to the in the amplitude of the spike. In fact the presynaptic Na+
mechanisms of action of the toxins themselves in the current was unchanged in the presence of 20 nM taipoxin
interaction with their presynaptic targets. Washout of the (notshown). Conversely, both theearly K+current, IA' and
preparation for up to 2 h did not allow the synapse to the delayed K+ current, IK, were largely increased by the
recover its control activity. Lower concentrations ofPLA2s toxin (Fig. 3A). These increases in IA and IK after
(0-2 and 2 nM) induced a lower decrease of transmitter application of taipoxin could induce a reduction of ACh
release (data not shown). The evolution ofthe presynaptic release by lowering the level and durationofdepolarization
action potential in the presence oftaipoxin (Fig. 1)differed of the nerve terminal during the action potential, thus
OS2
350 r 80
300
0
cn 250 ao0
c 0
0 0OO -°
rX 200
0)
40 E
o 150 o CO 0
000 0
CL *t o ° b
(>a- 100 0 0o a b%,
CL00
50 o0 00
120
0 L -0
-60 0 60 120 180
Time (min)
150 nS
a b c
mV
20
40 ms
Figure 2. ActionofOS2onAChrelease
The graph represents the evolution of the amplitude of the postsynaptic response (0) and of the
presynaptic action potential (0) before and after OS2 application (at time 0, arrow). Bottom recordings:
the upper trace represents the postsynaptic response evoked by a presynaptic action potential (lower
trace), before(a), 60min(b)and 130min(c)afterbathapplicationofOS2(20 nM).Therecordingsa, band
careindicated onthegraph. After2happlicationofOS2, theamplitudeofthepostsynapticresponsewas
reducedby90%andthatofthespikebylessthan 10%.
J. Physiol.489.1 Effects of neurotoxic PLA2s at an Aplysia synapse 33
secondarily reducing the presynaptic Ca2+ current. OS2, conditions, itwasobservedthatOS2(20 nM)inducedalarge
which had a small effect on the presynaptic action decrease (more than 30% after 1 h application) in the
potential, had practically no effect on the presynaptic K+ presynaptic Ca2' current (Fig. 4), which might be partly
currents when used at 20 nm (Fig. 3B). With a higher responsible for the decrease in ACh release induced by OS2.
concentration (200 nM) it was possible to detect a slight The action oftaipoxin on thepresynaptic Ca2Pcurrentwas
increase in both IA and IK. We never observed any also tested. Contrary to what was expected from the
inhibition ofanyoftheK+ currents, even atthe beginning decreasing effect of taipoxin on transmitter release,
of the experiment. As the important decrease in taipoxin (20 nM) increased the Ca2P current by 20-30%
transmitter release induced by OS2could notbetraced toa (Fig. 5). Thus, taipoxin appears to have multiple actions at
change in the characteristics of the action potential, we thepresynaptic level, theeffectsofwhichareopposite with
checked the possibility of an action of OS2 on the respecttoAChrelease.
presynapticCa2+current, thedirecttriggerofAChrelease.
Effects oftaipoxin andOS2ontransmitter release
Effects ofOS2andtaipoxin onthepresynaptic Ca+ evokedbyadirect depolarizationoftheterminalin
current theabsenceofanactionpotential
The presynaptic Ca2P current was recorded in the In the synaptic preparation studied in this paper, the
presynaptic neurone voltage clamped at -50 mV, under synapticareasareveryclosetothesoma(somehundredsof
conditions in which the other presynaptic voltage-gated micrometres) and connected to it by large processes (some
ionic currents were blocked (see Methods). Under these tens of micrometres in diameter) in such a way that it is
A B
Taipoxin OS2
2000 2000
1500 1500
S 1000 1000 _
500 500
0I-
-40 -30 -20 -10 0 10 -40 -30 -20 -10 0 10 20
6000 6000
5000 5000
4000 4000
c 3000 3000
.Y
2000 2000
1000 1000
0 I~
-20 -10 0 10 20 30 40 -30 -20 -10 0 10 20 30 40
V (mV) V(mV)
Figure3. ActionoftaipoxinandOS2(20nM)onpresynapticK+currentsIAandIK
A, the presynaptic neurone was voltage clamped at -50mV. *, control; +, after 50min application of
taipoxin. The recordings ofthe presynaptic currentare presented foradepolarization to-10mVofthe
presynaptic neurone before (a)and after (b) taipoxin application. In these recordings the increase in the
amplitudeofboththefastK+current(IA)andthelateK+current(IK)isveryclear. B, actionofOS2onIA
and IK.Thepresynapticneurone wasvoltageclamped at-50mV.*,control; +, after55minapplication
ofOS2.
34 Fossier, Lambeau, M. Lazdunski and Baux Physiol.489.1
P G. J
(nA)
I
-40 -20 20 40
V(mV)
AK
-200
-250
100 nA
-300
10 rms
Figure4. EffectsofOS2onpresynapticCa2+current
Current-voltage curves, leakage subtracted, represent the Ca2+ current recorded in the presynaptic
neurone in the presence ofTTX, TEAbromideand 4-APin ASWcontaining55mmCaCl2. Theneurone
wasvoltageclamped at -50mV. The control Ca2+ current (0)wasreduced with timebybathapplication
(A, 45min; 70min)of20nMOS2. Therecordings on the left(inset)represent theCa2+current at the
*,
peak(+20 mV)inthecontrol(0)andafter45(A)and70 min(*)ofOS2application.
possible to induce transmitter release directly by a we could calculate the number of ACh quanta released by
depolarization of the whole presynaptic neurone. For this the depolarization (see Methods). Figure 6 shows that,
purpose, the presynaptic neurone was voltage clamped at under these conditions, taipoxin increased transmitter
-50 mV in the presence of TTX toavoid presynapticspike release, a result completely opposite to that obtained when
generation in of the ganglion, and then long ACh release evoked by action potential (Fig. 1). In
neurones was an
duration depolarizations (3 s) evoked LDIPSC from which this experiment, the polarization level of the presynaptic
(nA)
I
-40 -20 20 40
V(mV)
-1'
-200
-250
-300
Figure5. EffectoftaipoxinonpresynapticCa2+current
Current-voltage curves, leakage subtracted, represent the Ca2+ current recorded in the presynaptic
neurone in thepresence ofTTX, TEAbromide and 4-APin ASWcontaining 55mmCaCl2. Theneurone
was voltage clamped at -50 mV. Thecontrol Ca2P current (0) was increased by the application of20nm
taipoxin(+,43 minapplication).
J Physiol.489.1 Effects of neurotoxic PLA2s at an Aplysia synapse 35
neurone was maintained by the voltage-clamped system at the concentration used, had practically no effect on
-50 mV and could not be modified by the changes in the outward K+ currents recorded in the voltage-clamped
presynaptic K+ currents, which nevertheless still took presynapticneurone(Fig. 7D).
place as shown by the increase in the presynaptic K+
In the presence of taipoxin and OS2, the mean amplitude
currents recorded in the voltage-clamped presynaptic
and decay time of miniature postsynaptic currents were
neurone(Fig. 6D).
not reduced with respect to the control, which indicates
In the presence of OS2 (20 nM), the amplitude of the thattheeffectsofthesePLA2sonsynaptictransmission are
LDIPSCand then thenumber ofAChquanta released were not due to an action on the postsynaptic neurone. Indeed,
decreased (Fig. 7). This result is in agreement with the the mean amplitude ofthe miniature postsynaptic current
decrease in presynaptic Ca2P current (Fig. 4) and the is a good index of the blocking effect of a drug on the
reduction in transmitter release evoked by an action postsynaptic receptor-channel complex (Baux & Tauc,
potential (Fig. 1). Thisexperiment alsoshowed thatOS2, at 1987).
Control Taipoxin
200 nS
50 nS
A is
_B
IIllj
B _, IrT171%<
~ III
i... +10 mV **.
C
L
___l -50 mV
D |
1 0-5,uAI
I I..... 100% .100%
I g T Q
Figure 6. Effect oftaipoxin (20nM) on ACh release evoked by a depolarization ofthe voltage-
clampedpresynapticneurone
A,traces represent LDIPSCs recorded in a postsynaptic cell voltage clamped at -80mV and induced by
presynaptic depolarizations to +10mV (shown in C) under control conditions (left column) and after
60 minapplicationoftaipoxin (rightcolumn). B, sameLDIPSCsrecordedthroughanACfilteratahigher
gain than in A. D, traces showing amplitude ofthe presynaptic current recorded in the voltage-clamped
presynaptic neurone. The bottom histograms represent, with respect to the control (100%), the changes
inducedbytaipoxin inthemeanamplitude (I)oftheLDIPSC(100%= 199 + 6nS: mean +S.D.), inthe
amplitude (g) of the calculated evoked miniature postsynaptic current (100%=0-87 +0-12nS), both
expressed asconductance, inthemeandecaytime(T)oftheevokedminiature(100%= 11-8ms), andin
the quantal content (Q) of the LDIPSC (100%= 58086 quanta). When the presynaptic neurone was
voltage clamped, taipoxin nolongerdecreased ACh release as itdid when ACh release wasevoked byan
action potential (see Fig. 1), but conversely induced an increase by about 30% in the number of ACh
quanta released by the presynapticdepolarization. Note the increase in the presynaptic current (mainly
K+)represented in D.
36 P Fossier, G. Lambeau, M. Lazdunski and G. Baux J Physiol.489.1
Control OS2 (20 nM) OS2 (200 nM)
A r 200 nS
_ 1 50 nS
i
B | a +1 mV l l
C +10 mV
L
___ ......-5 mv .....
D
0-5.uA
-L-- -1- L--
Figure 7. Effect of OS2 (20 and 200nM) on ACh release evoked by a depolarization of the
voltage-clampedpresynapticneurone
See legend to Fig. 6 for an explanation of A, B, Cand D. Left column, control; middle column, after
40min OS2 (20nM) application; right column, after 30min OS2 (200nM) application. The amplitude of
theLDIPSC(A)wasreducedbyincreasingtheconcentrationofOS2. Astheamplitudeandthedecaytime
ofthe miniature postsynaptic current were unmodified by OS2, the decrease in the LDIPSC is due to a
reduction in thenumberofAChquanta(Q)released bythe presynapticdepolarization. ValuesofQwere:
control, 50425; after 20nm OS2, 28891 (decreased by 43%); after 200nM OS2, 19206 (decreased by
62%). Note that the presynaptic current (mainly K+) represented in Dwas lessmodified byOS2than it
wasbytaipoxin(compareDinFig.6).
Effects ofother PLA2s onAChrelease (datanot shown) suggesting that OS2and bee venom PLA2
Application of 20 nM bee venom PLA2, another act by a common mechanism on the presynaptic terminal.
presynaptically active monochain PLA2 (Magazanik, Conversely, asubsequentapplication ofOS2after treatment
Gotgilf, Slavnova, Miroshnikov & Apsalon, 1979) resulted with OS1 or porcine pancreatic PLA2 led to an extensive
in a strong inhibition of ACh release (63 + 2%, inhibition of the ACh release (data not shown), similar to
mean + S.D., inhibition in 1 h; n= 3), very similar to that that measured when OS2 was applied alone (55 + 2 7%
obtained with taipoxin or OS2 under identical conditions (n= 3)comparedwith62 + 2-5%(n=4), respectively).
(65 + 4 and 62 + 2 5%, respectively; n=4; Fig. 8).
Detectionofsaturablebinding componentsfor
Conversely, OS1, a non-toxic snake venom PLA2 also
125I-OS2onmembranepreparationsfromgangliaof
purifiedfrom taipan snake(Lambeau et at. 1989)orporcine
Aplysia californica
pancreatic PLA2 produced a relatively weak inhibition
A crude membrane preparation of all ganglia from the
(21-8 ±3% (n= 3) and 18 + 6% (n= 2), respectively)
whole animal was used to demonstrate the presence of
when applied at the same concentration (Fig.8). Moreover,
125i-oS2 binding sites in Aplysia californica. Figure 9A
when OS2 (20 nM) was applied after treatment with bee
shows results obtained from a typical binding experiment
venom PLA2 (20 nM), no further inhibition was observed
with increasing concentrations of 125I-OS2. Scatchard plot
(D
()
c0aa)L 60 -
En
C4 4O Figure8. EffectsofvariousPLA2sonAChrelease
CC:O °C: 40 - AllPLA2swereappliedat20nmunderconditionsasdescribedin
Figs 1 and2.Meanamplitudesofthepostsynapticresponsewere
o- 20
o-C>-10c°), 0 measuredafter 1 happlicationofthePLA2s.Resultsareexpressed
° 20 - asapercentageofinhibitionofthepostsynapticresponsebefore
c applicationofthePLA2s(mean + S.D.).
......
:
O -
OS2 Taipoxin Bee
OSi Porcine
venom pancreatic
PLA2
PLA2
J Physiol.489.1 Effects of neurotoxic PLA2s at an Aplysia synapse 37
analysis of the specific binding revealed the presence of correction for experimental conditions. This latter value is
only one family of binding sites with an equilibrium close to the one measured by Scatchard plot analysis
binding constant (Kd) of 100 pM and a maximum binding (Fig.9A). Taipoxin and bee venom PLA2 also inhibited the
capacity (Bmax) of 55fmol (mg protein)-'. Similar binding binding of 1251-OS2 to membranes from ganglia with high
experiments were performed on membrane preparations affinity (Ko.5 values of 550 and 800 pM, respectively).
from dissected buccalganglia and resulted in an increase in Conversely, these binding sites were recognized by OS1 and
the Bmax value of up to 300-400 fmol (mg protein)-', the the porcine pancreatic PLA2 with much weaker affinity
Kdvalue beingthesame(datanotshown). (Ko.5valuesof130and500 nm, respectively).
The inhibitory effects of various PLA2s on the specific Figure 9C shows results from cross-linking experiments of
binding of 1251-OS2 to membranes from buccal ganglia are 125i-os2 to membranes of ganglia using the bifunctional
illustrated in Fig.9B. The concentration of unlabelled OS2 reagentDSS. Aftergelelectrophoresisandautoradiography,
that inhibited half of the specific binding (KO.5) was two bands were specifically labelled with an apparent
180 pM, corresponding to a Kd value of 140 pM after molecular mass of 140 and 18 kDa, respectively (after
A
-, 40
V
n
>C- 30 60-
= a) -0 60-
C---
cL.E 20 otoEa 30-
N 0 I ,,f o_
N- 0
Cl)
0 100 200
1251-OS2 free (pM)
B
-o
100
0
=0
O
aU)0
2,..o-
C,oD)0 50
0
N.
0
0 -11 -10 -9 -8 -7 -6
log [PLA2] (log M)
Figure 9. Characterization of125I-OS2bindingonAplysiaganglia
A, equilibrium binding of 125IOS2 to a membrane preparation from all ganglia of Aplysia californica.
Membranes (50jugprotein ml-') were incubated with increasing concentrations of'25I-0S2in theabsence
orpresence of100nmunlabelled OS2. Specificbinding(M)representsthedifference between total (T)and
non-specific(NS)binding. Inset, Scatchard plotofthespecific125I-0S2binding(B, specificallybound125I_
OS2 in fmol (mg protein)-'; F, free 125I-OS2 in pM). B, competition experiments with 1251-0S2 and
unlabelled PLA2s for binding to membranes from buccal ganglia. Membranes (50jugprotein ml-') were
incubatedwith30 pM'25I-OS2and variousconcentrationsofunlabelledPLA2s.Resultsareexpressedasa
percentage of the maximal specific binding measured in the absence of added competitor; 100%
corresponded to0 8pMof125I-OS2specifically bound. Non-specificbindingwasmeasured inthepresence
of100 nmunlabelled O82andaccounted for 20%ofthetotalbinding. *, OS2; 0, taipoxin;E, beevenom
PLA2; n, OSI; A, porcine pancreatic PLA2. C, autoradiogram pattern of SDS-polyacrylamide gel of
Aplysia ganglion membranes labelled with 125J-OS2 and cross-linked with DSS. Lane 1, no addition of
unlabelled PLA2; lane 2, addition of100 nm unlabelled OS2; lane 3, addition of100 nM taipoxin. 100#g
of protein was loaded in each lane. The position of the molecular mass markers is indicated (myosin,
200000Da;/3-galactosidase, 116200 Da; phosphorylaseB, 97400Da; bovineserumalbumin, 66200Da;
ovalbumin, 42700 Da; carbonic anhydrase, 31000 Da; soybean trypsin inhibitor, 21500 Da;
cytochrome C, 14400Da; Biorad). The gel (4-14%) was exposed on Kodak X-Omat AR film for 80h
withanintensifyingscreen.
38 P Fossier, G. Lambeau, M. Lazdunski and C. Baux J Physiol.489.1
subtraction of the contribution of the molecular of (ii) An inhibition of the phosphorylation of proteins
mass
125i-os2 assuming that one 1251-OS2 molecule (molecular involved in the mobilization of synaptic vesicles such as
mass, 14 kDa) is bound per molecule of binding synapsin I(Ueno&Rosenberg, 1992). (iii)Anactionon the
component). This labelling is specific it completely transmitter release mechanism itself. Neurotransmitter
as was
protected by the addition of 100 nm unlabelled OS2. secretion in response to a local depolarization-induced rise
Moreover, addition of 100 taipoxin also resulted in the in cytoplasmicCaW+requiresdockingandfusionofsynaptic
nM
protection ofthislabelling(Fig. 9C). vesicles (reviewed in Greengard, Valtorta, Czernik &
Benfenati, 1993; Rothman, 1994). The docking and fusion
particles contain proteins like NSF (N-ethylmaleimide
DISCUSSION
sensitive factor), SNAP (soluble NSF attachment protein),
Our results show that taipoxin and OS2 decrease the VAMP (vesicle-associated membrane protein)/synapto-
amplitudeofthepostsynaptic responseevokedbyanaction brevin, HPC1/syntaxin and SNAP-25 (synaptosomal-
potential. These decreases could be attributed to associated protein of 25 kDa), which have been identified
presynaptic effects of these PLA2s because the amplitude as targets for neurotoxins causing botulism or tetanus
and decay time ofevoked postsynaptic miniature currents (Siidhof, De Camilli, Niemann & Jahn, 1993; Pevsner &
wereunaffected. Scheller, 1994). These toxins are proteases thatcleave their
target proteins to block neurotransmitter release. It might
During the application oftaipoxin OS2, the presynaptic
or
very well be that neurotoxic PLA2s described in this work
neurone was polarized at -50 mV so that the decrease in
also have an effect on these processes via presently
transmitter release could not be attributed to a hyper-
unknown mechanisms of signal transduction associated
polarization of the presynaptic proposed in
neurone, as
with binding of these PLA2s to their specific neuronal
chick dorsal rootganglion cells in culture (Possani, Mochca-
receptor(s). Indeed, it is unlikely that PLA2s are
Morales, Amezcua, Martin, Prestipino&Nobile, 1992).
internalized in order to exert their action because it has
Contrary to some observations made at the mammalian been shown that bafilomycin, which blocks endocytosis via
neuromuscular junction with neurotoxic PLA2s including an inhibition of vacuolar adenosine triphosphatase,
taipoxin (Dreyer & Penner, 1987; Rowan & Harvey, 1988), prevents the action of clostridial neurotoxins but not that
we never observed any early transient increase in of PLA2s such as taipoxin, notexin, /1-bungarotoxin or
transmitter release after bath application of taipoxin or textilotoxin on neuromuscular transmission (Simpson,
OS2. Thisresult may be related tothe absence ofinhibition Coffield&Bakry, 1994).
of any ofthe K+ currents. Such an inhibition ofa specific Taken together, the binding data indicate that 1251-OS2 has
class ofK+ channels was proposed tobe responsible for the
specific high affinity binding sites (Kd= 100-140 pM) in
transient increase in transmitter release observed at the
ganglia from Aplysia californica. These OS2 receptors are
mammalian neuromuscular junction (Dreyer & Penner,
mainlycomposedofproteincomponentsof140and 18 kDa
1987; Rowan & Harvey, 1988) by secondarily
increasing
and are also recognized by other neurotoxic PLA2s
thepresynapticCa2+influx.
including taipoxin (Ko.5 = 550 pM). The subunit structure
In the presence of taipoxin, the increase in Ca2+ influx ofOS2receptors in Aplysia isdifferentfrom thatofN-type
should enhance ACh release. This was the case when ACh receptors identified in mammalian neuronal tissue
release was evoked by a controlled depolarization of the (Lambeau et al. 1989). There, the main subunits have a
voltage-clamped presynaptic neurone. When ACh release molecular mass of 40-50 kDa. This is more similar to
was triggered by an action potential, this increase in Ca2' M-type receptors identified inskeletal muscleforwhich the
influx was overcome by other mechanisms including the major subunit has a molecular mass of 180 kDa (Lambeau
enhancementofboth IAand IK insuch away thattaipoxin et al. 1990). However, the pharmacology of Aplysia
decreased ACh release. It isdifficult to know whether these neuronal OS2 receptors is more similar to that of N-type
opposite effects develop exactly concomitantly. One can mammalian OS2 receptors, which bind OS2, taipoxin and
imagine that, if the increase in presynaptic Ca2+ influx bee venom PLA2 with a high affinity but which display a
started first, an increase in transmitter release would be very low affinity for OS1 and pancreatic PLA2 (Lambeau et
detected at the beginning oftaipoxin application as shown al. 1989). The M-type receptor first identified in rabbit
attheneuromuscularjunction (Dreyer&Penner, 1987). muscle cells has a high affinity for OS2 and also binds OS,
One wonder ifthe large increase in the presynaptic K+ and pancreatic PLA2 but has no binding activity for bee
can
venom PLA2 (Lambeau et al. 1990). One intriguing
currents induced by taipoxin and the inhibition of the
question that still remains to be answered is the
presynaptic Ca current induced by OS2 may suffice to
contribution of these binding sites to the inhibitory effects
explain the reduction of transmitter release. Other intra-
of neurotoxic PLA2s, including OS2 and taipoxin, on ACh
cellular effects ofPLA2cannot be ruled out. (i) A reduction
release. These binding sites are probably essential to the
of the ACh content of the presynaptic neurone following
action ofboth toxins since other PLA2s such as OS1 (which
blockade of the high affinity choline uptake mechanism
is not neurotoxic; Lambeau et al. 1989)or pancreatic PLA2,
(Mollier, Brochier & Morot Gaudry-Talarmain, 1990).