Modulation of Acetylcholine Release at Mouse Neuromuscular Junctions by Interaction of Three Homologous Scorpion Toxins with K+ Channels 1H

Modulation of acetylcholine release at mouse neuromuscular junctions by interaction of three homologous scorpion toxins with K+ channels 1H. Vatanpour & 2A.L. Harvey

Department of Physiology and Pharmacology, University of Strathclyde, Glasgow GI IXW

1 The effects of three scorpion toxins, charybdotoxin (CTX), iberiotoxin (IbTX), and noxiustoxin (NTX) have been studied on acetylcholine release and on K+ channels by means of twitch tension and electrophysiological recording techniques using isolated skeletal muscle preparations and by a radioligand binding assay using 1251I-labelled dendrotoxin I (DpI) and rat brain synaptosomal membranes. 2 On chick biventer cervicis preparations, CTX and IbTX (125 nM) augmented the twitch responses to indirect muscle stimulation. Further, the increase (about 70-80% of control twitch height) was fast in onset, reaching a maximum within 25-30 min. NTX at 125 nm produced a slower augmentation of the twitch responses to indirect muscle stimulation, with the maximum response being seen after 40- 50 min. 3 On mouse triangularis sterni preparations, CTX (300 nM after 35-40 min) and IbTX (100 nM after 15 min) increased quantal content of the evoked endplate potentials (e.p.p.) by about two fold. However, NTX (300 nM) caused only a small increase in e.p.p. amplitude, which was followed by repetitive e.p.ps in response to single shock nerve stimulation after 40-50 min. 4 Extracellular recording of nerve terminal current waveforms in triangularis sterni preparations revealed that CTX and IbTX (3-100nM), but not NTX (100nM), blocked the Ca2"-activated K+ current, IK-Ca. However, there was no major change in the portion of the nerve terminal waveform associated with voltage-dependent K+ currents, IKV, 5 In the radioligand binding assay, NTX potently displaced labelled ['25I]-DpI, whereas CTX produced only partial displacement. However, IbTX did not displace ['25I]-DpI from its binding sites on rat brain synaptosomal membranes. 6 We conclude that these three structurally homologous scorpion toxins act on different K+ channels and that this leads to different patterns of facilitation of acetylcholine release. IbTX acts selectively on high conductance Ca2+-activated K+ channels, leading to an increase in the amplitude of e.p.ps without any other changes. NTX acts on voltage-dependent K+ channels that are sensitive to dendrotoxin and causes repetitive e.p.ps. CTX shares amino acid residues that exist in the structures of IbTX and NTX; CTX acts on both Ca2+- and voltage-dependent K+ channels. Keywords: Neurotoxins; potassium channels; neuromuscular junction; acetylcholine release; dendrotoxin; charybdotoxin; iberiotoxin; noxiustoxin

Introduction Potassium channels with distinctive properties and phar- increase in transmitter release from cholinergic nerve ter- macological sensitivites play critical physiological roles in minals. many types of cells. Although several different genes Noxiustoxin (NTX), a 39-residue-chain peptide toxin from encoding K+ channel proteins have been cloned and ex- the scorpion Centruroides noxius, is known to block neuronal pressed in oocytes, it is still unclear which gene products are K+ channels (Carbone et al., 1987). It facilitated acetyl- important in regulation of such processes as neurotransmitter choline release in chick biventer cervicis preparations and release. One approach to the study of channel function in situ displayed binding of a radiolabelled dendrotoxin from synap- is to examine the effects of highly selective channel blockers. tosomal membranes from rat brain (Harvey et al., 1992). Polypeptide toxins from several Old World scorpions block NTX probably blocks dendrotoxin-sensitive voltage- voltage-gated inactivating K+ channels (for review, see dependent K+ channels in rat brain synaptosomal mem- Harvey et al., 1993). Moreover, some toxins from Leiurus branes (Harvey et al., 1992) and at higher concentrations it quinquestriatus hebraeus (e.g. charybdotoxin, CTX) block also blocks Ca2"-activated K+ channels from T-tubules of high-conductance Ca2"-activated K+ channels (Miller et al., skeletal muscle (NTX's Kd = 450 nM) (Valdivia et al., 1985) as well as voltage-gated inactivating K+ channels 1988). (Schneider et al., 1989; Schweitz et al., 1989; Blaustein et al., Iberiotoxin (IbTX) from Buthus tamulus venom consists of 1991). In contrast, venoms from several New World scor- a single polypeptide chain (37 residues) that can block IK-Ca pions contain polypeptides that selectively block only selectively; it does not block other types of K+ channels voltage-gated, non-inactivating K+ channels (Blaustein et al., (Galvez et al., 1990). IbTX displays 68% sequence identity 1991). The present paper describes the interactions of three with charybdotoxin (CTX). However, IbTX possesses four homologous scorpion toxins with K+ channels that lead to more acidic and one less basic amino acid residue than does CTX, making this toxin much less positively charged than the other peptide (Galvez et al., 1990). IbTX interacts with 1 Present address: Department of Pharmacology and Toxicology, CTX at a distinct site on the channel and modulates CTX Shaheed Beheshti University of Medical Sciences, Tehran, Iran. binding by an allosteric mechanism (Galvez et al., 1990; 2Author for correspondence. Candia et al., 1992). IbTx has recently been reported to 1503 H. Vatanpour & A.L. Harvey- ScorpionScorpio toxins andr transmitter release 1503 increase acetylcholine release at frog neuromuscular junctions period. The same recording site was used throughout each (Robitaille et al., 1993). experiment. CTX is a 37-amino acid peptide from the venom of the For extracellular recording, presynaptic waveforms were scorpion Leiurus quinquestriatus hebraeus, which was recorded with a glass microelectrode (2 M NaCl, 16-30 originally described as a potent blocker of high-conductance Megohms) placed inside the perineural sheath (near endplate Ca2+-activated K+ channels (Miller et al., 1985). Subse- areas) of one of the branches of an intercostal nerve (Mallart, quently, it was shown that CTX can also block the inac- 1985; Penner & Dreyer, 1986; Anderson et al., 1988). Usually tivating voltage-dependent K+ channel that is present in rat 50-70 waveforms were recorded at each time period. Pre- brain and human T lymphocytes, but not other types of K+ parations were stimulated at 0.5 Hz. Recording sites were channels (Gimenez-Gallego et al., 1988; Stuhmer et al., 1989; rejected if signal amplitude varied by more than 10% during Price et al., 1989; Oliva et al., 1991). CTx also binds to the first 15 min prior to addition of the test substance. dendrotoxin-sensitive proteins from brain membranes, imply- Physiological salt solution (10-20ml) containing toxins was ing an action on voltage-dependent K+ channels (Schweitz et aerated and recirculated through the tissue bath during al., 1989; Harvey et al., 1989). CTx has been found to electrophysiological recording. increase acetylcholine release at frog neuromuscular junctions (Robitaille & Charlton, 1992), although an earlier study on a Radioligand binding assay mouse preparation reported no effect under normal recording conditions (Anderson et al., 1988). Synaptosomal membranes were prepared from the brains of Neuronal membranes contain several distinct K+ conduc- 200-250 g male Sprague-Dawley rats (Harvey et al., 1989). tances that can be distinguished by their voltage-dependency Each brain was homogenized in 10 ml homogenization buffer and interactions with blocking agents (Rogawski, 1985). (32 mM sucrose, 2 mM Tris.HCl; pH 7.4) at 4C. The suspen- Potassium channels contribute to the repolarization of the sion was centrifuged at 29000 r.p.m. for 10 min. The resulting membrane after an action potential and to the regulation of pellet was lysed by addition of 10 ml lysis buffer (5TnM the excitability of neurones, and they help to regulate the Tris.HCl; pH 8.1). The suspension was centrifuged at release of transmitter from nerve terminals. Their blocking by 20000 r.p.m. for a further 20 min, and the pellet was finally a K+ channel blocker (e.g., 3,4-diaminopyridine) can cause resuspended in 25 ml synaptosomal buffer of the following prolongation of the time course of depolarization and inc- composition (mM): NaCl 130, KCl 3, CaCl2 2, MgCl2 2, and rease transmitter release. The importance of particular sub- Tris.HCl 20, pH 7.4 Aliquots (5 x 5 ml) were stored frozen types of K+ channel to nerve terminal function cannot be until needed. For competition binding experiments, 200 iLl of examined by use of agents such as 3,4-diaminopyridine or membrane suspension was incubated at room temperature tetraethylammonium because of their low selectivity. It was (19-21°C) with a standard amount (1-3 nM) of '25I-toxin I hoped that a comparison of effects of the more selective and various amounts of competing toxin. The final volume blocking agents IbTx, CTX and NTX would reveal the was 0.5 ml. The membranes were collected by centrifugation physiological contributions of voltage- and Ca2"-dependent on a microfuge and radioactivity determined. K+ channels at motor nerve terminals in mouse skeletal muscle. Source of toxins NTX was isolated as described previously (Possani et al., 1982). CTX and IbTX were synthesized by Dr C. Vita Methods (DIEP, CEA Saclay, Gif sur Yvette, France). ['251]-DpI was prepared by F.A. De-Allie and Dr P.N. Strong (Neuromus- Hammersmith Hospital, London). Twitch tension recording cular Research Unit, Biventer cervicis nerve-muscle preparations (Ginsborg & Warriner, 1960) were isolated from 3-14 day old chicks and Results mounted with a resting tension of 0.5-1 g in 2 or 10 ml tissue baths containing physiological salt solution of the fol- Twitch tension experiments lowing composition (mM): NaCl 118.5, KCI 4.7, MgSO4 1.2, 11.1. The solu- CTX, IbTX and NTX were tested on indirectly stimulated KH2PO2 1.2, CaCl2 2.5, NaHCO3 25, glucose induced a was maintained at 33°C and was bubbled with 95% 02 chick biventer cervicis preparations. CTX (125 nM) tion large increase in the responses of indirectly stimulated nerve- plus 5% CO2 to buffer at pH 7.3. Muscle was indirectly after ring electrodes every 10 s with pulses of muscle preparations. Maximum twitch augmentation stimulated through 25 min was 85 ± 2% of control twitch height (Figure 1). 0.2 ms duration and a voltage greater than that which pro- twit- twitch. IbTX (125 nM) augmented indirectly stimulated muscle duced a maximal ches by 75 ± 7% of control twitch height. The maximum responses were observed after 30 min (Figure 1). NTX Electrophysiology (125 nM) augmented indirectly stimulated muscle twitches by 70 ± 5% of control twitch height. The maximum responses Experiments were performed at room temperature (20-25°C) min 1). on the left triangularis sterni nerve-muscle preparation were observed after 40-50 (Figure (McArdle et al., 1981) isolated from male mice (Balb C, 20-25 g). The complete dissection of the muscle, was per- Electrophysiology formed under continuous perfusion at a rate of 12-20 ml min'I with physiological solution of the above Extracellular recording experiments Under normal condi- composition. tions in the absence of 3,4-diaminopyridine (3,4-DAP), CTX To study evoked transmitter release, the muscle was (100-300 nM) did not change the first or second negative prevented from twitching by using low Ca2+ (0.5 mM) and deflection of the perineural waveform, which are associated high Mg2+ (1.5-5.0 mM) physiological solution. The record- with Na+ currents and voltage-dependent K+ currents, ing electrode (3 M KCI, 8-20 Megohms) was placed in a respectively (see Mallart, 1985). Adding 3,4-DAP (400 JiM) muscle cell at an endplate region, as judged by the revealed a positive component followed by a delayed negative appearance of miniature endplate potentials (m.e.p.ps) with a component, which can be seen to correspond to IKCa because rise time of 1 ms or less. In most experiments, of its sensitivity to tetraethylammonium (TEA) (Mallart, 150-250 m.e.p.ps and 80-150 e.p.ps evoked by stimulation 1985; Anderson et al., 1988) (Figure 2a). CTX, at 3-100 nM, of 0.5 Hz were recorded from each endplate, at each time induced a concentration-dependent blockade of IK-Ca within 1504 1504fH. Vatanpour & A.L. Harvey Scorpion toxins and transmitter release 4-5 min exposure (Figure 2b). IbTX (100 nM) did not dis- play any effects on the first and second negative deflection of the perineural waveform recorded in the absence of 3,4-DAP. In the presence of a maximally effective concentration of 160- 3,4-DAP (400 gM), IbTX (3-100 nM) reduced the amplitude ° 150 of the Ca2'-dependent K+ component of the perineural waveform in a concentration-dependent manner (Figure 2c). IbTX appeared to be slightly more active than CTX at co 14330j blocking IK-ca, and IbTX was faster in onset than CTX. NTX (up to 300 nM) blocked neither first and second negative .- deflections (recorded in the absence of 3,4-DAP), nor the IK-Ca (recorded in the presence of 3,4-DAP). Adding TEA (3 mM) or CTX (100 nM) to the recycling solution after 50 min exposure to NTX revealed a long positive deflection (Figure 2d). In the absence of 3,4-DAP, repetitive firings were detected in the presence of NTX. 10 20 30 40 50 Intracellular recording In Mg2+-paralysed preparations, Tme (m12) CTX (100-300 nM) and IbTX (100 nM) increased the amplitude of evoked with no Figure 1 Effects of three scorpion toxins on chick biventer cervicis e.p.ps significant change in e.p.p. nerve muscle preparations. Charybdotoxin (0), iberiotoxin (0), and time course (Figure 3a and b). As CTX and IbTX had no noxiustoxin (0) at 125 nM increased responses to indirect stimula- effect on the mean amplitude or time course of m.e.p.ps, tion. Points represent the means of 4-6 experiments with s.e.mean. CTX and IbTX increased quantal content of e.p.ps. The At the time of maximum twitch augmentation, responses to acetyl- increase in quantal content was observed after 15 min and choline, carbachol and KCl were unchanged (data not shown). reached a plateau, which was about two fold of control, after

a A In r TFI C- IV mIlM b CTX 100 nM t- TEA 3 mM

TEA 0.25 mM CTX3 nM /K-Ca control

j3mV 3 mV

5 ms 5 ms /Na '/Na IbTX 100 nM c d CTX 100 mM

NTX nM 'K-Ca control 300 /K-Ca control

J4mV 'Na 5 ms 2ms /Na * Figure 2 Effects of the three toxins on extracellulary recorded nerve terminal action potentials. Voltage-dependent K+-channels were blocked by 400;LM 3,4-diaminopyridine to reveal deflection corresponding to a Ca2l-activated K+ current (IKC.)- (a) Tetraethylammonium (TEA) progressively blocks the IK-Ca to reveal a Ca2l-dependent plateau current. (b) Charybdotoxin (CTX) also blocks IKca and induces a plateau current when added cumulatively from 3 to 100 nM. (c) Iberiotoxin (IbTX) blocks IKCa and induces a plateau current. (d) Noxiustoxin (NTX) up to 300 nm did not affect the IKca. In the presence of NTX, CTx (100 nM) could still induce a plateau response. The peak of the waveform corresponding to the Na+ current is indicated by the arrows marked INa, * indicates the stimulus artefacts. H. Vatanpour & A.L. Harvey Scorpion toxins and transmitter release 1505

a d NTX 300 nM 40 min c I1 2 mV 2 mV 1 mV 2 ms 2 ms IFi2 ms

b NTX 300 nM 40 min NTX 300 nM 20 min IbTX 100 nM I Control

* I Figure 3 Effects of the three toxins on intracellularly recorded endplate potentials and miniature endplate potentials. (a) Superimposed averaged e.p.ps in the absence (control) and presence of charybdotoxin (CTX) at 100 and 300 nm. Inset: superimposed averaged m.e.p.ps in absence and presence of CTX (100 and 300 nM). (b) Superimposed e.p.ps before and at different times after the addition of 100 nm iberiotoxin (IbTX). Inset: superimposed averaged m.e.p.ps before and in the presence of IbTX. (c) Superimposed e.p.ps before and in the presence of 300 nM noxiustoxin (NTX). Inset: aligned averaged m.e.p.ps before and after 20 and 40 min exposure to NTX (from left to right). (d) Four successive nerve stimulations after 40 min exposure to 300 nM NTX to show the repetitive activity after the evoked e.p.p. * Stimulus artefacts.

1001 Charybdotoxin ZFTNVSCTTSKECWSVCQRL Iberiotoxin ZFTDVDCSVSKE.CWSVCKDL Noxiustoxin T I I NVKCTSPKQC S KPCKEL 0 40 -H N T S R G K C M N K K C R C Y S -F G V D R G K C M G K K C R C YQ YG SSAGAKCM N G KCKCYN N 0 0. Cel Figure 5 Amino acid sequences of charybdotoxin, iberiotoxin and noxiustoxin. Standard single letter amino acid abbreviations used: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine, K, lysine, L, leucine, M, methionine; N, asparagine; P. proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, pyroglutamic acid.

I . - **g.. *-. -. g.F l-10 10-9 10-8 10-7 10-6 10- Concentration (M) tions. CTX was able to compete with ['251]-toxin I for its binding sites on rat brain synaptosomal membranes (Figure Figwe 4 Displacement of specific binding of [125I]-dendrotoxin I 4). The IC", value for CTX was about 50 nM, and the from rat brain synaptosomal membranes: (0) native dendrotoxin I; apparent K, was 3.8 ± 0.43 nM (n = 6). However, CTx dis- (@) charybdotoxin; (*) noxiustoxin; (U) iberiotoxin. placed only about 70% of the specific binding of [125I]-DpI. IbTX, up to 3 gM, was not able to displace labelled dendrotoxin from its binding sites (Figure 4). As previously shown, 30-45 min. IblX was faster in onset and more potent than NTX at 100 nM completely displaces the labelled dendrotoxin crx. from its binding sites (Harvey et al., 1992). The Kj for NTX NTX (300 nM) produced a small increase in the amplitude was about 0.5 x 10-1 M. of evoked e.p.ps with no significant change in the other characteristics of e.p.ps or m.e.p.ps after 40 min (Figure 3c). However, after 40-50min exposure to NTX, single shock Discussion stimulation gave rise to repetitive firing of e.p.ps (Figure 3d). The effects of three homologous scorpion toxins CTX, IbTX, and NTX (Figure 5) were studied on both Ca2+-activated K+ Competition binding assay studies current and dendrotoxin-sensitive voltage-dependent K+ currents at mouse motor nerve terminals. These toxins contain ['25IJ-DpI exhibits saturable binding to the dendrotoxin recep- 37-39 amino acid residues, with over 44% identity in tor protein in rat brain synaptosomal membrane prepara- sequence, and they have similar 3-dimensional conforma- 1506 H. Vatanpour & A.L. Harvey Scorpion toxins and transmitter release tions. Despite their structural homology, the three toxins had terminals (Anderson et al., 1988). Although a previous study different effects on neuromuscular transmission. IbTx caused failed to find effects of CTX on acetylcholine release at an immediate increase in quantal content, while CTX pro- neuromuscular junctions (Anderson et al., 1988), the present duced a similar increase with a slower onset. Neither IbTx results demonstrate that long exposures to a high concentra- nor CTX caused repetitive firing. In contrast, NTX tion of CTX increase quantal content at a mouse neuromus- augmented quantal content to a lesser effect than did IbTx cular junction. Previously, CTX had been shown to facilitate and CTX, but induced repetitive e.p.ps in response to single acetylcholine release at frog neuromuscular junctions shock stimulation. An earlier study with NTX (Harvey et al., (Robitaille & Charlton, 1992). 1992) had failed to find effects on acetylcholine release in From the effects of the three scorpion toxins, it appears mouse diaphragm preparations; however, higher concentra- that both Ca2+-activated and voltage-gated K+ channels are tions were effective in the present study. involved in regulating acetylcholine release at the mouse The effects on acetylcholine release appear to correlate motor nerve terminal. Although blockade of each channel with the effects of the toxins on different types of K+ chan- type causes an increase in transmitter release, blockade of nels in neurones. NTX binds with high affinity to dendro- IK-Ca is faster (as has been seen with IbTX) and increases the toxin-sensitive sites on synaptosomal membranes, and dend- amplitude of e.p.ps evoked by single nerve stimulation. rotoxins are known to induce repetitive e.p.ps at neuromus- Blockade of voltage-gated K+ channel in contrast is slower cular junctions (Anderson & Harvey, 1988). NTX had no to affect transmitter release, and leads to repetitive firing (as effect on IKQca recorded at motor nerve terminals, although it seen with NTX). can block other Ca2+-activated K+ currents (Valdivia et al., 1988). IbTx had no effect on dendrotoxin binding or on voltage-dependent K+ currents at the mouse motor nerve We thank Drs F.A. De-Allie and P.N. Strong (Royal Postgraduate terminal. However, IbTx was a potent blocker of IK-Ca at the Medical School, London) for the gift of [1251]-DpI, and Dr C. Vita motor nerve terminal. CTX was less specific in its effects: it (DIEP, CEN Saclay, France) for the gift of iberiotoxin and charyb- has an allosteric interaction at dendrotoxin binding sites dotoxin, and Dr L.D. Possani (UNAM, Cuernavaca, Mexico) for the (Harvey et al., 1989), and it blocks IK-Ca at motor nerve gift of noxiustoxin.

References

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