Chapter 4
Insect Sodium Channel as the Target for Insect-Selective Neurotoxins from Scorpion Venom 1
1
1
2
E. ZlotMn , H. Moskowitz , R. Herrmann , M . Pelhate , and D. Gordon
1
Molecular Action of Insecticides on Ion Channels Downloaded from pubs.acs.org by TUFTS UNIV on 08/20/18. For personal use only.
1
Department of Cell and Animal Biology, Life Sciences Institute, Hebrew University, Jerusalem 91904, Israel Départment de Neurophysiologie, Facultéde Médecine, Université d'Angers, Angers F-49045, France 2
1. 2. 3.
4. 5.
The combined employment of protein chemistry, electrophysiology and neurochemistry enabled the chemical and pharmacological characterization of two classes of neurotoxin polypeptides, the excitatory and the depressant, derived from the venom of Buthinae scorpions which selectively paralyze and kill insects. These insect selective neurotoxins: Affect insect neuronal sodium conductance; Serve as unique and exclusive probes of the insect voltage gated sodium channels; Bind to these channels through multipoint attachment sites which include segments of external loops in domains I, III and IV of the insect sodium channel; Distinguish among sodium channels of different groups of insects; Are employed as pharmacological tools for the study of insect excitability and the design of future selective insecticides.
Insect Selective Neurotoxins Derived from Scorpion Venoms Venom Neurotoxins. Venom is defined as a mixture of substances which are produced in specialized glandular tissues in the body of the venomous animal and injected, with the aid of a stinging-piercing apparatus, into the body of its prey in order to paralyze it. The majority of the venomous animals (such as snakes, spiders, scorpions, venomous snails, various coelenterates) are slow, and even static predators which feed on freshly killed prey of mobile and relatively vigorous animals. The locomotor/ inferiority of the venomous predator is largely compensated by the neurotoxic components of his venom, the neurotoxins, which are able to induce a rapid paralysis of the prey at a very low range of concentrations (10" -10" M). 9
12
NOTE: Abbreviations of scorpion toxin nomenclature are explained in the Figure 2 caption.
0097-6156/95A)591-0056$14.50A) © 1995 American Chemical Society
4.
ZLOTKIN E T AL.
Insect-Selective Neurotoxins from Scorpion Venom 57
In this context a neurotoxin is defined as a substance which interferes with the function of excitable tissues due to a specific 'recognition' through high affinity binding to given sites in these tissues (7). A very common characteristic of venom neurotoxins is their polypeptide nature. This chemical characteristic has a double advantage for the survival of the venomous animal. First, polypeptides are the most readily available structures for adaptive modifications by genetic mutations and selection. Second, polypeptides, through their high diversity of covalent structures and the resulting tridimensional conformations, may reveal a highly diverse array of functional specificities. When classified according to effect and site of action in the nervous system, venom neurotoxins are commonly divided into ion channel toxins which modify ion conductance, presynaptic toxins which affect neurotransmitter release, and postsynaptic toxins which block the neurotransmitter receptors (2). Each of these categories can be further classified according to more specific criteria. For example, the ion channel toxins are subdivide into sodium (such as the below-mentioned scorpion venom toxins), potassium and calcium channel toxins (2,77). Furthermore, some of the neurotoxins are able to distinguish between subtypes of ion channels such as the (D and \i venomous snail conotoxins which distinguish between the voltagesensitive axonal and muscle calcium and sodium channels, respectively (3). Where the vital prey-predator relationships serve as a target of preference for evolutionary selective pressure, additional specificities occur such as the animal group-selective toxins. Animal Group Specificity of Scorpion Venom Neurotoxins. Animal group specificity refers to the phenomenon where an animal venom and/or its derived toxins reveal toxicity to a given group of organisms but are not effective to other groups of organisms. In cases where a venomous organism feeds on a given and limited group of animals, the animal group specificity is manifested already on the level of the whole venom. For example, the venomous marine Conidae snails are subdivided according to their feeding habits into fish, mollusc and worm eaters. A series of early bioassays (4,5) showed that the above feeding preference is associated with the specific toxicity of their venoms aimed at the respective groups of prey animals. On the other hand in the venom of animals, such as scorpions, spiders, marine nemertine worms and sea anemones, the animal group specificity is revealed in venom fractions and purified toxins (1,6-8). In the past, when the study of Buthinae scorpion venoms was directed mainly by clinical and public health aspects, lethality to laboratory mice served as the exclusive method to monitor their fractionation and purification (9,10). This has resulted in the purification and chemical characterization of the so called a neurotoxins (Figure 2) responsible for strong toxicity to mammals and human envenomation (9-11). Zooecological considerations concerning the feeding and hunting behavior of scorpions in the field has motivated the choice of arthropods as test animals in
58
MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS
monitoring the fractionation of scorpion venom. Such an approach coupled with molecular exclusion column chromatography and starch gel electrophoresis enabled the isolation of factors from buthid scorpion venoms which specifically affect insects, vertebrates and Crustacea (12-14). The specific symptomatology revealed by blowfly larvae to the injection of various fractions of scorpion venom enabled the distinction among three categories of neurotoxins which affect insects: The first are the excitatory insect selective neurotoxins which induce in blowfly larvae an immediate fast and reversible paralysis (Figure 1 A). The second are the depressant insect selective neurotoxins which cause a slow progressively developing flaccid paralysis (Figure IB). The third group are the a toxins which include the extremely toxic neurotoxins to mammals (such as the AaH2, Figure 2), some of which possess a certain toxicity to insects (77, 18) and others (such as the LqhalT, Figure 2) are strongly, but however not exclusively, toxic to insects (16). The LqhalT induces in the blowfly larvae a syndrome defined as a delayed and sustained contraction paralysis (Figure 1C). It is noteworthy that the selective-exclusive neurotoxicity to insects of the excitatory and depressant toxins was established on three experimental levels: the intact animal, the neuromuscular preparation and binding assays to various neuronal and non-neuronal membranes (73). Chemistry of Scorpion Toxins: Scorpion toxins were isolated and purified by the aid of various methods of low and high pressure column chromatography and characterized by electrophoresis, amino acid composition and sequence analyses (16,19). Figure 2 demonstrates the primary structures of representatives of the three groups of toxins derived from the venom of Buthinae scorpions and, for comparative purposes, one toxin (Ts7) which represents the so-called P-toxins derived from scorpions from Central and South America. As shown (Figure 2), scorpion toxins are single chained polypeptides of about 6570 amino acids, including 8 cysteins of similar allocation and are all involved in disulphide bridges (16,19) (Figure 2). Effects on sodium conductance Neuromuscular Effects. The above (Figure 1) and other (18) symptomatological observations suggested that scorpion toxins are neurotoxic and paralyze the insects by affecting their skeletal musculature. Studies on various neuromuscular preparations (17,20,27-29) revealed that the insects skeletal muscles are indirectly affected through a presynaptic action (17,28,29) (Figure 3 A) on the peripheral branches of the motor neurons (17,30). This aspect is clearly exemplified in Figure 3 A revealing a synchrony between the axonal (action potentials) and muscular (junction potentials) activities induced by the AalT. In other words, the train of the junction potentials is caused by the repetitive firing of the motor axon (77) induced by the excitatory toxin. It has recently been shown (20) that the depressant insect selective neurotoxins induce in blowfly larvae a short transient phase of contraction preceding the prolonged flaccid paralysis. A study on a prepupal housefly neuromuscular
ZLOTKIN ET AL.
Insect-Selective Neurotoxins from Scorpion Venom
Figure 1. Responses of Sarcophaga falculata blowfly larva to the injection of various scorpion venoms and their derived insect toxins. A . A fast, immediate (seconds) and reversible contraction paralysis induced by excitatory insect selective neurotoxins such as AalT and LqqITl. B . Typical progressively developing (minutes) flaccid extended paralysis induced by depressant insect selective toxins such as BJIT2, LqqIT2 and LqhIT2. Bar corresponds to 3.8 mm. Taken from (75). C. Response at various time intervals to injection of a paralytic dose (PU =28ng) of a fraction including the LqhalT toxin, (a) Before injection; (b) 1 min after injection - mobile and without detectable change in body forms; (c) 5 min - obvious contraction and immobility; (d) 8 min - fully contracted and paralyzed; (e) 20 min - still contracted but with partial recovery of mobility. Taken from (76). 50
60
MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS
AMINO ACID SEQUENCES 1 2 3 4 5 6 7 0 0 0 0 0 0 0 LqhITZ ...DGYIKRR DGCKVACLIG NEG.CDKECK AYGG.SYGYC ...WTWGLAC WCEGLPDDET WK..SETNTC G LqqIT2 ...DGYIRKR DGCKLSCLFG NEG.CNKECK SYGG.SYGYC ...WTWGLAC WCEGLPDEKT WK..SETNTC G BjITZ ...DGYIRKK DGCKVSCIIG NEG CRKECV AHGG.SFGYC ...WTWGLAC WCENLPDAVT WK..SSTNTC G AalT .KKNGYAVDS SGKAPECLLS N..YCNNQCT KVHYADKGYC CLL SC YCFGLNDDKK VLEISDTRKS YCWTIIN LqqITI .KKNGYAVDS SGKAPECLLS N..YCYNECT KVHYADKGYC CLL SC YCNGLSDDKK VLEISDARKK YCDFVTIN LqhalT .VRDAYIAKN YNCVYEC.FR DA.YCNELCT KNGASS.GYC QWAGKYGNAC WCYALPDNVP IR...VPGKC R Lqq4 GVRDAYIADD KNCVYTC.GS NS.YCNTECT KNGAES.GYC QWLGKYGNAC WCIKLPDKVP IR...IPGKC R Lqq5 .LKDGYIVDD KNCTFFC.GR NA.YCNDECK KKGGES.GYC QWASPYGNAC WCYKLPDRVS IK...EKGRC N AaH2 .VKDGYIVDD VNCTYFC.GR NA.YCNEECT KLKGES.GYC QWASPYGNAC YCYKLPDHVR TK...GPGRC H Ts7 ..KEGYLMDH EGCKLSCFIR PSGYCGRECG .IKKGSSGYC AWP AC YCYGLPNMVK VWDRA.TNKC
PERCENT IDENTICAL RESIDUES LqhIT2 depressant depressant depressant excitatory excitatory a-insect a-mammaI a-mammal a-mammal p-toxin
-
LqhITZ LqqIT2 BJIT2 AalT LqqITI LqhalT Lqq4 Lqq5 AaHZ Ts7
100
LqqITZ
BJIT2
AalT
LqqITI
87 100
79 79 100
30 30 25 100
31 30 28 87 100
LqhalT
Lqq4
Lqq5
AaHZ
Ts7
34 39 38 27 25 100
38 38 38 29 29 75 100
43 44 43 33 33 56 83 100
38 39 39 34 34 59 83 78 100
39 41 41 31 33 38 38 43 44 100
Figure 2. Comparison of scorpion toxin amino acid sequences. The depressant insect selective toxins affecting insects, namely LqhIT2, LqqIT2, BJIT2 (20) are compared to excitatory toxins (AalT (21); LqqITI (22)). As shown the a toxin affecting insects (LqhalT (16)) closely resembles the a toxins affecting mammals (Lqq4 (23); Lqq5 (24) and AaH2 (25)). Ts7 is a P-toxin isolated form a South American scorpion (26). Abbreviation of scorpion venom nomenclature: Aa - the North African scorpion Androctonus australis Hector; Bj - the Israeli black scorpion Buthotus judaicus, Lqh - the Israeli yellow scorpion Leiurus quinquestriatus hebraeus; Lqq - the African scorpion Leiurus quinquestriatus quinquestriatus; Ts - the Brazilian scorpion Tityus serrulatus.
ZLOTKIN ET AL.
Insect-Selective Neurotoxins from Scorpion Venom
A
Figure 3. Neuromuscular effects of the excitatory (A) and depressant (B) insect selective neurotoxins. A . The excitatory toxin AalT (125 nM) was applied to the locust hindleg extensor tibiae preparation. Simultaneous recording form the motor nerve (upper trace, showing a train of action potentials) and muscle (lower trace, showing a train of junction potentials produced by an activated muscle). Calibration: 50 msec; upper 0.15 mV, lower 10 mV. Taken from (77). B. The depressant insect toxin LqhIT2 (40 nM) causes a brief phase of repetitive motor neuron activity (b) followed by gradual suppression (b,c,d) of excitatory junction potentials (a) in prepupalM domestica. Upon removal of toxin with a saline wash, repetitive activity reappears again only briefly (trace e) and the amplitude of the EJP partially recovers over a period of 30-60 min (trace f). Vertical calibration is 20 mV; horizontal calibration is 30 ms. Taken from (20).
62
MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS
preparation revealed that both phenomena, transient contraction and the prolonged flaccidity, follow from a presynaptic effect of the toxin. The effect of the depressant LqhIT2 toxin on a prepupal housefly neuromuscular preparation mimics the effects of the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission (Figure 3, 20). Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AalT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin (20). To summarize, the neuromuscular studies have pointed out that the insect neuronal membrane is the target of both the excitatory and the depressant insect selective toxins. This conclusion, which was also supported by binding studies (31,32) and microscopical autoradiography (30), has directed our attention to an isolated insect axon as an experimental preparation (37) to study the mode of action of the scorpion toxins (16,33,35,36). Studies on an Axonal Preparation. The isolated giant axon from the central nervous system of the cockroach Periplaneta americana in both current and voltage clamp experiments, using a double oil-gap, singlefibertechnique (37) was employed. Figure 4 presents a current clamp experiment which demonstrates the effects of the excitatory and depressant toxin on the action potentials of the cockroach giant axon, the excitatory toxin induces a small depolarization (Figure 4b) followed by either induced (Figure 4c) or spontaneous (Figure 4, upper trace) repetitive firing. The depressant toxin (BJIT2, Figure 4d-g) induces an obvious depolarization accompanied by a reduction of the action potential amplitude (Figure 4d,e) up to a complete blockage (Figure 4f). As shown (Figure 4g) the depolarization is prevented, in a reversible manner, by saxitoxin, indicating that it follows from an effect on sodium conductance. This depolarization, however, is not the cause or at least not the only cause for the action potential blockage (see below). Figure 5 presents voltage clamp experiments which demonstrate the effects of the excitatory (AalT) and depressant (LqhIT2) toxins on the sodium currents in the giant cockroach axon. The excitatory toxin does not effect the potassium current (Figure 5B). However, as revealed in Figure 5 A, AalT increases the sodium peak currents and slows their inactivation process (Figure 5 Ab) in a voltage dependent manner (not shown, 33). AalT slowed the N a current turnoff most effectively at low negative values of applied voltage and increased by 18% (S.D.±3, n=6) the peak sodium current (33). It was concluded (33,35) that the repetitive activity induced by AalT results from the voltage dependent modulation of sodium inactivation coupled with an increase in sodium permeability. Figure 5C reveals that the depressant toxin blocks the sodium current in a dose and time dependent manner. Thus, the blockage of the action potential (Figure 4f) by the depressant toxin is a consequence of two effects of sodium conductance: Firstly, an increase in the resting sodium permeability, +
ZLOTKIN ET AL.
Insect-Selective Neurotoxins from Scorpion Venom
I || I ter
•A
|40mV 1m«(a&b) 400ms fC) (40mV 2ms
9 II
|2
|
3
l50mV 90s Figure 4. The effect of the excitatory (AalT, BjITl) and depressant (BJIT2) toxins on the action potentials recorded fro the isolated axon of the cockroach. Upper trace: Spontaneous repetitive firing induced by 1.3 | i M AalT. Taken from (33). (a-c) Action of BjITl (8.5 MM): (a) control action potentials; (b) after 8 min of superfusion with BjITl the three superimposed records revealing depolarization and repetitive activity, (c) Burst of repetitive activity of action potentials, (d-g) Action of BJIT2 (15 |iM): (d) control action potential; (e) 2 min of superfusion with the toxin resulted in 5 mV depolarization accompanied by a progressive reduction in the amplitude of the action potential; (f) 2 min later an additional depolarization of 20 mV and a complete block of the evoked response were obtained; (g) continuous slow sweep recording indicating the gradual depolarization and blockage of the action potential induced by BJIT2 (applied at arrow 1). The depolarization was abolished by saxitoxin (applied at arrow 2) but has slowly reappeared after the removal of the saxitoxin by washing (applied at arrow 3). (Reproduced with permission from reference 34. Copyright 1981 Elsevier.)
64
MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS
A (*>
I=0.9±0.6 nM, E m a x l " - 1 * - pmol/mg of protein) and a second low affinity and high capacity site (K 2=185±13 nM, B = l 0.0+0.6 pmol/mg of protein) (Figure 10C). The nature of the low affinity and high capacity LqhIT2 binding sites is presently unknown. The above binding constants, however, can not be related to the ion channels. (3) The excitatory toxin, AalT, displaces the radioiodinated depressant toxin only from its high affinity sites (Figure 10D,E). We have employed sodium channel site-directed antibodies, shown to recognize the insect sodium channel (Figure 7), in order to clarify the phenomenon of the mutual displaceability among the insect selective neurotoxins. The specific question was whether the two categories of insect selective toxins, which differ in their induced symptomatology (Figure 1), effects on sodium conductance (Figures 3-5), and primary structure (Figure 2), compete for an identical binding site, located on the insect sodium channel. 0
D
0
0 7
m a x 2
4. ZLOTKIN ET AL.
Insect-Selective Neurotoxins from Scorpion Venom
A
0
02 0.6 1.0 1.4 Bound (pmol/mg)
5 10 15 t I ] A o I T (nM) C5
B
125
3
Figure 9. The binding of [ I]AaIT (A) and [ H]STX (B) to locust neuronal membrane preparation. Saturation equilibrium assays (left) and the respective Scatchard analysis (right) are presented, to determine the K and B , ^ . A : [ I]AaTT binding yielded a K =1.19 n M and B =1.37 pmol/mg of membrane protein. Taken from (61). B : [ H]STX binding yielded a K =0.14 n M and B = l .43 pmol/mg protein. (Reproduced with permission from reference 60. Copyright 1985 Elsevier.) D
125
D
max
3
D
m a x
72
MOLECULAR ACTION OF INSECTICIDES ON ION CHANNELS
o> '"D
i
100 ~
o
o
o
B
o
v
B
log
[Toxin] , M
Figure 10. Mutual binding displaceability among the excitatory and depressant insect selective neurotoxins. A , B : Displaceability of the [ I]AaIT (1.5 nM) by the excitatory and depressant insect toxins: (A) AalT (•); BjITl (•); BJIT2 (V); AaH2 ( • ) ; C. sqffusus P-mammal toxin 2 (o); (B) AalT (o); LqqITI (A); LqqIT2 (•). Taken from (61,67). C: Scatchard analysis of a saturation curve of the depressant toxin LqhIT2 binding to locust neuronal membranes. Scatchard plot analysis yielded the best fit (P
