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Home , Batrachotoxin, Histrionicotoxins, Neurotoxin

ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 5, No. 5 Copyright © 1975, Institute for Clinical Science

Mechanism of Action of

BARRY W. FESTOFF, M.D.

Medical Branch, National Institu te of Neurological Diseases and , National Institutes of Health, Bethesda, MD 20014

ABSTRACT This paper is a summary of studies over the past few years that pertain to animal neurotoxins. These are found throughout the animal kingdom. Homologies exist in the structures of these within classes and point to conservation of active sites throughout evolution. In the case of the peptides, invariant amino acids may be involved in the , be essential for maintaining the shape and conformation of the molecule or serve as a fulcrum for folding of the peptide chain after synthesis. At the nuclear or DNA-level, a constant base sequence may regulate gene operation sd that only a specific is coded. Physiologically, and with ultrastructural and biochemical correlation, the pre­ dominant mode of action of neurotoxins relate to one or the other of the major activities of the excitable ,—on conductile activity affecting Na+ or K+ per­ meabilities, on output or secretory activities affecting the release of neurotransmit­ ter or on input generator activities affecting the molecules for transmitter themselves. The future of these animal neurotoxins in neurobiological research is secure. The elucidation of molecular mechanisms, by which these various physio­ logical activities of excitable tissue are expressed, will surely involve one or more of these fascinating, naturally-occurring compounds.

Introduction or formation of analogs or metabolites A discussion of the effects of toxic agents bearing some chemical characteristics of the on the could easily occupy a native compound could all serve as great deal of time. In terms of ex­ mechanisms for effects. Addi­ posure, as a health hazard, all are becoming tionally, neural function, in vivo, could be acutely aware of the deleterious effects of impaired in a secondary or tertiary fashion inorganic and organic environmental by the effects of toxins on other systems pollutants. All age groups are at risk with in­ which may, in turn, impair its normal regu­ dustrial or accidental exposure in the high lation. risk bracket. Mechanistically, a toxic agent, Neurotoxins can be of mineral, or to be classified a neurotoxin, should express animal origin. In recent years, potent animal its adverse effects by interfering with some toxins have attracted steadily increasing at­ microphysiologic or biochemical reaction tention. New techniques have developed for generally specific for the nervous system. the isolation and purification of these Combination with neural receptor naturally occurring neurotoxic compounds molecules, blocking of enzyme active sites, which have resulted in a rapid rise in our

377 3 7 8 FESTOFF understanding of their . drophidae (sea-) have been widely These neurotoxic products, from a wide va­ used in research on transmission riety of animal sources, have become in the nervous system.34 valuable tools for neurobiological research. For this reason, this paper will describe Mechanism of Action some of the potent neurotoxins and the state To discuss the probable mechanism of of our knowledge concerning their mech­ action of a few select animal neurotoxins, it anism of action. Even with this restriction, would be profitable to separate sites of this presentation is only a short summary action both anatomically and physiologi­ and for further reading, proceedings of the cally. This is somewhat arbitrary but tends three recent major symposia and several to distinguish the various functions of ex­ review articles are strongly recom­ citable cells as outlined by Grundfest.26 mended.25,30,50 These functions, if not delineated geo­ Neurotoxins can be found throughout the graphically, can be separated effectively by animal kingdom. The cause of paralytic differing physiologic responses and prop- shellfish has been shown to be perties. In general, the , as the unit of caused by release of toxins by several pro­ activity of the nervous system, consists of tozoan species (expecially Gonyaulax ca- input or generator activity, conductile tenella) which accumulate in the siphons activity, and output or secretory activity. of the shellfish feeding on these dinoflag- The neurotoxins under consideration here ellates.49 In Arthropoda, the smallest have been shown to effect specific sites re­ neurotoxic peptide, , has been lating to these activities. They are, therefore, isolated from bee . A number of in their most predominant mechanism of neurotoxins have been isolated from various action: postsynaptic, axonal or presynaptic venoms15,42,46 and black widow neurotoxins. .15,37,44 In the , neurotoxic substances Axonal Neurotoxins have been shown in since antiquity. has been isolated in crystalline Tetrodotoxin (TTX), an amino-perhy- form from the puffer and its structure droquinazoline compound found in tissues elucidated.53 , such as , of fishes in the order toads and salamanders produce neurotoxins (puffer fish, , etc.) and in the California found largely in glands. Many of these newt, inhibits thè of nerve compounds are highly toxic steroidal al­ and muscle membranes.31,40 It has been kaloids and are used to dip arrows by shown that tetrodotoxin blocks the transient Indians of the Colombian rain forests. inward flow of sodium (blocks sodium (BTX) has been one such conductance) which prevents depolariza­ major toxic compound isolated.18 Recently, tion. (STX) having the same po­ another toxic compound, histrionicotoxin tency and action as TTX, is found in clams (HTX), has been shown to have apparently and mussels only when the shellfish have specific neural actions.4,5,17 been feeding on certain . It is In the , however, where socio­ responsible for the syndrome of paralytic logical interest has been present since .47,48,49 Since the conduc­ primeval times, the most widely known tile mechanisms ¡and properties of axonal neurotoxins are those isolated from the and muscle membranes are similar, it is not of poisonous snakes. In recent years, surprising that these neurotoxins have toxins found in snakes of the families Elap- similar effects on blocking both neuronal idae (cobras, , kraits, etc.) and Hy- and muscle action potentials. Recently, MECHANISM OF ACTION OF NEUROTOXINS 379 using 3H-labelled TTX partial characteriza­ external sodium concentration, increasing tion of a binding-component in membrane concentration, or by TTX.1 particles of unmyelinated garfish olfactory Recently, however, at peripheral adren­ nerve has been demonstrated.7,28 This ergic , it has been suggested that a component is presumed to be a protein from Leiurus quinquestriatus causes embedded in a phospholipid environment in transmitter release43 indicating a presynaptic the membrane. From previous studies, it had terminal mode of action. Earlier reports sug­ been shown that neither active Na+ gested this presynaptic mechanism at transport nor NaK ATPase activity31,38 is cholinergic neuromuscular junctions.19 This affected by TTX. type of mechanism has been determined for Batrachotoxin (BTX), a steroidal black widow venon (BWSV) by obtained from the , several laboratories.15,37,44 The specific toxin aurotaenia, causes of elec- has not been purified and analyzed as yet. Its trically-excitable membranes.3,7 BTX ap­ mechanism of action appears to be one of pears to exert its action on nerve and muscle interacting, somehow, with nerve terminal by irreversibly increasing permeability to membrane to affect “ avalanches” of trans­ Na+. Although both TTX and BTX influence mitter release until all stores are exhausted. permeability to Na+, both probably act on It is independent of Ca++ and terminal depo­ different receptor substances. It has been larization. Correlation of ultrastructure shown that denervated muscles become and physiological studies show depletion of insensitive to TTX but remain sensitive to presynaptic vesicles when no further release the action of BTX. It is possible, however, occurs. that denervation induces some structural or Venoms of several snakes contain toxins conformational changes in Na+ permeability which appear specific for presynaptic molecules which would dissociate sensitivity mechanisms. One such neurotoxin, isolated to these toxins. from the venom of the , Bun- garus multicinctus, has been named Beta- Presynaptic Neurotoxins (B-BuTX). This ca. 25,000 The most potent presynaptic neurotoxin is M.W. basic peptide appears to act exclu­ not of animal origin but is produced by the sively on the presynaptic side by inhibiting growth of a bacterium, Clostriduim bo- the release of (ACh).11,12 An tulinum. Mention is made here because in its initial burst of ACh release occurs and corre­ purified form, botulinus toxin is the most lates with depletion of synaptic vesicles.14 toxic biological substance known.51 Animal Recently, inhibition of Ca++ accumulation neurotoxins with a presynaptic mode of into rat mitochondria by B-BuTX has action can be found in scorpion venoms and been demonstrated.54 Since Ca++ influx is in the black widow spider venom as well as necessary for release of transmitter these from several venoms. authors conclude that the presynaptic action In Mexico, from 1940 to 1949 and 1957 to B-BuTX might be through alteration of 1958, 10 times as many people were killed by mitochondrial Ca++ . , scorpion stings than from snake bites.39 derived from the venom of the South Some 10 or 11 basic peptides have been American rattlesnake, is one of the first isolated from scorpion venom.42 These toxins neurotoxins isolated from .52 It have been shown to cause depolarization of is a complex of a non-toxic acidic protein nerve and muscle membranes,46 probably and a basic phospholipase. It is also some­ owing to increased sodium permeability. In what complex in its actions, having both pre- this respect they are similar to BTX since and post-junctional effects. It is of interest they are antagonized by either reducing the that presynaptic mechanisms predominate in 3 8 0 FESTOFF

junctions, while in mammalian neurotoxins in the majority of cobra species. systems the mechanisms are primarily Of the larger group, the best known are postsynaptic.10 alpha-bungarotoxin (alpha-BuTX) and the Recently, another snake toxin isolated cobrotoxin of N. naja naja and N. naja sia- from the Australian tiger snake (Notechis mensis. Chemically, the larger toxins, in ad­ scutatus) has been shown to block transmit­ dition to containing more amino acids and ter release.27 However, complete depletion the important fifth disulfide bridge, contain of vesicles was not found despite the physio­ more hydrophobic amino acids such as logical findings of an increase in miniature alanine, phenylalanine and valine.34 end-plate potential (MEPP) frequency and The larger and smaller curarimimetic then followed by a decline and ultimate neurotoxins differ in other respects. The cessation of MEPPs. These presynaptic affinity for the ACh receptor is greater for toxins isolated from scorpion venom, BWSV, the larger toxins than for the smaller. Alpha- and the several snake venoms all appear to BuTX binding has been referred to as support the vesicle hypothesis29 of synaptic “ essentially irreversible” .34 Antisera directed transmission. One must be cautious, how­ against one group is specific for the group ever, that although correlation of structure but doesn’t neutralize toxin from the other and function is of utmost importance in elu­ size group.9 Smaller postsynaptic snake cidating basic mechanisms, they may not be neurotoxins are also more susceptible to causally related. chemical degradation than are the larger ones.32 Postsynaptic Neurotoxins Utilizing labelled alpha-BuTX or larger In the search for receptor molecules in cobra toxins, receptor molecules have been neurobiology the use of postsynaptic-acting isolated, purified and solubilized from the snake neurotoxins has brought considerable electric organs of the eel (Electrophorus progress related to isolation and purification electricus) and the fish (Torpedo Spp). In ad­ of ACh receptors. As in the scorpion toxins, dition, the binding of 125I-BuTX to particu­ significant homologies exist in the structures late membranes and solubilized skeletal of these small basic peptides isolated from muscle membrane molecules identified as ni­ snakes of the families and Hy- cotinic cholinergic receptors by certain cri­ drophidae.13-3* These toxins are curarimi- teria, has been demonstrated by several metic but are more than 30 times as potent laboratories.6,8,23,24 Using a toxin-binding as . In spite of this, they are assay for identifying the receptor, anti­ significantly less toxic than the presynaptic receptor antibodies have been produced acting snake neurotoxins.33 They are small which resulted in a paralytic syndrome sug­ basic peptides tightly cross-linked by gestive of neuromuscular transmission disulfide bridges. The high degree of blockade.45 Recently, using 125I-BuTX, sep­ disulfide cross-linking accounts for stability aration of membranes enriched for acetyl­ at high temperatures or on exposure to 8 M (AChE) from the receptor urea.55 However, they are rapidly inacti­ was demonstrated in electric tissue21 and in vated by strong alkali and by reduction of membranes.24 The human the disulfide bonds. Within this class there disease myasthenia gravis (MG) has, at are two groups: smaller (Ca 7000 mol. wts.) times, been thought to be due to either a containing 60 to 62 amino acids and four presynaptic or postsynaptic defect. Suppor­ disulfide bridges, and larger (Ca 8000) with tive but not conclusive evidence for a 71 to 74 amino acids and five disulfides.34 postsynaptic mechanism has recently been Prototypes of the small type are the sea- reported. Muscle from biopsies of MG snake neurotoxins and the principal patients appeared to bind less 125I-BuTX MECHANISM OF ACTION OF NEUROTOXINS 38 1 than normals and disease controls.22 In ad­ garotoxin on neuromuscular transmission. Naunyn- Schmiedeberg’s Arch. Pharmacol. 282:129-142, dition, a serum globulin from MG patients 1974. has been reported to block 125I-BuTX finding 13. Chang, C. C. and Lee, C. Y.: Isolation of to solubilized, denervated (extrajunctional) neurotoxins from the venom of multicicntus and their modes of neuromuscular receptors but not to normal junctional blocking action. Arch. Int. Pharmacodyn. 144:241- receptors of rat membranes.6 It is, at pres­ 257, 1963. ent, not clear as to the significance of these 14. Chen, J. L. and Lee, C. Y.: Ultrastructural changes in the motor nerve terminals caused by B-bun­ findings in understanding the patho­ garotoxin. Vichows Arch. Abt. B. Zellpath. 6:318- physiology of the disorder. 325, 1970. 15. 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