Pharmac. Ther.Vol. 46, pp. 137-162, 1990 Printed in Great Britain.All rights reserved

0163-7258/90$0.00+ 0.50 © 1990PergamonPress plc

Associate Editor: A. L. HARVEY

POTASSIUM CHANNEL TOXINS P. N. STRONG Jerry Lewis Muscle Research Centre, Department of Paediatrics and Neonatal Medicine, Royal Postgraduate Medical School, London WI2 0NN, U.K. Abstract--Many venom toxins interfere with ion channel function. Toxins, as specific,high affinityligands, have played an important part in purifying and characterizing many ion channel proteins. Our knowledge of potassium ion channel structure is meager because until recently, no specific potassium channel toxins were known, or identified as such. This review summarizes the sudden explosion of research on potassium channel toxins that has occurred in recent years. Toxins are discussed in terms of their structure, physiological and pharmacological properties, and the characterization of toxin binding sites on different subtypes of potassium ion channels. CONTENTS 1. Introduction 2. A Family of Potassium Channels 3. A Family of Toxins 3.1. fl-Bungarotoxin 3.2. Dendrotoxins 3.3, MCD peptide 3.4. Inter-relationships between dendrotoxin, MCD peptide and fl-bungarotoxin 3.5. Apamin 3.6. Noxiustoxin 3.7. Charybdotoxin 3.8. Leiurotoxin 3.9. Other toxins 4. Conclusion Acknowledgements References

!. I N T R O D U C T I O N The toxins secreted by venomous animals, whether as an attack mechanism to immobilize prey or as a counter-defensive mechanism against predators, have long fascinated the biological scientist. As pharmacologists began to study the properties of crude venoms on isolated tissue preparations, and biochemists set about purifying the individual toxic components in the venoms, it became clear that many of these purified toxins were extremely potent molecules that blocked, both selectively and with high affinity, specific steps in a number of complex physiological processes. From these original studies, examining venoms and venom toxins for their own sake, the potential use of toxins as biochemical tools and pharmacological probes to study the physiologically important macromolecules with which these toxins interact, became apparent to a far wider scientific audience. For example, our knowledge of the structure of the acetylcholine receptor ion channel protein has been almost entirely due to the use of ~-bungarotoxin and other snake venom ct-neurotoxins to assay

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and purify the receptor. Similarly, tetrodotoxin, saxitoxin and many scorpion venom toxins have been instrumental in helping biochemists isolate and characterize the transmembrane protein that forms a specific sodium ion channel in nerve and muscle. The use of individual toxins as selective blocking agents allows the electrophysiologist to 'dissect' individual ionic currents in voltage clamp or patch clamp experiments. Most venom toxins appear to interfere with the function of ion channels and the development of our knowledge of ion channel proteins has, until recently, subsequently followed the isolation of specific channel toxins. The acetylcholine receptor ion channel protein was one of the first to be characterized because snake venom is relatively plentiful and cheap and the a-neurotoxins present in many snake venoms form a major component (up to 40% total protein for abungarotoxin in Bungarus multicinctus venom). Scientists then turned their attention to other venomous animals (notably scorpions) and this resulted in the isolation of many sodium channel toxins. However, the abundance of any one of these purified scorpion

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toxins is commonly on the order of 1-10%. Increasingly, more and more minor components from more and more exotic venomous creatures are being isolated, giving rise to toxins of new selectivity. For example, charybdotoxin (a blocker of calcium-activated potassium channels) is present in 0.2% abundance in the venom of the Israeli scorpion, and co-conotoxin, a calcium channel blocker, is isolated from the venom of marine hunting snails. Our knowledge of the biochemistry of potassium ion channel proteins is meager with respect to most other ion channel proteins, for the simple reason that until fairly recently, no specific potassium channel toxins were known, or identified as such. However, some previously well characterized toxins (at least from a structural and pharmacological point of view) have subsequently been shown to interact with various types of potassium channels at the molecular and cellular level (e.g. fl-bungarotoxin and dendrotoxin). Efforts to characterize increasingly minor venom components have recently led to the discoveries of many other potassium channel toxins (e.g. charybdotoxin, noxiustoxin, leiurotoxin); all those venom toxins that have been identified as affecting one or another type of potassium channel are the subject of this review.

2. A F A M I L Y OF POTASSIUM C H A N N E L S Potassium channels form a remarkably diverse group of ion channel structures (Rudy, 1988). The first type of potassium channel to be described was the classical voltage-activated (delayed rectifier) channel found in the squid giant axon (Hodgkin and Huxley, 1952). A second type of potassium channel is activated by a hyperpolarizing voltage (inward, or anomalous rectifier) and is often modulated by intracellular metabolites and second messengers (Kaczmarek and Levitan, 1987). In some cells (e.g. skeletal muscle), these channels determine the resting membrane potential (Adrian, 1969). A third class of potassium channels produce transient outward currents, or A-curents (Rogawski, 1985) and were originally described in molluscan neurons (Hagiwara et al., 1961). These channels are outward rectifiers and are inactivated at normal resting potentials; they can be distinguished from the delayed outward rectifier by differences in their voltage dependence and the former's insensitivity to tetraethylammonium ion. A-currents are thought to play a role in neurons that are spontaneously active or that fire repetitively in response to tonic depolarization. A fourth category of potassium channels are known as large conductance channels, BK channels or maxi-channels, with typical single channel conductance values often in excess of 200 pS (Marty, 1983). These are activated both by voltage and internal calcium ions and occur in both excitable and non-excitable cells. In neurons, calcium activated potassium currents contribute to spike repolarization and also aid in controlling the frequency of repetitive firing (Meech, 1978). In nonexcitable cells, they play an important role in controlling secretion (Petersen and Maruyama, 1984). The preceding classification of potassium channels is a biophysical one and more specifically, is a classification of ionic currents rather than channels

(see also Hille, 1984; Yellen, 1987), but it is also possible to characterize potassium channels on a pharmacological or regulatory basis (Cook, 1988). Some potassium channels are activated by neurotransmitters (e.g. serotonin; Siegelbaum et al., 1982), while others are modulated by a variety of specific intracellular agents such as ATP (Stanfield, 1987), second messengers (Belardetti and Siegelbaum, 1988) and G-proteins (Dunlap et al., 1987). Other potassium channels are activated by cations (e.g. sodiumactivated channels; Hartung, 1985) as well as calcium-activated channels mentioned earlier). More recently, potassium channels activated by changes in cell volume have been reported (Richards and Dawson, 1986). As indicated earlier, potassium channels are not restricted to excitable tissues such as nerve and muscle but are found in most secretory cells (chromaffin cells, pancreatic fl-cells, lachrimatory cells and salivary glands (see Petersen and Maruyama, 1984), as well as in kidney cells, fat cells, hepatocytes, lymphocytes and erythrocytes. With the cloning of more and more ion channel proteins, distinct family structures are emerging. Striking structural homologies have been identified between acetylcholine, GABA and glycine receptor channel proteins, forming a ligand-gated, ion channel family (Numa et al., 1983; Grenningloh et al., 1987; Schofield et al., 1987). Similarly, sodium and calcium channel proteins share different structural homologies and belong to a voltage-activated ion channel family (Noda et al., 1984, 1986; Tanabe et al., 1987). Electrophysiological studies on the Drosophila mutant, Shaker, have uncovered defective A-type potassium currents (Salkoff, 1983; Wu and Haughland, 1985) and recently several groups have shown that the Shaker gene locus encodes a family of voltagedependent potassium channel proteins (Baumann et al., 1987; Kamb et al., 1987; Papazian et al., 1987; Tempel et al., 1987; Schwarz et al., 1988; Pongs et al., 1988; Timpe et al., 1988). The structure of these proteins, as deduced from cDNA sequencing of the cloned Shaker gene and hydropathy analysis of the corresponding amino acid sequences, shows remarkable similarities with the dihydropyridine receptor and voltage-dependent sodium channels. Interestingly, low-stringency hybridization with Shaker cDNA clones has identified potassium channels in mammalian brain (Tempel et al., 1988; Baumann et al., 1988; Stfihmer et al., 1988). This suggests (maybe not surprisingly) that there are probably structural motifs common to different types of potassium channel, whether classified on a biophysical or a pharmacological basis. Further support for this idea is provided by a few examples given below, in which individual toxins appear to recognize more than one subtype of potassium channel. Although this review discusses individual toxins as specific markers for particular potassium channel subtypes, it is to be expected that in the future, increasing numbers of toxins affecting more than one category of potassium channel will be found. This might be dispiriting to those who wish to use one of these toxins as a specific means of identifying a particular potassium channel but should nevertheless stimulate others with new insights of structural relationships between potassium channel subtypes.

Potassium channel toxins 3. A F A M I L Y O F TOXINS 3.1. fl-BUNGAROTOXIN fl-Bungarotoxin, isolated from the venom of the Taiwan krait Bungarus multicinctus, belongs to a group of presynpatically acting neurotoxins (other members include crotoxin, notexin, taipoxin) that possess calcium-dependent phospholipase activity (Wernicke et al., 1975; Strong et al., 1976; Abe et al., 1977b; Howard and Gunderson, 1980; Strong, 1987). B-Bungarotoxin has two dissimilar chains, linked by a single disulfide bridge. The larger chain (Mr=13,500 Da) shares considerable sequence homology with pancreatic phospholipase A 2 and other snake venom phospholipase enzymes. The smaller chain (M r = 7000 Da) has some sequence homology with Kunitz-type trypsin inhibitors, although the toxin has no protease inhibitor activity. Reduction of the interchain disulfide bonds leads to a complete loss of activity and to date, it has not been possible to separate the two chains of flbungarotoxin in their native state and to demonstrate that either chain has any biological activity. Five isotoxins have been characterized and sequenced (Kondo et al., 1978, 1982a,b) and they conveniently fall into two groups based on sequence analysis. The two same subgroups can also be recognized on an immunological basis, using a panel of anti-B-bungarotoxin monoclonal antibodies (Strong et al., 1984). The properties of fl-bungarotoxin as a phospholipase A2 enzyme, are in most cases, indistinguishable from pancreatic phospholipase and another nontoxic phospholipase, also present in Bungarus venom (e.g. inhibition by active site directed inhibitors, activation by calcium and inhibition by strontium ions) (Strong, 1985, 1987). However, fl-bungarotoxin, in common with crotoxin, notexin and taipoxin, and unlike the vast majority of other phospholipase A2 enzymes, selectively inhibits the release of acetylcholine at the neuromuscular junction. At frog endplates, fl-bungarotoxin causes complete destruction of the motor nerve terminal (Abe et al., 1976; Strong et al., 1977). In the peripheral nervous system, this inhibition of transmitter release appears to be specific for cholinergic synapses. The mechanism of transmitter blockade by fl-bungarotoxin at frog nerve terminals (IC50 = 100 nM) is a complex process; there is a primary phospholipase-independent inhibitory phase, followed by a phospholipase-dependent stimulatory phase and subsequent complete inhibition of transmitter release (Abe et aL, 1977a; Abe and Miledi, 1978; Caratsch et aL, 1981, 1985). Similar effects are seen with both nerve-evoked and spontaneous transmitter release, although the time course of events produced by nerve stimulation occurs much more rapidly. In rat and mouse skeletal muscle nerve terminals however, the primary inhibitory phase is extremely small in comparison to a dominant, secondary facilitatory phase (Chang et al., 1973; Kelly and Brown, 1974; Oberg and Kelly, 1976a). To further complicate matters the secondary facilitatory phase seen in the mouse motor nerve terminal is not enzyme dependent (Chang, 1985) and interestingly in this regard, mammalian nerve terminals are not

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irreversibly disrupted by the toxin (Landon et aL, 1980). The facilitatory effects of fl-bungarotoxin at the mouse neuromuscular junction have recently been suggested to be due to an inhibition of potassium currents at motor nerve endings (Dreyer and Penner, 1987; Rowan and Harvey, 1988). Nerve terminal spikes were recorded with an extracellular microelectrode and the potassium current contribution to the observed perineural waveform was rapidly and irreversibly inhibited by fl-bungarotoxin (5-500nM) (Fig. 1). These effects were unchanged if strontium was substituted for calcium ions in the bathing medium suggesting that potassium channel block was independent of the toxin's phospholipase activity (Rowan and Harvey, 1988). The results also indicate that the toxin does not block calcium-activated potassium currents, which are suppressed in zero calcium conditions. Only a fraction of the potassium currents (defined by their susceptibility to block by 3,4diaminopyridine) were inhibited by fl-bungarotoxin. Other presynaptic phospholipase neurotoxins, at similar concentrations (but not nontoxic phospholipase enzymes, at concentrations up to 1500 riM), appeared to inhibit the same component of the potassium current as did fl-bungarotoxin. However,

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FIG, I. Top: Effect of fl-bungarotoxin on the perineural waveform of a mouse triangularis sterni preparation. (a) Control waveform; (b) waveform 45 min after addition of fl-bungarotoxin (0.15 #M). Similar waveforms were seen in the presence of calcium (2.5/~M), or in its absence. Bottom: Effect of 3,4-diaminopyridine and tetraethylammonium after exposure to fl-bungarotoxin. (c) After 0.2mM 3,4-diaminopyridine; (d) after 1 mM tetraethylammonium in the continued presence of the aminopyridine. (From Rowan and Harvey 1988.) Reprinted with the permission of the authors and the copyright holder, The Macmillan Press Ltd, Hampshire.

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these last observations are in contrast with other pharmacological and electrophysiological data which indicate a complete lack of mutual antagonism between fl-bungarotoxin and crotoxin, notexin and taipoxin (Chang and Su, 1980). Similarly, these other toxins have no effect on 3H-fl-bungarotoxin binding to rat brain membranes (see later). Interestingly,/~-bungarotoxin (up to 5000 riM) had no effect on perineural waveforms of frog motor nerves (Lee and Chang, 1966; Rowan and Harvey, 1988). The subtle differences in the manner in which fl-bungarotoxin inhibits transmitter release at amphibian as compared with mammalian nerve terminals have already been commented upon. One is forced to conclude that either frog nerve terminals do not possess E-toxin binding sites associated with potassium channels or else the toxin's effects on mammalian potassium channels is via an indirect mechanism, not directly associated with toxin binding. Further evidence supporting the idea that flbungarotoxin blocks mammalian potassium channels comes from a voltage clamp study on guinea-pig dorsal root ganglion neurons (Petersen et al., 1986) where it has been shown that the toxin (0.45-45 nM) selectively blocks a subtype of the noninactivating, slow outward potassium current. This current is also (and more completely) blocked by dendrotoxin (see below). Whether all the complex effects of flbungarotoxin on neurotransmitter release at the motor endplate can be attributed to a simple blockade of nerve terminal potassium channels, remains to be shown. fl-Bungarotoxin acts on the autonomic nervous system in a random fashion and in a species-dependent manner (Muramatsu et al., 1980; Miura et al., 1981), although only in the rat superior cervical ganglion has blockade been unequivocally demonstrated (Kato et al., 1977). fl-Bungarotoxin is also active in the central nervous system. Upon intraventricular injection, toxicity is increased by three orders of magnitude (Hanley and Emson, 1979; Othman et al., 1982). The toxin produces an irreversible blockade of neurotransmission in nerve terminals of the cerebellum, olfactory cortex and hippocampus, using both in vivo injections and incubation (200 riM) with isolated slice preparations (Halliwell and Dolly, 1982; Halliwell et al., 1982; Gulya et al., 1984). Using autoradiographic techniques on brain sections, ~25I-fl-bungarotoxin binding sites have been found enriched in grey matter areas and synaptic regions, in a manner consistent with the pharmacological data (Pelchen-Matthews and Dolly, 1988)./~-Bungarotoxin appears to inhibit a number of different transmitters from primary afferent systems, without affecting postsynaptic sensitivity to neurotransmitters such as glutamate and aspartate. In the developing chick retina, the toxin preferentially targets and destroys cholinergic and GABAergic cells in the amacrine and ganglionic cell layers, at concentrations as low as 0.1 nM (Rehm et al., 1982). There is also selective cell death of target neurons in chick embryos (Hirokawa 1977, 1978; Pittman et al., 1978) and this has led to the toxin being used as an experimental tool for general destruction of peripheral nerve axons, in studying their functional role during embryogenesis (McCaig et al.,

1987). However, not all developing cells show sensitivity to fl-bungarotoxin; in contrast, the cholinergically innervated neonatal rat diaphragm is less sensitive to fl-bungarotoxin than the adult rat diaphragm (Gundersen, 1981). Neuromuscular transmission in adult rat diaphragm innervated by 'amputated' motoneurons also shows a marked resistance to blockade by the toxin (Harris, 1976). Experiments with brain synaptosomes, using a wide range of fl-bungarotoxin concentrations (0.4-1000 nM), indicate that the toxin perturbs many membrane functions, including the enhancement of transmitter release (Wernicke et aL, 1975; Smith et al., 1980) and the inhibition of transport systems (Sen et al., 1976; Dowdall et aL, 1977). The toxin also induces the release of cytoplasmic markers from synaptosomes (Rugolo et al., 1986) and causes the depolarization and eventual collapse of the synaptosomal membrane potential (Ng and Howard, 1978; Nicolls et aL, 1985). Care, however, should be taken with the interpretation of some of these studies that use high toxin concentrations, when one considers that the KD for toxin binding to brain membranes is less than 1 nM (Othman et al., 1982; Rehm and Betz, 1982). Electron microscope studies using iodinated flbungarotoxin and horseradish peroxidase conjugated toxin (Esquerda et aL, 1982; Strong et al., 1977) provide evidence that the toxin itself might become internalized. Once inside the nerve terminal, the toxin attacks synaptic vesicles, most probably by hydrolyzing regions of disorganized phospholipid (Strong and Kelly, 1977), and uncouples both ATPase and acetylcholine transport systems within the vesicles (Anderson and Parsons, 1986; Noremberg and Parsons, 1986). Attempts to identify B-bungarotoxin-binding proteins have had a long and checkered history, primarily because of the difficulties in obtaining a biologically active radioligand of high enough specific activity. In light of more recent data (Othman et al., 1982; Rehm and Betz, 1982), it is likely that earlier studies detected low affinity binding sites (Oberg and Kelly, 1976b; MacDermot et aL, 1978). The best available data suggest that toxin binding sites on mammalian brain membrane homogenates are few ( B m ~ x = 5 0 - 1 5 O f m o l / m g protein) but of very high affinity (KD = 0.47-0.6 riM); binding is destroyed by pronase or bee venom phospholipase A2 but is insensitive to lectins (Othman et al., 1982; Rehm and Betz, 1982). The binding properties of different forms of labeled fl-bungarotoxin vary in their dependence on calcium; calcium induces a conformational change in the toxin (Abe et aL, 1977b; Ikeda and Hayashi, 1983) and therefore the stimulation of ~25I-flbungarotoxin binding by calcium (Rehm and Beth, 1982) probably reflects changes in toxin rather than in toxin-binding protein. On the other hand, 3H-flbungarotoxin, which binds in a calcium-independent manner (Othman et al., 1982) probably binds to a population of lower affinity sites. It is interesting to note in this regard that while the iodinated toxin retains full neurotoxicity, the toxicity of the tritiated derivative is reduced to one fifth of native toxin. Two populations of ~25I-fl-bungarotoxin binding sites, including one of low affinity (KD = 6 nM) can also be

Potassium channel toxins uniquely observed in imidazole buffers (Breeze and Dolly, 1989). Electrophysiological studies with native fl-bungarotoxin at the neuromuscular junction suggest that calcium is required for binding as well as for full expression of neurotoxicity (Caratsch et al., 1981, 1985). Dendrotoxin and toxin I, homologous, facilitatory presynaptic neurotoxins which also block potassium channels (Harvey and Karlsson, 1980; Harvey and Anderson, 1985) compete for flbungarotoxin binding sites (Othman et al., 1982; Mehraban et al., 1985; Black et al., 1988); the rather complex interaction between /3-bungarotoxin and dendrotoxin is discussed in Section 3.4. The other presynaptic phospholipase neurotoxins, crotoxin, taipoxin and notexin do not compete for fl-bungarotoxin binding sites (on brain membranes) even though they too have been shown to block potassium currents in motor nerve terminals. Triton X-100 successfully solubilizes the flbungarotoxin binding protein (Rehm and Betz, 1984; Black et al., 1988); interestingly, 100-250 mM potassium ions were required to stabilize toxin-binding activity. Gel filtration and sedimentation analysis indicated that the solubilized binding protein had a molecular mass of ~430,000 Da. The affinity of the toxin for its acceptor was marginally affected (KD = 0.5-2 riM) upon solubilization, and the acceptor protein retained its sensitivity to protease. Dendrotoxin also inhibited binding of fl-bungarotoxin to the solubilized component (Black et al., 1988). A fl-bungarotoxin binding protein of 95,000 Da has been identified using an arylazido-succinimidyl ester as a photoactivatable cross-linking agent (Rehm and Betz, 1983). Since the same toxin-binding polypeptide was identified under reducing and nonreducing conditions, it is unlikely that the solubilized binding component mentioned earlier is made up of identical Mr = 95,000 subunits, but rather the M r = 95,000 toxin-binding polypeptide is one subunit of a hetero-oligomeric complex. It is, however, unclear how the molecular weight of this binding protein relates to the different molecular weights obtained for dendrotoxin binding proteins (see Section 3.2). In summary, the consensus of these experiments is in agreement with the hypothesis (Kelly et al., 1979) that fl-bungarotoxin binds to a specific site (potassium channel?) on nerve terminals and there follows a highly selective permeabilization of the nerve terminal membrane in the immediate vicinity of this binding site. However, the relationship between the effects of fl-bungarotoxin at peripheral cholinergic nerve terminals and in the central nervous system is not clear because in the brain the effects of the toxin are not transmitter specific. Whether these discrepancies can be accounted for simply by invoking subtle differences in potassium channel structure remains to be ascertained. In addition to the existence of specific binding proteins, there is much evidence to suggest that the physical state of the membrane phospholipid may be an important parameter in determining flbungarotoxin specificity. Correlations between low cholesterol/phospholipid ratios and an increased susceptibility of membranes to attack by presynaptic phospholipase toxins have been observed biochemi-

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cally (Strong and Kelly, 1977; Napias and Heilbronn, 1980; Fischer et al., 1983). Electron microscopy studies using filipin as a cytochemical marker of sterols have shown that the presynaptic active zones at the neuromuscular junction are devoid of cholesterol (Nakajima and Bridgeman, 1981). Phospholipid head-group charge might also play a role in governing specificity; fl-bungarotoxin and crotoxin bind selectively to negatively charged phospholipid micellar structures, in distinction to other nontoxic phospholipases, which do not discriminate (Radvanyi et al., 1987). Thus, as well as invoking an infinite variety of voltage-sensitive, potassium channel genes to account for both the transmitter and species specificities of fl-bungarotoxin for selected nerve terminals, differences in the physical state of the phospholipid membrane, surrounding just one channel subtype, might provide an alternative explanation for the toxin's selectivity. 3.2. DENDROTOXINS Another group of snake toxins (dendrotoxins) which most probably act on potassium channels in presynaptic nerve terminals has been isolated from African mambas (Harvey and Anderson, 1985). Unlike fl-bungarotoxin, dendrotoxins enhance, rather than block, neuromuscular transmission. Dendrotoxin, isolated from the venom of the green mamba Dendroaspis angusticeps (Harvey and Karlsson, 1980; Joubert and Taljaard, 1980) has been the most intensively studied member of this group; other members include toxin I and toxin K from the black mamba Dendroaspis polylepis (Strydom, 1972), as well as three or four others. Dendrotoxin and its homologs all have 57~51 amino acid residues including six half-cysteines. All are highly homologous to Kunitz-type protease inhibitors, e.g. bovine pancreatic trypsin inhibitor (Dufton, 1985), although dendrotoxins have been reported to be unable to inhibit the protease activity of either trypsin or chymotrypsin. Similarly, protease inhibitors do not possess dendrotoxin-like activity nor inhibit radiolabeled dendrotoxin binding to brain membranes. Again in contrast to fl-bungarotoxin, the dendrotoxins are not phospholipase A s enzymes. This makes the dendrotoxins more useful than fl-bungarotoxin as specific tools for studying neurotransmitter release, since the enzymatic properties of the latter toxin can mask toxin-acceptor interactions by hydrolyzing phospholipids of the nerve terminal membrane (Strong et al., 1977; Strong and Kelly, 1977; Kelly et al., 1979; Rugolo et al., 1986). Dendrotoxin and its homologs increase acetylcholine released in response to motor nerve stimulation at the mammalian neuromuscular junction, without causing spontaneous contractions. There is both an increase in quantal content and the development of repetitive e.p.p.'s in response to a single stimulus. These effects have a slow onset (at least 1 hr for maximum augmentation) and are irreversible. Binding is independent of external calcium ions (Harvey and Karlsson, 1980; Harvey et al., 1984; Anderson and Harvey, 1985, 1988). Many of these effects are also seen with 3,4-diaminopyridine, but at

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FIG. 2. Channel blockade by dendrotoxin in an outside-out patch from cultured sensory neurons in physiological K + gradient. The 3 columns in each case represent the currents evoked by 4 successivejumps to - 3 5 mV from - 7 0 mV (leakage subtracted) made at 10sec intervals. The traces show K channel activity in control conditions (left), immediately following an application of dendrotoxin of less than I 0 sec duration (middle), and after washing for more than 30 min (right). Currents of 0.3 pA were reversibly suppressed. Sampling frequency, 400 Hz. (From Stansfeld and Feltz, 1988). Reprinted with the permission of the authors and the copyright holder, Elsevier Scientific Publishers Ireland, Ltd, Sjannon. one thousand-fold higher concentrations. Experiments monitoring nerve terminal activity with extracellular electrodes (Anderson and Harvey, 1988) indicate that dendrotoxins can decrease waveform components associated with potassium currents and also induce repetitive nerve terminal activation. Although these results described above are consistent with a direct effect of dendrotoxins on motor nerve terminal potassium channels, they are not conclusive. More compelling evidence has been obtained from studying the effects of dendrotoxins at the frog node of Ranvier. Dendrotoxin (0.1 riM) prolonged the duration of the action potential in a partial blockade of the potassium current but had no effect on sodium currents (Weller et al., 1985). Three separate nodal potassium currents (two fast and one slow) have been identified (Dubois, 1981) and one of the dendrotoxin homologs, toxin I, selectively inhibits the ft component of the fast (completely inactivating) potassium conductance, in a voltage-dependent manner (Benoit and Dubois, 1986), with an approximate KD of 0.4 nM. Toxin I's affinity for the f, component of the fast conductance or the slowly activating, noninactivating s component is three orders of magnitude less than for the fj component. Dendrotoxin and toxin I also affect transmitter release from the mouse vas deferens in a selective presynaptic manner (Anderson, 1985). There is no evidence for the toxins interfering with any of the postulated presynaptic feedback mechanisms that might possibly influence transmitter release. The major difference between these effects on the autonomic nervous system and those on the somatic motor system described earlier, is that effects on the former are more rapid and more readily reversible. Dendrotoxin blocks potassium currents in peripheral sensory neurons. In guinea pig dorsal root ganglion cells, the toxin (0.14--1.4riM) irreversibly blocked a part of the delayed noninactivating outward potassium current whereas the fast-inactivating potassium current in these cells was not blocked (Penner et al., 1986); this is in direct contrast to the effects at the frog node of Ranvier. In rat nodose ganglia, single A cells fired repetitively when stimulated in the presence of dendrotoxin (3-10nM). Voltage clamp studies indicate that the toxin reversibly blocks a slowly inactivating potassium

current (Stansfeld et al., 1986). The dendrotoxinsensitive current in dorsal root ganglia has recently been shown to have a maximum single channel conductance of between 5-10 pS in a physiological potassium gradient (Stansfeld and Feltz, 1988) (Fig. 2). Blockade appears to be by a direct action on the channel. Although the peripheral toxicity of dendrotoxin is not high (LDs0 = 23/~g/g mouse; Joubert and Taljaard, 1980), this increases ten thousand-fold upon direct administration to the central nervous system. Intracerebroventricular injection produces severe convulsions and death within 30rain (Mehraban et al., 1985; Silveira et al., 1988). Using a hippocampal slice preparation, and recording from CA1 pyramidal cells, the cellular basis for these convulsive effects has been studied (Dolly et al., 1984; Halliwell et al., 1986). Periods of enhanced synaptic activity alternated with periods of depressed activity, and bicuculline and picrotoxin-sensitive i.p.s.p.'s were enhanced by dendrotoxin. After i.p.s.p, block, toxininduced underlying e.p.s.p.'s were observed, sufficiently large to cause spontaneous firing of action potentials. These results are consistent with a toxininduced generalized increase in neuronal activity, affecting the release of both excitatory and inhibitory neurotransmitters. Using a single electrode voltage clamp on hippocampal pyramidal cells, relatively high concentrations of dendrotoxin (300 nM) caused a selective 70% block of the inactivating outward (A-type) potassium current. The toxin had no effect on calcium-activated potassium currents, mixed sodium/potassium currents and noninactivating (M type) potassium currents (Halliwell et al., 1986). The convulsive effects of dendrotoxins can therefore probably be ascribed to the inhibition o f a hyperpolarizing current which normally suppresses excitability. These seizures induced by dendrotoxin (as well as MCD peptide, see Section 3.3) can be inhibited by three different types of [,-type calcium channel blockers (Gandolfo et al., 1989a). Potassium channel inactivation has been proposed to be a significant factor in the action potential prolongation and facilitation of secretion which occur during high frequency firing in neurosecretory neurons (Bondy et al., 1987) and in support of this idea, dendrotoxin (1 riM) has been shown to enhance

Potassium channel toxins the electrically evoked secretion of vasopressin and oxytocin from isolated rat neurointermediate lobes (Bondy and Russell, 1988). What particular type of dendrotoxin-sensitive potassium channel this is, remains to be established. With the aid of the lipophilic cation, tetraphenylphosphonium, dendrotoxin has been shown to cause a slight depolarization of both rat and guinea-pig synaptosomes (Weller et al., 1985; Nicholls et al., 1985). The toxin causes release of both GABA and glutamate from synaptosomes in a calcium-dependent manner (Weller et al., 1985; Tibbs et al., 1989). All these results are analogous to those obtained with 4-aminopyridine and are consistent with the notion that dendrotoxin blocks some type of potassium channel in nerve endings. Using 86Rb+ flux through rat brain synaptosomes as an assay, dendrotoxin and three other homologs have recently been purified and characterised from D. angusticeps venom (Benishin et al., 1988). Dendrotoxin (100100nM) preferentially blocked an inactivating voltage-gated potassium channel, while two new toxin homologs (fl- and 7-dendrotoxin, 10-100nM) preferentially blocked a noninactivating voltage-gated potassium channel. Although nearly all available data indicate that the dendrotoxin family is specific for one or more different types of voltage-sensitive potassium channels, one anomalous report has indicated that dendrotoxin also slows sodium inactivation in Myxicola giant axons (ICs0=300n~), as well as affecting potassium currents (ICs0 = 150 nM; Sehauf, 1987). Dendrotoxin and its homolog toxin I can be successfully iodinated to a specific activity of 300-400 Ci/mmol without loss of biological activity, using either the chloramine-T method or one of its solidphase variants (Dolly et al., 1984; Harvey et al., 1984; Black and Dolly, 1986; Rehm et al., 1988). Autoradiographic studies show that ~25I-dendrotoxin binding sites are widely distributed in the brain, with high densities being observed in most grey matter (especially in synapse rich areas of the hippocampus and cerebellum) and along nerve tracts (Halliwell et al., 1986; Pelchen-Matthews and Dolly, 1989.) Labeled toxin binds to a single class of binding sites (KD = 0.3-1.1 riM; B,~,x = 0.51-1.17 pmol/mg protein) on both hippocampal and cerebral cortex membrane preparations from the rat (Black et al., 1986) and on brain cryostat sections (Pelchen-Matthews and Dolly, 1989). Dendrotoxin binding was not displaced by apamin, a blocker of calcium-activated potassium channels but was partially reduced by fl-bungarotoxin (see Section 3.4). Binding did not require calcium ions (an analogous situation to that which had previously been found at the neuromuscular junction) and was sensitive to proteases. With chick brain membranes, a second, lower affinity binding site for ~25I-dendrotoxin was found (KD= 15nM; Bm,x = 400 fmol/mg protein) in addition to the high affinity site. The toxin I homolog, on the other hand, completely inhibited 3H-/3-bungarotoxin binding to rat brain membranes (Othman et al., 1982) (see Section 3.4). Recently, two dendrotoxin binding sites have been identified in rat brain, using a flbungarotoxin affinity column (Rehm and Lazdunski, 1988a).

143

Cross-linking studies with dimethylsuberimidate have identified a 65,000 Da, 125I-dendrotoxin binding polypeptide in rat brain (Mehraban et al., 1984) and two binding polypeptides (M r = 68,000 and 75,000 Da) in chick brain. (A 76,000 Da peptide, as part of the solubilized toxin binding protein, has recently been identified in both bovine and rat brains, see later.) Dendrotoxin and toxin I binding sites can be solubilized in the presence of most detergents (Mehraban et al., 1985; Rehm et al., 1988; Black et al., 1988; Rehm and Lazdunski, 1988b) with retention of toxin binding and no loss of binding affinity (KD=0.084).4nM). Binding sites are remarkably stable (half life > 7 days at 4°C) in the presence of Lubrol PX or the zwitterionic detergent CHAPS (Parcej and Dolly, 1989). Omission of potassium ions from the solubilization medium results in a complete loss of specific binding, an analogous situation to that discussed previously with the solubilization of the fl-bungarotoxin binding protein. An estimate for the molecular weight of the solubilized dendrotoxin complex of M r = 405,000--465,000 Da has been obtained from gel filtration and sedimentation analysis (Black et al., 1988), and this compares with estimates of Mr = 240,000°265,000 Da obtained from radiation inactivation experiments on the toxinbound membranes (Dolly et al., 1984). Using sequential toxin I and wheat germ lectin affinity columns, a several thousand-fold enrichment of the solubilized putative channel protein has been obtained (Rehm and Lazdunski, 1988b). The specific activity of these preparations (0.4-1.6nmol 125I-toxin binding sites per mg) compares extremely favorably with the calculated value of 2.2 nmol/mg for the pure binding protein. The purified channel complex appears to be a multimeric protein; subunits of 76,000 Da, 38,000 Da and 35,000 Da have been identified (Rehm and Lazdunski, 1988b; Parcej and Dolly, 1989). Interestingly, the A-type potassium channel cloned from Drosophila contains peptides of 70,000 Da (Tempel et al., 1987) and 35,000 Da (Baumann et al., 1987). 3.3. MCD PEPTIDE MCD peptide (mast cell degranulating peptide, peptide 401) is a peptide isolated from the venom of the European honey bee, Apis mellifera. MCD peptide (M r = 2600 Da) is extremely basic: 7 out of 22 amino acids are either arginine or lysine residues and there are no aspartic or glutamic acid residues (Fig. 10). The peptide possesses two disulfide bridges and an amidated C-terminal carboxyl group. The structure of MCD peptide in solution has recently been determined by two-dimensional N M R spectroscopy and has been shown to have a conformation very similar to that of apamin, with an N-terminal fl-turn and a C-terminal ~t-helix (Kumar et al., 1988). MCD peptide was originally found to be a potent anti-inflammatory agent (ICs0 = 1/~g/g rat; Billingham et al., 1973) and to trigger histamine release from mast cells (Habermann, 1972), although it was later shown that much lower concentrations caused convulsions upon direct injection (10 ng/g mouse) into the brain (Haberman, 1977). The peptide can be readily iodinated and high affinity binding sites

144

P. N. STRONG

(KD = 50-150 pM; B m a x = 60-200 fmol/mg) have been described, both in rat brain (Taylor et al., ] 984), and more specifically, on hippocampal membranes (Bidard et al., 1987b). Other mast cell degranulating agents do not displace MCD peptide and most probably the ability of MCD peptide to trigger histamine release is rather nonspecific and is due to its polycationic structure, a feature shared by other degranulating agents. Selective chemical modification studies have also shown that neurotoxic and mast cell degranulating properties are not related (Banks et al., 1978). It is interesting to note that tertiapin, another bee venom peptide, highly homologous to MCD peptide but with no identified biological activity to date, can displace ~25I-MCD peptide from rat brain membranes (1(o5 = l O 0 - 1 5 0 n M , Taylor el al., 1984). MCD peptide initially produces long-lasting hippocampal theta rhythms associated with an increased level of wakefullness; this is followed by the induction of epileptic discharges, especially at higher doses (Bidard et al., 1987a). The epileptic discharges can, in turn, be inhibited by potassium channel openers such as cromakalim (Gandolfo et al., 1989b). The state of arousal produced by MCD peptide appears to be associated with a long-term potentiation of hippocampal synaptic transmission, which can be observed on application of the toxin (0.5-2/~M) to the CA1 region of hippocampal slices (Cherubini et al., 1987). The density of MCD peptide binding sites is very low during the perinatal period in rat brain, but increases rapidly one week after birth and thereafter maintains a steady rise for almost six weeks. During this period, there is a six-fold decrease in the affinity of the peptide for its binding site. The dramatic increase in the density of MCD peptide binding sites corresponds to the development of hippocampal theta activity and to the increased sensitivity of rat brain to MCD toxicity (Bidard et al., 1987a). The similar effects of convulsions and hyperactivity that are seen in rats injected with either dendrotoxin or MCD peptide suggest that both toxins might have

control

MCDP 3 0 0

125

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;- , I

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) ]

FIG. 3. Effects of MCD peptide and toxin 1 on specific peptide binding of ~251-toxin I and ~25I-MCD peptide to Triton X-100 solubilized extract of rat brain membranes. Binding 1:sI-toxin I (84pM) (top) or ~25I-MCD peptide (79pM) (bottom) to the detergent extract (270,ug/m[ protein) in the absence or presence of toxin I (O) and MCD peptide (O). (From Rehm et al., 1988.) Reprinted with the permission of the authors and the copyright holder, American Chemical Society, Washington, D.C.

similar cellular targets. Electrophysiological and biochemical experiments, as well as an autoradiographic study, show this to be the case. Dendrotoxin is a noncompetitive inhibitor of ~25I-MCD peptide binding sites (K0.~= 160 pM) on synaptosomal membranes (Bidard et al., 1987b). An MCD peptide-binding protein, M r = 77,000 Da (identical in size to a dendrotoxin-bindingprotein) has also been identified on rat brain synaptosomes. The dendrotoxin-binding protein discussed in the previous section maintained its ability to bind 125I-MCD peptide throughout various stages of purification, including

nM

recovery

i, . . . . . . . . . . . . . . . . . . . .

.,

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.

,

_

50 mV 2.5 nA ~150 ms FIG. 4(a). Intracellular recording from a nodose A cell showing the response to MCD peptide superfusion. Records (voltage lower and current upper) are superimposed oscilloscope traces showing the membrane response to depolarizing (upwards) or hyperpolarizing current passed through the recording electrode; resting membrane potential - 59 mV. In the control trace, the current is subthreshold for spike generation; yet in the presence of MCD peptide, continuous firing is generated in response to the same current. Recovery required washing for more than I hr and here it is still incomplete. (From Stansfeld et al., 1987.) Reprinted with the permission of the authors and the copyright holder, Pergamon Press, Oxford.

Potassium channel toxins

145

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FIG. 4(b). Current-voltage curves from a nodose A cell showing the effects of either MCD peptide or toxin I on the same cell and demonstrating that the action of toxin I can be occluded by MCD peptide. Measurements were made at the end of 1 sec voltage steps, Left: 300 nM MCD peptide reduces outward rectification at potentials positive to - 50 mV ( 0 ) and subsequent addition of 11 mM toxin I (C)) has little effect. Initial control curve (O). Right: Original control curve (Q). After 2 hr washing, 11 nM toxin I reapplied ((3). Relative to the control values, the changes produced by toxin I alone or together with MCD peptide are indicated by the shaded areas; these are essentially identical. (Adapted from Stansfeld et al., 1987.) Repinted with the permission of the authors and the copyright holder, Pergamon Press, Oxford. solubilization in detergent (Rehm et al., 1988) (Fig. 3). Toxin I (2 n~) also selectively inhibits ~25I - M C D peptide binding in the hippocampus. Qualitatively similar results have been obtained with autoradiographic studies on rat brain (Bidard et al., 1987b), Brain sections show a heterogeneous location of ~25I-MCD peptide binding sites; high densities of binding sites are found in areas rich in neuronal connections (e.g. pons, neocortex and parts of the hippocampus), while low densities are found in white matter and the granular area of the brain (Mourre et al., 1988). Electrophysiological experiments have shown that M C D peptide blocks (IC50 = 37 nM) the same fast activating potassium current in nodose ganglia as dendrotoxin (Stansfeld et al., 1987), albeit with a slightly lower affinity than dendrotoxin (IC50 = 2 nM) (Fig. 4). In contrast, both toxins (at concentrations up to 500 nM for M C D peptide) were unable to block outward transient potassium currents in superior cervical ganglion cells.

The most elegant proof that M C D peptide (and dendrotoxin) block a noninactivating delayed rectifier potassium channel has recently come from an experiment expressing potassium channel genes in Xenopus oocytes (Stiihmer et al., 1988). Using low stringency hybridization techniques with a Drosophila Shaker c D N A probe (see Section 2), a homologous c D N A was isolated from a rat cerebral cortex library. In vitro transcription of this isolated rat brain c D N A into m R N A and subsequent injection into Xenopus oocytes resulted in the expression of a functional potassium channel with delayed rectifier properties which was inhibited by both M C D peptide and dendrotoxin (Fig. 5). Using an M C D peptide antiserum as an assay, an endogenous MCD-like peptide has been purified from pig brain, 250 brains yielding 60 pmol of endogenous peptide, M r = 2900 Da (c.f. M C D peptide, M r = 2600 Da). The endogenous peptide inhibits L25IM C D peptide binding to synaptosomal membranes

-II

1.0 O8 x

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0.4 0.21 ii

, 0.1

1 I Inhibitor I I mM

10

/I

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10 [ Inhibitor]

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FIG. 5. Sensitivity of outward currents elicited after injection of RCKI cRNA into oocytes toward K channel blockers added to the bathing solution (normal frog Ringer's). (A) Concentration dependence of tetraethylammonium ( 0 ) and of 4-aminopyridine ( I ) block. (B) Concentration dependence of dendrotoxin (V) and of MCD peptide (A) block. Corresponding open symbols indicate the recovery of outward current when the bathing solution with the highest blocker concentrations was replaced with normal frog Ringer's. Peak currents at + 20 mV test potentials were 15.5 #A ((3), 15.7 # A (Zl), 5.1 # A ( ~ ) and 2.2 p A (A) respectively. (From St/ihmer et al., 1988.) Reprinted with the permission of authors and the copyright holder, Elsevier Science Publishers B.V., Amsterdam.

146

P. N. STRONG

(Cherubini et al., 1987). The role of this endogenous peptide in memory function becomes a fascinating speculation in the light of the effects of MCD peptide itself upon injection into the brain. 3.4. INTER-RELATIONSHIPS BETWEEN DENDROTOXIN, MCD PEPTIDE AND fl-BUNGAROTOXIN There is increasing evidence to suggest that there are mutual interactions between discrete binding sites for dendrotoxin, MCD peptide and fl-bungarotoxin. In some cases the physiological effects of one toxin can be antagonized by another (e.g. the effects of fl-bungarotoxin on both motor nerve terminals and brain synaptosomes can be partially inhibited by dendrotoxin; Harvey and Karlsson, 1982; Rugolo et al., 1986) (Fig. 6). However, it is clear that all these interactions are complex, both in terms of stoichiometry and competition. For example, although dendrotoxin can compete with fl-bungarotoxin and effectively displace all ~25Ifl-bungarotoxin from its binding protein, the efficacy of fl-bungarotoxin as an inhibitor of ~25I-dendrotoxin binding is rather poor, suggesting that there are two populations of dendrotoxin binding sites. Both forms of mutual inhibition are noncompetitive (Breeze and Dolly, 1989). Further evidence for heterogeneity in dendrotoxin binding sites can be seen in autoradiographical studies of ~25I-dendrotoxin binding to brain slices; fl-bungarotoxin can effectively block dendrotoxin binding sites in grey matter, but is much less able to block dendrotoxin binding sites in white matter (Pelchen-Matthews and Dolly, 1989). Two dendrotoxin binding sites have been identified in rat brain, using a fl-bungarotoxin affinity column (Rehm and Lazdunski, 1988a). One subtype (60-70% of dendrotoxin binding proteins) has a low affinity for fl-bungarotoxin (ICs0= 560riM) while the second

70

~

50

,o

Control, DTx

.

I

3'0

6o Time (min)

FIG. 6. Dendrotoxin protects synaptosomes against fl-bungarotoxin-induced plasma membrane depolarization. Synaptosomes were incubated in the presence of 1.3 mM Ca2+ and 160#M albumin. At 32min, 14nM dendrotoxin and 2.3 nM fl-bungarotoxin were added separately or in combination. (C)), control; (A), dendrotoxin; (I--1)/~-bungarotoxin; (A) dendrotoxin plus fl-bungarotoxin. (From Rugolo et aL, 1986.) Reprinted with the permission of the authors and the copyright holder, the Biochemical Society, London.

subtype (30-40% of dendrotoxin binding proteins) has a high affinity for fl-bungarotoxin (ICs0 = 16 riM). Although it is extremely difficult to provide an accurate estimate, present data would suggest that the ratio of high affinity binding sites for dendrotoxin: flbungarotoxin is approximately 3:1. MCD peptide inhibits 125I-fl-bungarotoxin binding to chick and rat brain membranes (K~= 180riM, 1100 nM respectively; Schmidt et al., 1988) in a noncompetitive manner and also inhibits the binding of 125I-dendrotoxin in a noncompetitive fashion. Similarly dendrotoxin and fl-bungarotoxin inhibit 125IMCD peptide binding (Rehm et al., 1988). The dendrotoxin binding protein (subtype with low affinity for fl-bungarotoxin) binds MCD peptide, while the second subtype (high affinity for flbungarotoxin) has negligible affinity for MCD peptide (Rehm and Lazdunski, 1988a). The estimated ratio of dendrotoxin:MCD peptide binding sites is 2:1. It appears that MCD peptide, dendrotoxin and fl-bungarotoxin probably all bind to the same ancestral voltage-sensitive potassium channel. Since each toxin can allosterically inhibit the binding of the others, it is likely that each toxin possesses a different binding site on the same macromolecular assembly (Schmidt et al., 1988; Rehm et al., 1988; Breeze and Dolly, 1989). Differences in voltage-sensitive potassium channel subtypes at the biophysical level will probably be reflected in the differences in affinity that these three toxins have evolved for an individual channel subtype and consequently subtle differences in their allosteric relationships. It is also fruitful to discuss structural relationships between the toxins themselves, in order to try and ascertain common themes that might be involved in binding to voltage-sensitive potassium channels. The sequence homologies between the B chain of flbungarotoxin and dendrotoxin and its homologs have been put forward to argue for the involvement of the B chain in toxin binding (Harvey and Karlsson, 1982) (Fig. 7). However, the loss of toxin binding on removal of calcium or by toxin modification with p-bromophenacyl bromide (Rehm and Betz, 1982), both disturbances to the toxin structure involving the A chain of fl-bungarotoxin, have been used as counter arguments. Minor sequence similarities between dendrotoxin homologs, MCD peptide and the A chain of fl-bungarotoxin exist (Schmidt et al., 1988) (Fig. 8); their significance has not been established. A comparison between dendrotoxin homologs and their nontoxic protease inhibitor counterparts indicates considerable similarities in the C-terminal regions of these proteins (residues 32-59) (Fig. 9). This suggests that the other N-terminal halves of these molecules may, determine neurotoxicity (Benishin et al., 1988). Of interest are the sequence homologies between the bee venom peptides, apamin, MCD peptide and tertiapin. Apamin does not compete for MCD peptide binding sites whereas tertiapin does (Taylor et al., 1984). All three peptides are structurally similar in their N-terminal regions, whereas at the C-terminal, MCD peptide and tertiapin share structural features not possessed by apamin. The two adjacent

147

Potassium channel toxins

Dendrotoxin Toxin I Bt

.,

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,,,

o,

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o o ,IyI,l

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-Bun~orotoxin BPTI F10. 7. Comparison of amino acid sequences of dendrotoxin, toxin I, B chain of flrbungarotoxin and bovine pancreatic trypsin inhibitor (BPTI) (see Anderson and Harvey, 1985). 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. Invariant residues boxed in.

Oendrotoxin I'~'lQ['fflI PA F Y Y N Q [ ' ~ , H, OF[~]WS ~' D~'~Iy GD A'¢~KK.IHIKr]N P K S, "~' MCDI~ptide I~C[NC~KRHVIK~JPIHIIC~RKIIGKN

~,-,ungorotox,n l~.lV H

FlG. 8. Comparison of partial amino acid sequences of dendrotoxin and A chain of flcbungarotoxin with that of MCD peptide (see Schmidt et al., 1988). See Fig. 7 for further details.

arginine residues in apamin (Arg 13, 14), critically dependent for toxic activity, are also in this C-terminal region (Fig. 10). 3.5. APAMIN Another toxin found in bee venom is apamin (Habermann, 1984). Its purification is an extremely complex and lengthy process (Gauldie et al., 1976) but procedures have been improved with the discovery that immobilized heparin can act as an affinity support to bind selectively both apamin and MCD peptide (Banks et al., 1981). Like MCD peptide, apamin is also extremely basic. It is an octadecapeptide (M r = 2000 Da), slightly smaller than MCD peptide but of remarkably similar structure, with two intramolecular disulphide bridges and an amidated C-terminal carboxyl group (Gauldie et al., 1976). Apamin is a remarkably stable molecule and its structure is insensitive to its environment (pH, salt effects, etc). Synthetic apamin (Cosland and Merrifield, 1977; Granier et al., 1978) folds correctly to exhibit native biological activity and CD structure, suggesting that information for the tertiary structure

is contained in the primary sequence. In the absence of any X-ray data, such properties have encouraged many theoretical studies of three-dimensional structure prediction, based on amino acid sequence (Hider and Ragnarsson, 1981; Freeman et al., 1986). Spectroscopic studies (nuclear magnetic resonance and circular dichroism) have also been used to predict the tertiary structure of apamin (Bystrov et al., 1980; Hider and Ragnarsson, 1980; Wemmer and Kallenbach, 1983; Dempsey, 1986) (Fig. 11). One consensus of all these studies is that apamin possesses an ~t-helical core with a pair of arginine residues (Arg 13, 14) protruding from the surface of the molecule. These two residues are essential for the maintenance of toxicity (Vincent et al., 1975) and immunological studies indicate that they are also part of the dominant epitope of the toxin (Kommissarenko et al., 1981). The only other chemical modification that completely destroys biological activity is perhaps not surprisingly, disulfide bridge reduction. All the structure-activity studies have been confirmed by synthesizing derivatives of apamin, using solid-phase methods (Cosland and Merrifield, 1977; Granier et al., 1978).

R F E T ' E E e RR T a--Dendrotoxin Z P R R~] L~'~l ~ p R N "~RI'~¥ D ~ - - - ~ A ~ Q M K ~ W ~ I q ~--Dendrotoxin- - A A Y K V R Y P K KIK I PISIF Y Y KIW[KIAIK Q C L P F DIYIS G C G G N A N R F K T I ~ E C R R T C V " FL~FIFS GC GG[N.IA[~IF,TIGE I--I [ B- Dendrotoxin P~L~]p: : . ~ G T--,E NS ~ ~ L Y--Dendrotoxin o ,,-El;-F o e o° I:EF-I, o,, o, FIG. 9. Comparison of amino acid sequences of dendrotoxin homologs. Horizontal lines indicate nonidentified residues (see Benishin et al., 1988). See Fig. 7 for further details.

~-~-

Apamin

MCDpeptide Tertlopin

-A P E T ^ L~AR R~QQ S-~ 2

I A L[C N CINL~-- I ~I]I P~_~MICIWIEIK[C G KIK

FIG. 10. Comparison of amino acid sequences of apamin, MCD peptide and tertiapin (see Habermann, 1984). See Fig. 7 for further details.

148

P.N. STRONG

~COOH 15

18

FIG. 11. Structural features of apamin based on nmr data of Wemmer and Kallenbach (1983). Reprinted with the permission of the authors and the copyright holder, American Chemical Society, Washington, D.C. Apamin is a centrally acting peptide neurotoxin that produces motor hyperactivity and convulsions; the toxin is remarkable for a peptide of its size, in being able to cross the blood-brain barrier, although it is three orders of magnitude more toxic when injected intravascularly (LDs0= 1.5pg/kg) as opposed to intravenously (LDs0 = 4 mg/kg). The mechanism of intoxication is independent of the route of injection (Habermann and Cheng-Raude, 1975). Although apamin was originally described as a centrally-acting neurotoxin, the target site for its highly specific action proved to be elusive until it was shown that nanomolar concentrations of the toxin blocked 'purinergic' inhibition on smooth muscle (Vladimirova and Shuba, 1978; Baidan et al., 1978). However, when the toxin was found also to block the inhibitory effects of noradrenaline on smooth muscle, it was quickly established that apamin blocked neither the P2-purinergic nor ~-adrenoceptors but

"I ..........

specifically the increase in potassium permeability which results from receptor activation and underlies the ensuing inhibition (Banks et al., 1979). Apamin has subsequently been shown to block receptor-mediated, calcium-activated potassium permeabilities (ICs0 = 1-7 riM) in intestinal smooth muscle (Maas et al., 1980; Shuba and Vladimirova, 1980; Hugues et al., 1982b; Weir and Weston, 1986) and hepatocytes (Burgess et al., 1981; Cook and Haylett, 1985). Although apamin does not affect the contractility of arterial or portal vein smooth muscle, apamin-sensitive, calcium-activated potassium channels are probably involved in blood pressure regulation. It is thought that the channels open during hormone receptor activation to hyperpolarize the membrane and thereby terminate vasoconstrictor responses by closing voltage-sensitive calcium channels (Coats, 1983; Cook and Hot', 1988). However, cromakalim, a recently discovered vasodilator drug which acts by opening smooth muscle potassium channels (Hamilton et al., 1986), has no effect on apamin-sensitive channels in either vascular or intestinal smooth muscle (Weir and Weston, 1986; Cook and Hof, 1988). Apamin also blocks calcium ionophore-induced potassium tracer efflux (ICs0 = 0.5-2 riM) in primary neuronal cultures (Seagar et al., 1984) and brown adipocytes (Nanberg et al., 1985). The slow afterhyperpolarization (AHP) in many cells, following bursts of action potentials, results from activation of calcium-dependent potassium conductances; apamin (0.5-100riM) has been shown to block AHPs in cultured rat myotubes (Hugues et al., 1982c) (Fig. 12), denervated rat skeletal muscle (SchmidAntomarchi et al., 1985), adult frog skeletal muscle (Cognard et al., 1984; Traore et al., 1986), both frog and rat sympathetic ganglia (Pennefather et al., 1985; Kawai and Watanabe, 1986; Tanaka et al., 1986; Goh and Pennefather, 1987), neuroblastoma cells and

0

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m 0.2sec

mV .~

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FIG. 12. Upper: Apamin blocks AHP in cultured rat muscle cells. Left: Reversal of slow AHP. The superimposed voltage traces show action potentials and after potentials evoked in a rat myosac during the passage of a steady hyperpolarizingcurrent of increasing intensity. Right: Same myosac, selective block of AHP after 10 min incubation with 10 nM apamin. Zero voltage line indicated. Lower: Voltage clamp analysis. Families of membrane currents associated with different step depolarizations from a holding potential of - 9 0 mV. Left: control currents. Right: currents 10 min after the addition of 10 nM apamin. The fast inward current is not affected; the slow outward current is strongly depressed. (,~) indicates peak inward Na + current. (Adapted from Hugues et al., 1982e.) Reprinted with the permission of the authors and the copyright holder, IRL Press, Oxford.

Potassium channel toxins neuroblastoma/glioma hybrids (Hugues et al., 1982e; Brown and Higashida, 1988), spinal motoneurons (Zhang and Krnjevic, 1987) and also neurosecretory neurons (Bourque and Brown, 1987). Most recently, the apamin-sensitive, calcium-activated potassium channel has been identified on both rat myotubes and guinea-pig hepatocytes in culture, using single channel recording techniques. The amplitude of single channel currents through apamin-sensitive channels, recorded from inside-out patches of rat myotube membranes, increases linearly with membrane potential ( - 2 0 to - 6 0 m V ) , giving a single channel conductance of 12pS in symmetrical (140 mM) potassium solutions. The half-maximal response to calcium was estimated to be between 0.2 and 0.5/~M Ca :+ (internal) (Blatz and Magleby, 1986). In guinea-pig hepatocytes (inside-out or outside-out patches), a single channel conductance of 20 pS was obtained in symmetrical high potassium solutions but this was reduced to 6 pS in the presence of a physiological potassium gradient (5mM KL ..... j135 mM Ki+ternal) (Capiod and Ogden, 1989). The reduction in unitary current on reducing Kexternal + suggests that besides an outward flow of potassium ions, there is a regulatory action of potassium on the external face of the channel. It should be noted here that apamin binding to both intact neurons and to brain membranes is stimulated by occupation of a potassium ion binding site and that the solubilized putative calcium-activated potassium ion channel protein has a critical requirement for potassium in order to retain its apamin-binding ability (see later). Apamin can be readily labeled with ~25Iby several variants of the basic chloramine-T procedure and a monoiodoapamin derivative (modified at His-18) can be purified (Hugues et al., 1982a). This labeled molecule is surprisingly stable for an iodohistidine derivative and is biologically active. Saturable, high affinity [125I]monoiodoapamin binding sites (K D = 10-400 pM) have been identified both on intact cells and on isolated membrane fractions from a variety of tissues, including liver (Cook et al., 1983; Strong and Evans, 1987; Marqueze et al., 1987), embryonic skeletal muscle (Hugues et al., 1982e), embryonic neurons (Seagar et al., 1984), neuroblastoma cells (Hugues et al., 1982c), smooth muscle (Hugues et al., 1982b), heart (Marqueze et al., 1987) and brain synaptosomes (Hugues et al., 1982a; Wu et al., 1985). Binding affinity is extremely sensitive to cations and is optimal in the presence of 5mM potassium ions (Seagar et al., 1984; Hughes, 1982a). Physiological concentrations of sodium ions inhibit apamin binding to membrane homogenates and this probably explains why there is such a great range of published dissociation constants; dissociation binding constants to intact cells in physiological, isotonic saline are an order of magnitude higher than similar constants determined on isolated membrane preparations in hypotonic solutions. One can speculate that it is the positive charges of two essential arginine residues on apamin that compete with these cations; interestingly, other molecules which have a similar spatial separation of two positive charges (e.g. tubocurarine, dequalinium and other bisquaternary neuromuscular blocking agents) can also compete effectively for [12sI]monoiodoapamin binding sites on

149

isolated hepatocytes (Cook and Haylett, 1985; Castle, 1987), as well as for similar binding sites on the solubilized apamin-binding protein from rat brain (Seagar et al., 1987b). These neuromuscular blocking agents can themselves also block calcium-activated potassium channels in smooth muscle and hepatocytes (Cook and Haylett, 1985; Gater et al., 1985). Although apamin has a very high affinity for its putative potassium channel acceptor protein, the density of binding sites found on all these former tissues is extremely low (Bmax < 30 fmol/mg protein), and up to two orders of magnitude less than that found for tetrodotoxin-sensitive sodium channels. This has made purification and characterization of the apamin acceptor protein a formidable task; however, the discovery that some PC12 pheochromocytoma cell lines over-express the acceptor protein (~ 600 fmol/mg protein) (Schmid-Antomarchi et al., 1986) suggest these cells might prove a useful source for isolation studies in the future. Three groups of apamin binding proteins (23,000°33,000 Da; 57,000059,000 Da; 85,000087,000 Da) have been identified by a variety of cross-linking procedures, using either chemical cross-linking or photoaffinity labeling. The first group (Mr= 33,000 Da) is seen by straightforward cross-linking with disuccinimidyl suberate (Hugues et al., 1982d; Wu et al., 1985; Schmid-Antomarchi et al., 1984) in the presence of protease inhibitors. Using photoactivatable apamin derivatives, modified with arylazide groups at either Lys-4 (e-amino) or Cys-1 (a-amino), the pattern of labeling was complex, being dependent on photolabile derivative and tissue labeled. Thus the ~-amino derivative labeled two proteins, (Mr= 33,000 and 22,000 Da) in both cultured neurons and brain membranes, whereas the ~-amino derivative labeled an 86,000 Da protein in cultured neurons and both the 86,000 Da protein and a third protein of Mr = 59,000 Da in brain membranes (Seagar et al., 1985, 1986). Photoaffinity labeling of heart, smooth muscle and liver membranes also identified an 85,000-87,000Da polypeptide. The 57,000-59,000 Da polypeptide was detected in liver but not in heart or smooth muscle (Marqueze et al., 1987). Since the 59,000 Da peptide was also found in astrocytes (Seagar et al., 1987a) but is not present in neurons, this suggests that the 59,000 Da binding component originally detected in brain membranes is of glial origin. How many of these identified polypeptides are bona fide components of a putative ion channel complex remains to be seen and the possible oligomeric nature of the channel is still unresolved. Initial attempts to purify apamin-binding proteins have used rat brain (Schmid-Antomarchi et al., 1984; Seagar et al., 1987b). Binding proteins are effectively solubilized in either sodium cholate or CHAPS detergents and binding constants for the solubilized protein (Ko = 25-40 pM) are similar to those found for the membrane-bound protein. The best preparations reported have binding capacities of 17 fmol/mg protein and a half life of 50 hr at I°C (Seagar et al., 1987b). From density gradient sedimentation analysis, the detergent/protein complex has an S2o, w of 20, which corresponds to Mr = 700,000 Da, although as much as half of this size could be contributed by detergent. Assuming a molecular weight of

150

P.N. STRONG

350,000 Da, the homogeneous protein should have a specific activity of 3 nmol/mg protein and therefore a purification factor of a further five orders of magnitude is necessary! The solubifized binding protein nevertheless still retains other pharmacological properties such as a requirement for potassium ions and the ability of neuromuscular blockers to compete for the binding site. Radiation inactivation analysis studies have also been employed to try and determine the molecular weight of the intact ion channel protein. Using [~25I]monoiodoapamin binding to lyophilized and rehydrated rat brain membranes, a value of M r=250,000Da has been obtained (SchmidAntomarchi et al., 1984), while using a frozen rat brain membrane preparation, the molecular weight of the ion channel protein has been estimated at M r = 84,000-115,000 Da (Seagar et al., 1986). Apamin-sensitive potassium channels have been shown to be regulated during differentiation and development. PC12 cells lose apamin-binding properties after the cells have been made to differentiate and sprout neuronal processes in the presence of N G F (Schmid-Antomarchi et al., 1986). In addition to apamin-sensitive channels being present in embryonic muscle cells in culture, apamin binding sites are also present in rat fetal muscle. They increase in utero until 15 days p o s t c o i t u m and thereafter there is a steady decline during subsequent maturation; binding sites have disappeared by the end of the first week of postnatal life (Schmid-Antomarchi et al., 1985). This disappearance during muscle development closely corresponds to the elimination of multi-innervation. If, however, adult rat muscle is denervated, apaminsensitive binding sites return two days post denervation and reach a maximum after ten days. Action potentials from denervated muscle fibres (in distinction to normally innervated fibers) are characterized by a following A H P (Thesleff and Ward, 1974) and this is also apamin-sensitive. Apamin binding sites can also be detected in adult human muscle under certain pathological conditions, e.g. myotonic muscular dystrophy (Renaud et al., 1986). This observation seems to be relatively specific--there is no evidence for apamin-binding sites on muscle biopsy samples associated with other muscular dystrophies or anterior horn disorders. Although it is presently unknown why apamin-sensitive potassium channels should be regulated in this manner and what are the neuronal factors that control this mechanism, it has been shown that, in liver cells, apamin-sensitive channels can be regulated by recycling from the cell surface to the cell interior, through a process resembling receptor-mediated endocytosis (Strong and Evans, 1987). The structures in the brain that are functionally affected after apamin intoxication remain unclear. Radiolabeled toxin binding sites are widely but unevenly dispersed in the brain (Habermann, 1984; Mourre et al., 1986), with high receptor densities lbund in the limbic system and to a slightly lesser extent in the motor system. Toxin binding sites are however absent in regions that are rich in myelin (Mourre et al., 1987a). The forebrain and basal ganglia seem to be relatively insensitive to microinjections of the toxin (Habermann and Cheng-Raude,

1975). The autoradiographic [~4C]deoxyglucose technique has recently been used in an ingenious attempt to provide further evidence for brain areas functionally disturbed after apamin intoxication, by measuring local cerebral glucose utilization (Mourre et al., 1987b). Unfortunately, the results have not been clear cut, with rates of energy metabolism increased in two thirds of the areas examined. Although glucose utilization did not correlate precisely with the distribution of apamin binding sites, certain limbic areas (notably the habenulo-interpeduncular tract and the interpeduncular nucleus) had both high levels of apamin binding and increased glucose utilization. It should be pointed out that these effects also differ with both the amount of apamin injected and the time after injection. Using an apamin radioimmunoassay (Schweitz and Lazdunski, 1984), apamin-like immunoreactivity has been discovered in pig brain (Fosset et al., 1984). With this assay, as well as a toxin-binding assay and the ability of test fractions to block the contractile activity of guinea pig taenia coli, an endogenous apamin-like molecule has been purified. The purified apamin-like molecule also inhibits the A H P in rat muscle cells in culture. The levels of this peptide are vanishingly small, estimated at less than 1.5ng (0.75 pmol) per pig brain. The role of an endogenous apamin-like molecule in the brain is intriguing. One would assume that regions of high apamin binding activity would probably also be regions containing the putative endogenous apamin molecule. In one of these regions, the hypothalamic supraoptic nucleus, neurohypophysial secretion of oxytocin and vasopressin is controlled by the electrical activity of magnocellular neurosecretory cells. These cells in turn, produce AHPs after bursts of action potentials and perhaps not surprisingly, the AHPs are blocked by apamin (Bourque and Brown, 1987). Thus, one function for an endogenous apamin-like molecule would be to control the secretion of oxytocin and vasopressin in the neural lobe (Bourque and Brown, 1987). Apamin has recently been shown to inhibit the electrogenic effect of the sodium/potassium pump (IC50 = 10 riM; Zemkova et al., 1988). The intriguing aspect of this study is that the toxin does not affect [3H]ouabain binding. It is therefore unlikely to affect the cation binding site and suggests that apamin might decrease the turnover of the pump. 3.6. NOXIUSTOXIN

All the toxins described previously are relatively abundant venom proteins or polypeptides which were purified and sequenced before their potassium channel blocking activity was discovered. In contrast, the remainder of the toxins discussed in this review are much less abundant proteins (often present at less than 0.5% of total venom protein) and they have been identified and subsequently purified on the basis of their pharmacological ability to block specific types of potassium channel. Although scorpion venoms are classic sources of sodium channel toxins, even some of the earliest studies with these venoms suggested that there might be toxins present which affected potassium channels

151

Potassium channel toxins C .100 mV

I K m A . c r n "2

FIG. 13(a). Selective action of noxiustoxin on potassium currents. Membrane currents associated with step depolarizations to 0 and + 100 mV before (C) and after (T) 13 min application of noxiustoxin (5/~M). Potassium currents are strongly depressed, while inward (0mV) and outward (+100mV) peak sodium currents are almost unaltered. Bars 2mA/cm2 and 2msec. (From Carbone et al., 1987.) Reprinted with the permission of the authors and the copyright holder, Springer-Verlag, Berlin. (Koppenhofer and Schmidt, 1968; Narahashi et al., 1972). The first scorpion venom shown to have a direct effect on potassium channels was the Mexican scorpion C e n t r u r o i d e s n o x i u s Hoffman, which reversibly blocked the delayed rectifier potassium current in the squid giant axon (Carbone et al., 1982). The major active component of this venom, noxiustoxin, has been extracted and purified by conventional gel exclusion and ion exchange chromatography (Possani et al., 1981b, 1982). Sequence studies revealed that noxiustoxin is a 39 amino acid polypeptide (Mr = 4,200 Da), containing six cysteine residues (Fig. 14), and structurally unrelated to the larger 57~56 amino acid sodium channel toxins found in scorpion venoms which possess eight cysteine residues (Possani et al., 1982). A noxiustoxin homolog has been isolated from the same venom and another homolog has been found in the venom of the Brazilian scorpion, T i t y u s serrulatus (Possani et al., 1981a). Purified noxiustoxin blocks potassium currents in the squid giant axon in a complex manner (Carbone et al., 1987) (Fig. 13). At concentrations less than 1.5/~M, block is independent of membrane potential, while at higher concentrations, toxin block becomes voltage-dependent and the toxin probably dissociates when the preparation is repetitively depolarized. These effects are similar to the voltage-dependent properties of some sodium channel scorpion toxins. Noxiustoxin is unable to block all potassium currents in the giant axon, suggesting that the toxin either allosterically inhibits channel function in a partial manner or else there are

FIG. 13(b). Effect of noxiustoxin on the instantaneous I-V relationship. I-V curves in 300 K-sea water in control (A) and after addition of 1.5 #M (O) and 3 #M noxiustoxin (I-q). At the bottom right-hand and top left-hand are shown potassium currents recorded before (C) and after (T) addition of 1.5/~Mnoxiustoxin to the bath. The fiber was left at its resting potential ( - 4 mV) and repolarized to - 7 0 mV, 2 min before applying a step depolarization to + 100 mV. Repolarizations to the voltages given on the abscissa. Bars: 2mA/cm2 and 2msec. (From Carbone et al., 1987.) Reprinted with the permission of the authors and the copyright holder, Springer-Verlag, Berlin. two subtypes of potassium currents, only one of which is toxin sensitive. Noxiustoxin has also been shown to block skeletal muscle T-tubular calciumactivated potassium channels incorporated into planar bilayers (IC50 = 450 riM; Valdivia et al., 1988). The toxin also affects potassium permeabilities in mouse synaptosomes, as indicated by its ability to stimulate [3H]GABA release and inhibit 86Rb+ efflux (Ki = 3 riM, Sitges et al., 1986). These results suggest that the toxin is much more potent (by two orders of magnitude) in blocking potassium currents in the brain as distinguished from peripheral potassium currents in squid axon or calcium-activated potassium currents. A synthetic nine amino acid peptide corresponding to the N-terminal sequence of noxiustoxin (see Fig. 14), has recently been shown to be lethal, either on intraperitoneal or intraventricular injection (Gurrola et al., 1989), although it is an order of magnitude less toxic than native protein (noxiustoxin lethal dose ~ 20 #g/g mouse). The peptide stimulates [3H]GABA release, at a similarly reduced potency. Another synthetic peptide, corresponding to the nine residues at the C-terminus of noxiustoxin was completely inactive in both assays. These results suggest that the N-terminal region of noxiustoxin (corresponding to 25% of the total linear sequence) is responsible for toxin interaction with voltage-sensitive potassium channels. This N-terminal sequence

Charybdotoxin Charybdotoxin-2 Noxiustoxin Leiurotoxin

A FC~N L - R HCLCJQL SCLC]RS L~-G~L L -[O~- K ~ I

G D K ~ E C~]V K a

FIG. 14. Comparison of amino acid sequences of charybdotoxin, charybdotoxin-2, noxiustoxin and leiurotoxin (see Possani et al., 1982; Chicchi eta/., 1988; Lucchesi and Moczydolowski, 1989). See Fig. 7 for further details. -K

152

P.N. STRONG

must show remarkable conformational independence from the rest of the molecule in order to maintain recognition for its biological target, considering the high structural constraints that must be placed upon the cysteine-rich native toxin.

3.7. CHARYBDOTOXIN The anomalously high single channel conductance values characteristic of certain calcium-activated potassium channels (100-250 pS) make these channels relatively easy to identify in planar bilayers and patch-clamped cells. Rat skeletal muscle T-tubular membranes incorporated into planar phospholipid bilayers contain these 'maxi-channels' and provide an elegant bioassay for screening venoms for toxins active in blocking these channels. Many scorpion venoms are indeed active in this assay (Miller et al., 1985), different venoms having a broad range of relative potencies and mean times of channel block (Moczydlowski et al., 1988). The first toxin to be isolated and characterized on the basis of blocking large conductance calcium-activated potassium channels was charybdotoxin, from the old world scorpion Leiurus quinquestriatus (Miller et al., 1985; Smith et al., 1986). Charybdotoxin is present in extremely low abundance, accounting for about 0.2% of total venom protein. It can be purified by conventional cationic ion exchange chromatography, making use of its extremely basic nature (pl > 10) followed by a final step on a reverse phase column. Charybdotoxin is another low molecular weight polypeptide (37 amino acid residues, M r = 4 , 3 0 0 D a ) with six cysteine residues and a blocked N-terminus (pyroglutamate); its sequence is

strikingly homologous to noxiustoxin (GimenezGallego et al., 1988) (Fig. 14). Charybdotoxin has no sequence homology with either apamin or with the dendrotoxins. There are evident similarities between the hydropathic pattern of charybdotoxin and the long chain snake ~-neurotoxins that irreversibly block acetylcholine receptor-operated ion channels at the motor endplate; a three-dimensional structure of charybdotoxin has been proposed based on the structure of one of these ~-neurotoxins, ~-bungarotoxin (Gimenez-Gallego et al., 1988). The biological significance of these structural similarities is unclear at present, since charybdotoxin has no effect on m.e.p.p. amplitude (Anderson, A. J. et aL, 1988) and is therefore unable to block endplate receptors. A second isoform of charybdotoxin has recently been isolated from the same venom (Lucchesi and Moczydlowski, 1989); it has a lower affinity in the planar bilayer assay. The amino acid sequences of the two isoforms differ in 8 out of the total 37 residues, although 4 of these 8 are isofunctional substitutions (Fig. 14). Most differences occur in the N-terminal half of the two molecules. In the same abstract, the properties of iodinated charybdotoxin isoforms have been reported (Lucchesi and Moczydlowski, 1989). Iodinated toxins have an impaired affinity in the standard planar bilayer assay (by a factor of ~ 50). [125I]Monoiodocharybdotoxin binds to rat muscle microsomes with a reduced affinity (Ko = 30-50 rim); only 50% of binding is displaceable with unlabeled toxin. Calcium-activated potassium channels are present in an extraordinarily wide range of cells. Other cells in which high conductance channels have been identified and which are blocked by low concentrations of

HP +50mV

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FIG. 15. Effect of charybdotoxin on calcium-activated potassium channels in an outside-out patch from a rabbit portal vein smooth muscle cell. Patch held at + 50 mV. The arrows mark the unitary current levels for the fully open state(s) of the channels and the continuous line marks the current when no channels are open. The block is reversible on washout. Signal digitized at 0.4 Hz. Note the presence of a second, small conductance channel (of unknown identity) which is not blocked by charybdotoxin. (From Strong et al., 1989.) Reprinted with the permission of the copyright holder, The Macmillan Press Ltd, Hampshire.

Potassium channel toxins charybdotoxin (2-50nM) include: cultured kidney epithelial cells (Guggino et al., 1987), rat aortic smooth muscle cells (Talvenheimo et al., 1988), dissociated rabbit portal vein smooth muscle cells (Beech et al., 1987; Strong et al., 1989) (Fig. 15), human cultured macrophages (Gallin, 1987), the pheochromocytoma cell line PC12 (Hoshi and Aldrich, 1986), the anterior pituitary cell line GHa (GimenezGallego et al., 1988) and the rat glioma cell line C6 (Tas et al., 1988; Reiser et al., 1989). The toxin also blocks, at similar concentrations, calcium-activated potassium currents in rat hippocampal neurons (Lancaster and Nicoll, 1987), bullfrog sympathetic ganglia (Adams et al., 1986; Goh and Pennefather, 1987), mouse and frog motor nerve terminals (Anderson, A. J. et al., 1987, 1988), as well as the calcium-activated potassium current in Drosophila longitudinal flight muscle that is abolished in the Slowpoke mutant (Elkins et al., 1986). The kinetics of charybdotoxin block (studied on channels identified in rat skeletal muscle plasma membranes inserted into planar bilayers) indicates that toxin binding to the channel is dependent on both the membrane potential and on the open or closed state of the channel (Anderson, C. S. et al., 1988), The on rate constant is seven times faster when the channel is open than when it is closed, whereas the off rate constant is the same. Increasing the ionic strength of the external medium with sodium or potassium ions from 20 to 300mM also selectively reduces the on rate, by a factor of one hundred whereas membrane depolarization increases the off rate, if the channel's open probability is maintained constant. Increasing the internal potassium ion concentration relieves the block achieved by externally applied toxin (MacKinnon and Miller, 1988) (Fig. 16). These studies have prompted the suggestion that charybdotoxin binds to a site near the external mouth of the channel, acting as a direct plug. The observed voltage dependence of toxin binding appears to be an indirect effect, the primary cause being voltagedependent binding of internal potassium ions. Further evidence for the location of the toxin binding site

153

comes from competition experiments with tetraethylammonium ions, the latter being known to bind to the external side of the channel (Villarroel et al., 1989). Since tetraethylammonium decreases the on rate of charybdotoxin in direct proportion to its blocking of the single channel current, this indicates that tetraethylammonium and charybdotoxin bind in a mutually exclusive fashion to the channel (Miller, 1988). Not all calcium-activated potassium currents blocked by charybdotoxin are of the high conductance type and not all high conductance potassium channels are blocked by charybdotoxin. For example, the toxin blocks a 35 pS calcium-activated potassium channel in various identified ganglia in Aplysia (Hermann and Erxleben, 1987) and also blocks calcium-activated potassium fluxes in human erythrocytes (Beech, et al., 1987; Wolff et al., 1988; Strong et al., 1989), which have a single channel conductance of 25 pS (Grygorczyk et al., 1984). Recently, multiple types of calcium-activated potassium channels have been identified in rat brain synaptosomal membranes inserted into planar phospholipid bilayers (Farley and Rudy, 1988; Reinhart et al., 1989). As well as blocking classical 'maxi-channels', charybdotoxin also blocked intermediate (75-140 pS) conductance channels. On the other hand, a slow-gating, large conductance (225 pS) channel was also identified in these experiments, that was not blocked by charybdotoxin (Reinhart et al., 1989). A large conductance, cromakalim-sensitive, calcium-activated potassium channel has recently been characterized in human vascular myocytes (Trieschmann et al., 1988); cromakalim-activated channels are most probably not blocked by charybdotoxin (Weir and Strong, 1988; Strong et al., 1989). None of the low conductance channels blocked by charybdotoxin are blocked by apamin; our present state of knowledge would suggest that the individual channels acted upon by these two toxins are mutually exclusive. There are probably, however, some structural homologies between high conductance calcium-activated potassium channels and certain voltage sensitive potassium channels•

ox (raM1 17

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20 pA 20= FIG. 16. Relief of charybdotoxin block by internal potassium. A single channel was observed at a holding voltage of + 30 mV. The solution on the external side of the channel contained 100 mM KCI and 5 nM charybdotoxin, while KC1 activity on the inside was increased, in a stepwise fashion, from 17 to 382 mM. Recordings displayed at low chart speed, so that individual opening and closing events cannot be discerned. (From MacKinnon and Miller, 1988.) Reprinted with the permission of the authors and the copyright holder, the Rockefeller University Press, New York.

154

P. N. STRONG

For example, 5 nM charybdotoxin blocks voltagegated potassium channels in subsets of developing T cells in mouse thymocytes (Lewis and Cahalan, 1988) and the toxin also blocks (KD = 3.6 riM)Drosophila Shaker A-type potassium channels expressed in Xenopus oocytes (MacKinnon et al., 1988). On the other hand, high concentrations of noxiustoxin (450 riM) block high conductance calcium-activated channels in rat skeletal T-tubules (Valdivia et al., 1988). 3.8. LEIUROTOXIN Leiurus quinquestriatus hebraeus scorpion venom contains other toxins which block potassium ion channels, distinct from those sensitive to charybdotoxin. Crude venom also blocks apamin-sensitive, calcium-activated potassium permeabilities in guineapig hepatocytes (Abia et al., 1986; Castle and Strong, 1986) and 86Rb+ efflux stimulated by the potassium channel opener, cromakalim in rat portal vein (Quast and Cook, 1988) (Fig. 17). Fractionation of crude venom has verified that the apamin-like toxin, active on blocking angiotensin II-stimulated potassium efflux from guinea-pig hepatocytes and able to inhibit [~25i]monoiodoapamin binding to the same preparation, is distinct from charybdotoxin (and indeed charybdotoxin does not block these apamin-sensitive ~ooo °

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channels in hepatocytes) (Castle and Strong, 1986; Strong et al., 1989). This apamin-like toxin is also structurally unrelated to apamin, since antibodies against apamin do not cross-react with any component of crude Leiurus venom (Castle and Strong, 1986). The toxin, present in the venom as 0.02% of total protein, has recently been both purified to homogeneity and sequenced (Chicchi et al., 1988). It is even smaller (M r = 3,400 Da) than charybdotoxin, with some homology to charybdotoxin and noxiustoxin (Fig. 14). Although leiurotoxin has little sequence homology with apamin, it completely inhibits [~25I]monoiodoapamin binding to rat brain synaptosomes (apparent Ki = 75 pM), although in a complex manner, affecting both K D and Bm~x. Like apamin, purified leiurotoxin blocks epinephrine-induced relaxation of guinea-pig taenia coli (IC50 = 6.5 nM), although it is 5-10-fold less potent (Chicchi et al., 1988). It is too early to ascertain how specific leiurotoxin is for apamin-like, low conductance calciumactivated potassium channels. Several chromatographic procedures, including a reverse-phase chromatography step, have not been able to separate leiurotoxin from the component in Leiurus venom which blocks cromakalim-activated potassium channels (Weir and Strong, 1988; Strong et al., 1989). 3.9. OTHER TOXINS The African scorpion, Pandinus imperator, also contains toxins active on potassium channels. Crude venom partially, but irreversibly, blocks voltage-activated potassium currents in frog myelinated nerve (but not in frog skeletal muscle) and in GH3 pituitary cells (Pappone and Cahalan, 1987; Pappone and Lucero, 1988). The venom also facilitates release of acetylcholine at the neuromuscular junction by selectively blocking extracellularly recorded potassium currents, sensitive to 3,4-diaminopyridine (Marshall and Harvey, 1989). The toxin(s) responsible for these effects have not yet been purified. The marine snails Conus have been shown to possess a large number of peptides that affect ion channels; conotoxins specific for muscle sodium ion channels and for neuronal calcium ion channels have been described (Olivera et al., 1985). Crude Conus striatus venom has been shown to block delayed rectifier potassium currents in Aplysia (Cheshunt, et al., 1987). The venom (and probably more than one toxic component) affects peak current, activation and inactivation kinetics. A screen of several Conus venoms for charybdotoxin-like activity on skeletal muscle has not yielded any positive results (Moczydlowski et al., 1988).

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FIG. 17. Effect of Leiurus quinquestriatus venom (LQV) on spontaneous activity (upper traces) and the rate constant, k, of 86Rb+ efflux (lower traces) in rat portal vein. A: Excitatory effect ofa 4 min superfusion with LQV (0.3 mg/ml) and subsequent washout of the effect. B: Inhibition of cromakalim (6/~M) stimulated 86Rb+ efflux by LQV in the presence of the dihydropyridine calcium antagonist PN200 ll0 (0.5/tM).

4. CONCLUSION The structural and pharmacological characterization of potassium channels at a molecular level has been slow to develop, primarily because of the lack of readily available toxins which specifically block potassium channels. Indeed, some toxins which had been purified and sequenced many years ago, have only just been recognized as having potassium channels as their primary cellular target. The ability to

Potassium channel toxins purify v e n o m c o m p o n e n t s representing less t h a n 0.5% o f total protein in the small a m o u n t s of crude v e n o m available, coupled with sophisticated a n d sensitive electrophysiological assay systems to characterize these toxins, have resulted in a d r a m a t i c increase in the n u m b e r o f such molecules which we n o w k n o w are able to selectively block potassium channels. As more a n d more v e n o m o u s creatures are studied, one can confidently predict t h a t this n u m b e r will multiply. It is b e c o m i n g increasingly clear t h a t individual toxins have a r e m a r k a b l e selectivity for different p o t a s s i u m c h a n n e l subtypes a n d one o f the most p r o m i s i n g aspects o f p o t a s s i u m c h a n n e l toxin research will be to use these toxins to study physiological a n d structural relationships between different m e m b e r s o f the p o t a s s i u m channel family. Acknowledgements--I thank Professor Alan Harvey for his comments during the preparation of this manuscript and the many colleagues who made available papers prior to publication. 1 also acknowledge the MRC, the Wellcome Trust and the Muscular Dystrophy Group of Great Britain for financial support.

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NOTE ADDED

IN P R O O F

The following full papers have appeared which amplify results referenced only as abstracts: GALLIN, E. K. and McKENNEY, L. W. (1988) Patch-clamp studies in human macrophages: single-channel studies and whole-cell characterization of two K ÷ conductances. J. Membr. Biol. 103, 55~56. Hosm, T. and ALDRICH, R. W. (1988) Voltage dependent K ÷ currents and underlying single K ÷ channels in pheochromocytoma cells. J. gen. Physiol. 91, 73-106. LUCCHESl, K. J., RAVINDRAN,A., YOUNG, H. and MOCZYDLOWSKI, E. (1989) Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca 2+ activated K ÷ channels. J. Membr. Biol. 109, 269 281.

Potassium channel toxins.

Many venom toxins interfere with ion channel function. Toxins, as specific, high affinity ligands, have played an important part in purifying and char...
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