Polyamine FTX-3.3 and Polyamine Amide sFTX-3.3 Inhibit Presynaptic Calcium Currents and Acetylcholine Release at Mouse Motor Nerve Terminals

Neuropharmacology, Vol. 36, No. 2, pp. 185–194, 1997 Copyright 01997 Elsevier Science Ltd. AU rights reserved Printed in Great Britain 0028-3908/97 $17.00 + 0.00

Pergamon @ PII: S0028-3908(96)00146-3

Polyamine FTX-3.3 and Polyamine Amide sFTX-3.3 Inhibit Presynaptic Calcium Currents and Acetylcholine Release at Mouse Motor Nerve Terminals M. FATEHI,l E. G. ROWAN,l A. L. HARVEY,l* E. MOYA2 and I. S. BLAGBROUGH2 IDepar~ent of phy~ioloo and pharmacoloe, Universityof Strathclyde,204 George Street, Glasgow, ‘1 1~, U.K. and ‘Departmentofkfedicinal Chemist~, Schoolof Pharmacyand Pharmacology,Universityof Bath, ClavertonDown, Bath,BA2 7A~ U.K. (Accepted5 September1996) Summary-FTX-3.3 is the proposedstructureof a calcium-channelblockingtoxin that has been isolated from the funnel web spider (Agelenopsisaperta). The effects of FTX-3.3 and one of its analogues, sPTX-3.3, on acetylcholine release, on presynaptic currents at mouse motor nerve terminals and on whole-cell sodium currents in SK.N.SH cells (a human neuroblastoma cell line) have been studied. FIX-3.3 (10-30 pM) and slWX-3.3(100-300 PM) reversibly reduced release of acetylcholineby approximately70-90% and 40-60%, respectively. FTX-3.3 (10 pM) blocked the fast componentof presynapticcalcium currents by approximately 60%. slWX-3.3(100 ,uM)reducedthe durationof the slow componentof presynapticcalcium currentsby about 50% of the control and also reduced presynaptic sodium current by approximately20% of the control. sFIX3.3 (100 pM) reducedwhole-cellsodiumcurrentrecordedfrom SK.N.SHcells by approximately15%,whereas IWX-3.3,even at 200 pM, did not affect this current. Since the only difference in chemical structuresof these toxins is that sFTX-3.3 has an amide function which is absent in IWX-3.3, the amide function may be responsiblefor the reducedpotencyand selectivityof sFTX-3.3.This study alsoprovidesfurthersupportfor the existence of P-type calcium channels at mouse motor nerve terminals. G 1997Elsevier Science Ltd. All rights reserved. Keywords-Spider toxins, polyamides, polyamine amides, calcium currents (presynaptic), neuromuscular transmission, sodium current.

Selective calcium channel blockers are required in order to determine the physiological functions mediated by different subtypes of calcium channels. A naturally occurring arthropod polyamine toxin, whose structure has yet to be completely agreed upon (called FTX) from the venom of the American funnel-web spider, AgeZenopsis aperta, and a synthetic analogue, thought to closely resemble the native toxin (called sFTX or sFTX3.3), have been used as pharmacological tools for the identification, characterization and isolation of P-type voltage-dependent calcium channels (Cherksey et al., 1991;Lliruiset al., 1992;Scott et al., 1993).FTX (whose “concentration” is usually expressed as a dilution relative to the crude venom) apparently blocks P-type calcium channels in various preparations at anywhere between 1:600and 1:200000 dilutionof the crude venom (Uchitel et al., 1992; Usowicz et al., 1992; Duarte et al., 1993; Moulian and Morot Gaudry-Talarmain, 1993; *To whom correspondenceshould be addressed 185

Williams et al., 1993; Fossier et al., 1994; Brown et al., 1994; Frittoli et al., 1994; Gonzalez et al., 1995). However, there are now reports of FIX interacting with other sub-classes of calcium channels, such as coconotoxin GVIA-sensitive channels on frog nerve terminals and co-conotoxin-sensitivetransient calcium currents on rat neurohypophysialnerve terminals (Wang and Lemos, 1994;Katz et al., 1995).sFTX-3.3 and other analoguesof FTX (10 nM–3 mM) have also been used to identify and characterizeP-type calcium channels.These analogueshad an inhibitoryprofilesimilar to that of FTX (Cherksey et al., 1991; Llintis et al., 1992; Gandia et al., 1993;Scott et al., 1993;Sutton et al., 1993;Frittoli et al., 1994; Gonzalez et al., 1995). Nevertheless, sFTX-3.3 blocks both low threshold and-lfigh threshold voltageactivated calcium currents and can enhance, as well as block voltage-dependentpotassiumchannels and ligandgated ion channels in cultured sensory neurons (Scott et al., 1992; Sutton et al., 1993). Therefore, the selectivity

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-+Y’-:TNH, -:++-:TNH, NH,

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Fig. 1. Structuresof sFIX-3.3andFI’X-3.3.

of these polyamides and polyamine amides remains in question. Previously, IWX isolated from the funnel-web spider venom and a synthetic analogue, sIWX were shown to reduce quantal content of endplate potentials, recorded from mouse nerve-muscle preparations, without affecting other ionic currents (Uchitel et al., 1992). Nevertheless, whether the effects of ITX and sFTX on neurotransmitter release can be attributed to the block of only one class of ion channel is still open to interpretation.Recently, FTX-3.3 (reputed to be structurally identical to the native FTX) and its amide-containing analogue, sFTX-3.3, (Fig. 1) have been synthesized (Blagbrough and Moya, 1994; Moya and Blagbrough, 1994) and have been shown to block P-type calcium channels in rat cerebella Purkinje cells (Dupere et al., 1996).Therefore, the objectivesof the presentwork were to compare the effects of FTX-3.3 and sFTX-3.3 on release of acetylcholine at mouse neuromuscularjunctions and to explore more fully the mechanisms underlying their effects on neurotransmitter release. A preliminarycommunicationon the effects of these toxins has recently appeared (Fatehi et al., 1995). For clarity it should be noted that where FTX is mentioned, the compound isolated from the venom of Agelenopsis aperta is being referred to. sFTX denotes a putative syntheticanalogueof FTX (see Cherksey et al. (1991)for the structuresof several FTX analogies). However,IWX3.3 and sFTX-3.3 refer to the compounds of known structuresgiven in Fig. 1. These compoundsare identical to those described in the previous studies (Llin& et al., 1992; Scott et al., 1992, 1993). MATERIALSAND METHODS Materials

Chemicals for the synthesisof ITX-3.3 and sFTX-3.3 were purchased from Aldrich Chemical Company (Gillingham, Dorset, U.K.) except for (CBZ)@g.OH which was purchased from NovaBiochem (Nottingham, U.K.). FTX-3.3 and sFTX-3.3 were synthesized as described previously (Blagbrough and Moya, 1994; Moya and Blagbrough, 1994). p-Conotoxin GIIIB and saxitoxin were purchased from Peptide Institute through Scientific Marketing Associates (Barnet, U.K.) and Calbiochem .ovabiochem (U.K.) Ltd., respectively.

Tetrodotoxin and all other chemicals were purchased from Sigma Chemical Co. Ltd. (Poole, Dorset, U.K.). Methods Electrophysiological recordings. Intracellular and extracellular electrophysiological recording techniques were employed to examine the effects of FTX-3.3 and sFTX-3.3 on neuromusculartransmission and presynaptic calcium currents at mouse motor nerve terminals, using the mouse triangularis sterni nerve-muscle preparation (McArdle et al., 1981). After dissection,,the preparationswere pinnedthoracic side downwards,to the base of a 5 ml Sylgard-coatedtissue bath and superfused continuouslyat a rate of 10-12 ml/min with a physiological solutionof the following composition(mM): NaCl 118.4, KH2P04 1.2, glucose 11.1, NaHC03 25, CaC121, MgS04 1.4 and KC1 4.7. The physiological solution (20 ml) was aerated with a mixture of 95% OJ5% CO, (to maintainpH at 7.2-7.4) and circulated throughoutthe experimentsusing a peristaltic pump. Acetylcholine release was measured by recording nerve evoked endplate potentials (e.p.ps) and spontaneous miniature endplate potentials (m.e.p.ps), using standard microelectrode techniques (Fatt and Katz, 1951). Preparations were paralyzed either by adding MgC12(to reduce quantal content to <10) or by the addition of p-conotoxin (300400 nM) to block muscle sodium channels. Presynaptic calcium currents were recorded using extracellular microelectrodes (Mallart, 1985) in the continuedpresence of 100 WMtubocurarine (to prevent the muscle from twitching), 400 PM 3,4diaminopyridine and 3 mM tetraethylammonium (to block potassium channels, because presynaptic calcium currents can only be revealed under these conditions).In general, if the resting membrane potential of muscle fiberswas more positive than –70 mV, or the amplitude of the signals being recorded decreased by more than 10%within the first30 min before addition of toxins, the recording site was considered to be unsuitable and was rejected. While adopting this protocol, stable recordings of over 180 min for both intracellular and extracellular recording could be achieved (Fatehi et al., 1994). All drugs and the toxins were applied to the preparationsvia adding to the 20 ml of the physiologicalsolution, which was circulated during the experiments by a peristaltic pump. The concentrations of the drugs and toxins

Polyamine toxins and neurotransmission

mentioned in the text indicate the final concentrationsin the physiologicalsolution. In preliminary experimentsit was found that maximum inhibition occurred within 4-5 min exposure to the toxins. Therefore, a toxin application time of 5 min was used throughout these experiments. Experiments were carried out at room temperature (22–25”C). Analysis of electrophysiological data. For intracellular and extracellularrecordings,the signalswere recordedon video tape using a video tape recorder (Sharp VC-A36) and Sony PCM-701ES,adaptedfor wide bandwidth(DC20 kHz) analoguesignalrecording.The SynapticCurrent Analysis(SCAN) programprovidedby Dr J. Dempsterat University of Strathclyde, was used to visualize and analyze the digitized signals (Dempster, 1988), At each time period 30-120 endplatepotentials(e.p.ps), 100-300 miniature endplate potentials (m.e.p.ps) and 20-30 perineural waveforms were stored. Abnormal signals due to electrical interference, summation of events, or spiking were rejected and accepted signals were averaged. Quantal content was then calculated as the average e.p.p divided by the average m.e.p.p. Because of the small size of the endplate potentials in Mg2+-paralyzed preparations, correction for non-linear summation was not required (McLachlan and Martin, 1981). Cell culture. Human neuroblastoma cells SK.N.SH were obtained from the European Collection of Animal Cell Cultures, Department of Animal Cell Resources, Centre for Applied Microbiology (Porton Down, Salisbury, U.K.) and were grown routinely in RPMI medium supplemented with heat inactivated fetal calf serum (10% v/v), sodium bicarbonate 0.075%, fungizone (2.5 PM) and penicillin (50 units/ml)/streptomycin (50 #g/ml) (pH adjusted to 7.3-7.4 with 1 M NaOH) in a humidified atmosphere of 5% C02/95% air at 37”C. Cells were used for patch-clamp experiments 1–5 days after replating on to collagen-coated coverslips. Cells were regularly maintained in the laboratory by replating trypsin-dissociated SK.N.SH cells weekly in Falcon culture flasks. Whole-cell patch-clamp recording. The whole-cell variant of the tight-seal patch-clamp technique (Hamill et al., 1981) was used to record sodium currents in SK.N.SH cells. The cells were initially superfused continuously at a rate of 1–2 ml/min, but in order to reduce the amount of toxin used per experiment,all toxin experiments and control experimentswere carried out in a static bath containing lml of the following external solution (in mM): 4-(hydroxyethyl)-l-piperazine ethane sulfonic acid (HEPES) 10, tetraethylammoniumchloride (TEA-Cl) 20, NaCl 135, NaH2P04 0.06, glucose 10, CaC120.01, MgC121.2, COC121 and CSC15, adjusted to pH 7.3 with NaOH. Experimentswere carried out at room temperature (23–25”C) and the toxins or the equivalent amount of toxin vehicle (H20) was added directly into the bath. In pilot experiments,it was establishedthat the responses to sFTX-3.3 and FTX-3.3 achieved equili-

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brium within 34 min exposure to the toxins. Thus, an exposure time of 5 min was applied throughout these experiments. Micropipetteswere fabricated from borosilicatecapillary tubing (Clark ElectromedicalInstruments,Reading, Berkshire, U.K.) and had tip resistances of 1.5–2 Mf2 (when filled with an internal solution of following composition (mM): EGTA 10, CSC1 120, NaCl 10, MgC122,TEA Cl 25 and HEPES 10, adjusted to pH 7.3 with 1 M KOH. Voltage clamp was achieved using an EPC-7 patchclamp amplifier (List Electronic, Darmstadt, Germany). Voltage-clamp pulses and data acquisition were controlled by a Viglen 486-DX computer running Whole Cell Program (WCP) software (Dempster, 1988), interfaced to the EPC-7 patch-clamp amplifiervia a National Instruments Labmaster PC/PC+ board. Cells were maintained at a holding potential of —80mV between pulse protocols.Capacitativecurrentswere compensated using analogue circuitry, combined with subtraction of leakage currents using P/–4 subtraction protocol. The inward currents recorded from SK.N.SH cells under above mentioned conditionswere designatedas voltagedependentsodium currents, since they were inhibitedby 100 nM saxitoxin. Statistics. The results are expressed as the mean~SE. The statistical significance was evaluated by the Mann– Whitney U-test. Values of p< 0.05 were taken as

significant. RESULTS Effects on endplate potential amplitude and quantal content of endplate potentials Effects on Mg2+-paralyzedpreparations. FTX-3.3 at 1 and 5 pM did not significantly (’p>0.05) affect nerveevoked or spontaneous release of acetylcholine from mouse motor nerve terminals. The amplitude of e.p.ps before exposure to the toxin, 5 min and 20 min after exposure to 5 VM FTX-3.3 were 4.8 t 0.6, 4.8 ~ 0.7 and 4.7 ~ 0.6 mV, respectively(n = 3). The amplitudeof m.e.p.ps before exposure to the toxin, 5 min and 20 min after exposure to 5 pM FTX-3.3 were 0.77 ~ 0.19, 0.75 ~ 0.18 and 0.74 f 0.19 mV, respectively (n= 3). The frequency of m.e.p.ps before exposure to the toxin, 5 min and 20 min after exposure to 5 VM FTX-3.3 were 1.7 t 0.2, 1.5 ~ 0.2 and 1.6 ~ 0.2 per see, respectively (n= 3). However, 10PM FTX-3.3 significantly (p c 0.05) reduced the average amplitude of endplate potentials within 4 min exposure. Figure 2 shows the time-course of the effects of ITX-3.3 (10 KM) on the amplitudeof e.p.ps (n = 4). It is obvious from this figure that maximum reduction in e.p.ps amplitude induced by the toxin reached a plateau within 4 min exposure. At a higher concentration (30 #M), FTX-3.3 reduced the amplitude of e.p.ps by 79 ~ 2% of control (from 7.2 ~ 1.1 mV before the toxin to 1.5 f 0.2 mV, 5 min after exposure to the toxin) (n= 4), without changing

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postsynaptic sensitivity, as determined by the lack of effect on m.e.p.p. amplitude (Fig. 3A). The quantal contents of e.p.ps were reduced by 64 f 3% (n= 4) and 79 * 2% (n= 4) of control after 5 min exposure to 10 and 30 PM FTX-3.3, respectively (Fig. 3A). No further decrease in e.p.ps amplitudewas observed up to 30 min after exposureto the toxin. FTX-3.3 (10 and 30 PM) did z 40 not alter muscle resting membranepotential(Fig. 3A), or i the time courses of rising and falling phases of m.e.p.ps (Fig. 3B). The changes to quantal content were reversed (to 99 t 5% (n=4) of control) 5 min after continuous washing of the preparationswith a toxin-free solution. ( I sFTX-3.3 at 10 and 50PM did not significantly 10 012345 (P> O.05)alter nerve-evoked release of acetylcholine. Time (rein) The amplitudes of e.p.ps before exposure to the toxin, 10 min and 20 min after exposure to 50PM sFTX-3.3 Fig. 2. The time-course of the effects of 10 PM FTX-3.3 (~) and 100 IJMsFI’X-3.3(0) on the average amplitude of e.p.ps were 3.8 t 1.0, 3.7 & 1.0 and 3.6 * 0.9 mv, respecrecorded from Mg2+-paralysed mouse triangularis sterni tively (n= 4). The amplitudesof m.e.p.psbefore exposure preparations. Points represent the means of values obtained to the toxin, 10 min and 20 min after exposure to 50PM from four experiments and standard errors of means are SFTX-3.3 were 0.73 + 0.11, 0.73 ~ 0.11 and indicated by the bars. 0.71 i 0.12 mV, respectively (n= 4). The frequency of 1

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amplitudeofendplatepotentials(e.p~ps), amplitudeofminiatureendplatepotentials(m.e.p.ps)andquantal&ntent (QC)at Mg2+-paralysed mousetriangularissternipreparations.Opencolumnsin (A) representthe results of time-

matched control experiments.Crossed and solid columnsin (A) representthe effects of FIX-3.3 at 10 and 30 jJM, respectively. Crossed columns in (C) represent the effects of 100vM sFTX-3.3 after 5 min exposure. Data are expressed as means and upward bars represent the’SEM, *significantat p <0.05, (n = 4). (B),(D)Superimposed

computeraveragedendplatepotentials‘inMgz+-paralysed preparations.Controlendplatepotential(a) and5 min after 10 PM FIX-3.3 (B,b) and .100PM sFTX-3.3 (D,b). The panel to the right of the endplate potentials shows superimposedaveraged of miniature endplate potentials before and 5 min after the toxins.

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Polyamine toxins and neurotransmission

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Fig. 4. (A), (C) Effects of FrX-3.3 (10 and 30.uM)and sFTX (100 and 300PM) on resting membranePotentials (r.;.psj, amplitude of endplate potentials (e.p.psj, amplitude of miniature endplate potentials (m.e.p.ps) and quantal content (QC) at p-conotoxin-paralyzedmousetriangularisstemi preparations.Opencolumnsrepresentthe results of time-matched control experiments. Crossed and solid columns represent the effects of the toxins at 10 pM (n = 4) and 30 PM (n = 3) for FTX-3.3, 100 pM (n = 3) and 300 PM (n = 3) for s~X, respectively.Data are expressedas means and upwardbars representthe SEM, *significantatp <0.05. (B), (D) Superimposedcomputer averagedendplate potentials in K-conotoxin-paralyzedpreparations.Controlendplatepotential (a) and 5 min after 10PM FIX-3.3 (B,b) and 100PM sFI’X-3.3 (D,b). The panel to the right of the endplate potentials shows superimposedaveraged of miniature endplate potentials before and 5 min after the toxins.

m.e.p.ps before exposureto the toxin, 10 min and 20 min after exposure to 50PM sFTX-3.3 were 1.3 ~ 0.4, 1.3 ~ 0.4 and 1.2 f 0.4 per see, respectively (n=4). Nevertheless, 100 PM sFTX-3.3 significantly(p c 0.05) decreased the average amplitudeof e.p.ps within a 4 min exposure.Figure 2 showsthe time-courseof the effectsof sFTX-3.3 (100 #M) on the amplitudeof e.p.ps (n= 4). A maximum inhibition of the evoked release of acetylcholine occurred within a 4 min exposure. sFTX-3.3 (100 pM) did not affect muscle resting membrane potential and the average amplitude of m.e.p.ps (Fig. 3C). The time-courses of rising and falling phases of m.e.p.ps remained unchanged after exposure to the toxin (Fig. 3D as a representative trace). Therefore, 100MM sFTX-3.3 decreased quantal content of e.p.ps by 36 ~ 1% (n=4) of control after 5 min exposure (Fig. 3C). No further reduction in e.p.p. amplitude was observed up to 30 min after exposure to the toxin. The changes to quantal content were reversed (to 97 f 4% (n =4) of control) 5 min after continuouswashing of the preparationswith a toxin-free solution.

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Effects on p-conotoxin-paralyzed preparations. In an attemptto examinewhether FTX-3.3 and sFTX-3.3could reduce nerve-evoked acetylcholine release in preparations where acetylcholinerelease was not decreased by excessive extracellular Mg2+ ions, experiments were carried out in the continued presence of the muscle sodiumchannelblocker, ~-conotoxin.FTX-3.3 at 10 and 30PM significantly(p c 0.05) reduced the amplitude of e.p.ps by 67 ~ 7% of control (from 25.3 i 6.0 mV before adding the toxin to 8.0 t 1.3 mV, 5 min after exposure to the toxin) (n= 4) and 90 t 270 of control (from 50.1 & 6.9 mV before adding the toxin to 5.1 f 1.0 mV, 5 min after exposure to the toxin) (n=3), respectively, without significantly (p >0.05) changing postsynaptic sensitivity, as determined by the lack of effect on m.e.p.p. amplitude (Fig. 4A, B). ~X3.3 did not significantly (p> 0.05) alter muscle resting membranepotential(Fig. 4A). The time-coursesof rising and falling phases of m.e.p.ps were not affected by the toxin (Fig. 4B). Therefore, IWX-3.3 (10 and 30 vM) significantly (p< 0.05) decreased quantal content of

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e.p.ps by 66 f 7!% (n =4) and 90 f 2$%(n= 3) of control, respectively (Fig. 4A). sFTX-3.3 at 100 and 300PM significantly(p c 0.05) reduced the average amplitude of e.p.ps by 38 f 670 of control (from 23.1 ~ 6.1 mV before adding the toxin to 14.4 ~ 4.1 mV, 5 min after exposureto the toxin) (n=3) and 55 ~ 570 of control (from 31.2 ~ 6.3 mV before adding the toxin to 14.3 ~ 3.9 mV, 5 min after exposure to the toxin) (n= 3), without changing muscle resting membranepotential,or m.e.p.p. amplitude(Fig. 4C). The time coursesof rising and falling phasesof m.e.p.pswere not altered (Fig. 4D as a typical trace). Therefore, sFTX3.3 at 100 and 300 pM significantly(p< 0.05) decreased quantal content by 38 f 6% (n=3) and 58 t 3% (n=3) of control, respectively (Fig. 4C). Eflects on presynaptic calcium currents

The waveforms recorded from the perineural sheath of motor nerves, close to the axon terminals, are associated with the movement of ions that produces terminal depolarization and depolarization(Mallart, 1985). These extracellular waveforms are composed of two negative components. The first negative component is associated with the inward sodium current at the heminode and nodes of Ranvier, and the second negative componentis the net local circuit current that is generated by the movement of both calcium ions (inward) and potassium ions (outward) at the motor nerve terminals (Fig. 5A). After application of 3,4-diaminopyridine(400 PM) and tetraethylammonium (3 mM), voltage- and calciumdependent potassium channels are blocked and a long lasting positive deflection,called the calcium plateau, is generated.As previouslydescribedby Penner and Dreyer (1986), the calcium plateau has two components (a fast initial phase and a slow, long-lasting plateau) that are pharmacologically distinct from each other (Fig. 5B). Both the fast rising phase and the slower long-lasting plateau phase can be blockedby cadmium.Verapamilhas a selective effect on presynaptic calcium currents. The slow, long-lastingplateau phase is blockedby verapamil, but the fast rising phase remains unaffected upon application of a relatively high concentration of verapamil (10 PM) (Penner and Dreyer, 1986). The effects of FTX-3.3 and sFTX-3.3at concentrations sufficient to reduce acetylcholine release (10 PM for FTX-3.3 and 100PM for slTX-3.3) were studied on calcium currents at motor nerve terminals. ITPX-3.3(10 pM) significantly(p c 0.05) reduced the average amplitude of the fast calcium current recorded extracellularly from motor nerve terminals by 55 ~ 7Y0 (n=3) of control 5 min after exposure (Fig. 6, as a representative trace). The negative component of the waveform, which is associated with inward sodium currents, was not significantly (p> 0.05) affected after 5 min continuous exposure of preparations to the toxin (Fig. 6, as a typical trace). sFTX-3.3 (100 PM) significantly (p c 0.05) reduced the duration of the calcium plateau by 50 ~ 10% (n =3)

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Fig. 5. (A) Computer averaged perineural waveform recorded extracellularly from the pre-terminal part of a mouse motor nerve. The first negative component is associated with Na+ current and the second negative component is associated with K+ current at the nerve terminals. (B) A computer averaged perineural recorded from the pre-terminal part of a mouse motor nerve after applicationof 3,4-diaminopyridine(400 pM) and tetraethylammonium (3 mM). Note the different time scales in (A) and (B).

of control 5 min after additioninto the tissue bath. It had little effect on the fast calcium current within 5 min (Fig. 7 as a typical trace). In addition, the first negative componentof the waveform (associatedwith the influxof sodium)was significantly(p c 0.05) reducedby 20 ~ 670 (n= 3) of control, after 5 min exposure to the toxin (Fig. 7, as a representativetrace). All of the effects of I?TX-3.3 and sFTX-3.3 on presynaptic waveforms were fully reversed 5 min after continuous washing of the tissue, with toxin-free physiologicalsolution (Figs 6 and 7). In order to investigate the effects of partial block of presynaptic sodium currents on the presynaptic waveforms, particularly on the positive signal related to presynaptic calcium currents, the effects of 1 PM

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‘ Fig. 6. Computer-averagedwaveforms representing the effect of FTX-3.3 (10 #M) on the presynaptic perineural waveforms, recorded from mouse motor nerve terminals in the presence of 3,4-diaminopyridine (400 PM) and tetraethylammonium (3 mM): (a) control; (b) 5 min after exposure to the toxin; and (c) 5 min after washout of IWX-3.3.The inset shows the lack of effect of IWX-3.3on presynaptic sodium current. Note the different scales in the figure.

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Fig. 7. Computer-averagedwaveformsrepresentingthe effects of sFTX-3.3 (100 PM) on the presynaptic perineural waveforms recorded from mouse motor nerve terminals in the presence of 3,4-diaminopyridine (400 PM) and tetraethylammonium (3 mM): (a) control; (b) 5 min after exposure to the toxin; and (c) 5 min after washout of sFTX-3.3. The inset shows the effect of sFTX-3.3 on presynaptic sodium current. Note the different scales in the figure.

terminal, a reduction of axonal sodium currents would reduce the depolarizationof the nerve terminal, resulting in a reduction in voltage-dependentcalcium currents in the nerve terminal. Hence, it was decided to determine more directly the effects of sFTX-3.3 and FTX-3.3 on sodium currents by whole-cell recording, from a neuroblastomacell line (SK.N.SH). Figure 9 shows the lack of effect of FTX-3.3 and the blocking effect of sFTX-3.3 on whole-cell sodium currents of SK.N.SH cells, elicited by step depolarizations from a holding potential of –80 to –20 mV. Bath applicationof FI’X3.3 (30 PM, the highest concentration used and which significantlyreduced e.p.p. amplitude)did not reduce the Effects on whole-cell sodium currents recorded from inward current associated with the opening of sodium SK.N.SH cells channels (Fig. 9A). Higher concentrations of FTX-3.3 Since the perineural recording technique is an indirect (200 uM) also had no effect on sodiumcurrents(Fig. 9C). measurement of the ionic conductance at the nerve sFTX-3.3 (100 PM), however, significantly (p c 0.05)

tetrodotoxin (a classical sodium channel blocker) on the presynapticwaveformswere tested. Tetrodotoxin(1 PM) reduced the first negative component of the waveforms by 14 t 5% (n= 3) of control after 5 min exposure.This block of the presynaptic sodium current was accompanied by an apparentinhibitionof calciumcurrent at motor nerve terminals. Tetrodotoxin (1 PM) decreased the amplitude of the fast phase and the duration of the plateau phase of the positive signal related to the presynaptic calcium currents by 23 ~ 3% (n= 3) and 34 t 7% (n= 3) of control, respectively (Fig. 8, as a representativetrace).

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1 ms Fig. 8. Computer-averagedwaveformsrepresentingthe effects of tetrodotoxin (1 PM) on the presynaptic perineural waveforms recorded from mouse motor nerve terminals in the presence of 3,4-diaminopyridine (400 PM) and tetraethylammonium (3 mM): (a) control; and (b) 5 min after exposure to the toxin. The inset shows the effect of tetrodotoxin on the first negative component of the waveform. Note the different scales in the figure.

reduced the inward sodiumcurrentby 16 f 4% (n= 4) of control (Fig. 9A, D). There was a complete reversal (to 102 i 5% (n =4) of control) 5 min after washing the cells with toxin-free external solution. DISCUSSION

The present study demonstrates that acetylcholine release (in both Mg2+- and ,wconotoxin-paralyzed preparations) from mouse motor nerve terminals is mediated by calcium influx through calcium channels sensitive to a polyamine, thought to be present in Agelenopsis aperta venom (FTX-3.3). It was observed that 10PM FTX-3.3 reduced acetylcholine release by about 64%. In mouse diaphragm nerve-muscle preparations, Uchitel et al. (1992) found half-inhibition of quantal content upon application of native FTX at 1:200000 dilution. In addition, Moulian and Morot Gaudry-Talarmain (1993) reported a similar inhibition of acetylcholine release from Torpedo synaptosomes, induced by native FIX at 1:100000 dilution. sFTX-3.3

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was more than 10 times less effective than FTX-3.3, at decreasing quantal content of endplate potentials. The difference in potency of FIX-3.3 and sFTX-3.3 is consistentwith other observations.For example,Moulian and Morot Gaudry-Talarmain (1993) reported that a high concentration of sFTX-3.3 (3 mM) was necessary to produce the same level of inhibition of acetylcholine release induced by native FTX at 100000 times dilution. The effects of FIX-3.3 and sFTX-3.3 on sodium and calcium currents recorded from the pre-terminal part of mouse motor nerve were examined in the continued presence of 3,4-diaminopyridine (400 ~M) and tetraethylammonium(3 mM). Under these conditions,abroad positive electrical signal can be recorded, which reflects presynaptic calcium currents. The fast rising phase and the long-lasting phase (plateau part) of this positive signal of perineural waveforms were designated as transient (fast) and sustained (slow) calcium currents, respectively (Penner and Dreyer, 1986), because both phaseswere blockedby cadmium.The fast componentof the positive signal is diminished by cadmium, but remains unaffected by organic calcium-channelblockers (verapamil and diltiazem). However, verapamil and diltiazem, but not the l,4-dihydropyridines, block the slow componentof presynapticcalcium currents (Penner and Dreyer, 1986). FTX-3.3 and sFTX-3.3 affect these calcium currentsin differentways. FTX-3.3 causes a marked reductionin the amplitude of the calcium plateau, with no significant change in the duration of the calcium current, whereas sFTX-3.3 reduces the duration of the calcium plateau, without affecting the amplitude of the fast calcium current. Effects of other calcium-channel neurotoxins, such as co-conotoxinGVIA (Andersonand Harvey, 1987) and co-agatoxin IVA (Protti and Uchitel, 1993) on presynaptic calcium currents at mammalian neuromuscularjunctions have alreadybeen characterized.Previous experience with m-conotoxinGVIA (an N-type calcium channel blocker) showed that the calcium currents at mouse motor nerve terminals were insensitive to the toxin. However, these voltage-activated currents were shownby Protti and Uchitel(1993)to be very sensitiveto co-agatoxin IVA (a P-type calcium channel blocker). Considering these observations and the present data related to the effect of FTX-3.3 on the fast componentof presynapticcalcium currents, it seems very unlikely that this current passes through N-type or L-type calcium channels. Since both FTX-3.3 and co-agatoxin IVA, which are known as P-type calcium channel blockers (Uchitel et al., 1992; Mintz et al., 1992; Dupere et al., 1996),exert a dramatic inhibitionof the fast rising phase of calcium currents, it can be suggested that P-type channels mediate this current at mouse motor nerve terminals. From the differentialeffects of l?lX-3.3 and sFTX-3.3 on presynapticcalcium currents, one would assume that the toxinsblock differentpopulationsof calcium currents

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Fig. 9. (A) Effects of FIX-3.3 (30 #M) and sFTX-3.3(100 #M) the amplitudeof sodium currents from SK.N.SH neuroblastomacells elicited after a voltage step of +60 mV from a holdingpotential of —80mV. Open column is the result of time-matchedcontrol experiments,the crossedand solidcolumnsrepresentthe effectsof FTX-3.3and sI?IX-3.3, respectively.Data are expressedas means and upwardbars representthe SEM, *significantatp c 0.05, (n =4). (B), (C), (D) Illustrate the lack of effect of FI’X-3.3(200 PM) and the effect of sFTX-3.3 (100 KM)on sodium currents elicited after a voltage step of +60 mV, from a holding potential of –80 mV.

at mouse motor nerve terminals. However, partial block of sodiumcurrents at the terminal portion of motor axons induced by sFTX-3.3 resulted in some ambiguity about the specific action of the toxin on calcium channels. Inhibition of the presynaptic sodium currents can indirectly affect both quantal content and the nerve terminalwaveform (Braga et al., 1992).It is evidentfrom the experimentswith tetrodotoxin,that a small reduction in the first negative component of the presynaptic waveform (associated with the influx of sodium) is accompanied by a considerable reduction in the presynaptic calcium signal. In order to determine whether FTX-3.3 and sFTX-3.3 could affect sodium channels, their effects were examined directly on whole-cell sodium currents of neuroblastorna cells (SK.N.SH). sFTX-3.3,but not FTX-3.3, significantlyreduced sodium currents in SK.N.SH, at concentrations able to reduce release of acetylcholine.Therefore, the inhibitory effect of sFTX-3.3 on the presynaptic calcium currents and transmitter release from mouse motor nerve endings could be due to a reduction in nerve terminal depolarization, as a consequenceof sodium channelblock. Overall, the present study providesno strong evidence to indicate that acetylcholine release is modulated by sFTX-3.3sensitive calcium channels at mouse motor nerve terminals. Acknowledgements—Theauthors would like to thank AFRC/ BBSRC (AG86/521)for a fellowship grant to E. M. I. S. B. is the recipient of a Nuffield FoundationScience Lectures award (SCI/180/91/15/G).

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