Insect selective toxins derived from scorpion venoms: An approach to insect neuropharmacology

Insect Biochem., Vol. 13, No. 3, pp. 219 236, 1983 Printed in Great Britain.

0020-1790/83/030219-18508.50/0 Pergamon Press Ltd

S H O R T REVIEW

INSECT SELECTIVE TOXINS D E R I V E D F R O M S C O R P I O N V E N O M S : AN A P P R O A C H TO INSECT N E U R O P H A R M A C O L O G Y ELIAHUZLOTKIN Department of Zoology, The Hebrew University of Jerusalem, Jerusalem, Israel (Received 21 July 1982)

1. INTRODUCTION As IN MANYother topics in the field of toxinology, the study of the chemistry and pharmacology of scorpion venoms was mainly motivated by medical aspects, thus employing vertebrates and mainly mammals as experimental animals. The capacity of a scorpion sting to kill a human being is astonishing when taking into consideration the relatively small amount of active substance injected (YAHEL-NIv and ZLOTgIN, 1979), which is about three orders of magnitude less than that released, for example, by the bite of an average viperid or elapid snake. From the background of the accumulated and recently reviewed (ZLoTKIN et al., 1978) information, it may be concluded that the different cardio-vascular, muscular and respiratory manifestations associated with the pathology of scorpion envenomation, are a consequence of a general sympathetic and parasympathetic discharge due to the peripheral axonal excitatory effect of scorpion venoms. In Nature, however, the rendezvous between scorpions and humans is coincidental. The stinging activity of scorpions, in the field, is mainly directed to arthropods and especially soft-bodied insects which serve as their prey food. From an ecological point of view the usage of chemical means for defence or food obtaining purposes may be considered as an efficient and energy saving method. The venom apparatus of scorpions serves as a perfect example of this principle. As predators, scorpions appear to lack certain essential anatomical and physiological qualities. They move relatively slowly, are practically blind and have a rather undeveloped sense of smell. Scorpions do not seek their food actively, but rather wait for the prey to approach their lairs (BEAR~, 1961; S~AHNKE, 1966). Thus, a device for paralyzing the prey at the earliest moment of contact is essential. This is achieved by the Abbreviations: AalT, the insect toxin from the venom of Androctonus australis: AaCT, the crustacean toxin from the venom of Androctonus australis: AaMT2, the mammal toxin II from the venom of Androctonus australis, AmlT the insect toxin from the venom of Androctonus mauretanicus; BjlT l and 2, The insect toxins 1 and 2 from the venom of Buthotus juadaicus, SmlT 1 and 2, The insect toxins 1 and 2 derived from the venom of Scorpio maurus palmatus.

use of venoms which have highly specified chemical adaptations. 2. FACTORS SPECIFICALLY AFFECTING ARTHROPODS The symptoms of scorpion envenomation in arthropods mainly indicate a stimulatory effect on the skeletal musculature which in the adult locust (Locusta), injected with 2-3/lg of the venom of L. quinquestriatus, is expressed in uncoordinated leg movements, trembling of hindlegs and spasmodic contractions of the genital valves (KAMON and SHULOV, 1963). Similarly, injection of 2LDs0 (23/~g per 100 mg of body wt) of A. australis scorpion venom in isopods (Armadillium vulyare, a terrestrial crustacean) causes a paralysis within 2-3 min preceded by irregular and uncoordinated movements of the legs and a curvature of the body due to a sustained contracture of the ventral body musculature (ZLOTKIN et al., 1972a). In blowfly larvae, injection of small amounts of different scorpion venoms (see Table 1) causes an immediate and sustained contraction of body musculature, expressed in drastic thickening and shortening of the body accompanied by complete paralysis (Fig. la), the degree and duration of which are dosage dependent. This contraction paralysis of blowfly larvae was employed as a quick, convenient and sensitive bioassay for the evaluation of the paralytic potency of scorpion venoms. The paralytic potency of different scorpion venoms to blowfly larvae compared to their mice lethality is presented in Table 1 (ZLOTKINe t al., 1971a). The marked resistance of an intact nerve to scorpion venom, as previously shown in vertebrate preparations (HoussAv, 1919; DEL Pozo and ANGUIANO, 1947; ADAM and WEISS, 1959; LAGRANGE and RUSSELL, 1971), was also demonstrated by the application of concentrated solutions of L. quinquestriatus venom to the tympanic nerve (ZLOTKINet al., 1970) as well as the ventral nerve cord of Locusta (PARNASet al., 1970). Thus one may assume that the excitatory paralysis symptoms of scorpion envenomation in arthropods should represent a peripheral action on the skeletal musculature, either directly or through the neuromuscular junction. The latter point was clarified by PARNASand RUSSELL(1967) and PARNASet al. (1970),

219

220

ELIAHU ZLOTKIN

Table 1. The 500o lethal dose to mice (LDso) and the contraction paralysis unit (CPU) to blowfly larwte of different scorpion venoms* Scorpion venom Leiurus quimt.estriatus A~ldro¢'fOllllS ¢1~,11~1s (1¢11¢)~ts AlldroctOllllS lTltlltrc{~lllicllS IIRlltr~2f¢llliClIS

Androctcmus australis Centruroides ,gcttlltl I~l¢lri~l Alldroclolllls crossi¢'~llldct

Tityus serrulcmcs Bulhisctts hicalcarattts Centruroides liml~idus tecomantts AlldrocfotlllS ~tltlor('llvi

Buthactts leptochelis Bllthlts occil~llllts flltT¢'l¢lllllS Buthacus arolicoht Bttllltt,s occit~m.s paris t'Jlttlloflts miltaY Parabuthtts transraalicus

LD5o (l~g/mouse)?

CPU (/*g,larva)~_

5,1 6.2 6.3 7.0 7.7 ~.0 ~.6 2.0 3.7 5.{) 5.3 7.9 9.S 83.0 ,~5.0 S5.0

0.22 0.05 0.47 0.29 3.$2 0.6~ 2.17 {).{)7 0.22 O.36 0.09 0.05 0.07 0.03 0.30 O.35

* Taken from ZLOTKIN et ul. (1971a). The venoms were obtained by electrical milking followed by lyophilization. f Subcutaneous injection into mice of both sexes weighing about 20 g. + Tested on Sarcophagalidculata larvae weighing about 100 mg. with crustacean and insect neuromuscular preparations employing Centruroides and Leiurus scorpion venoms, respectively. The muscle stimulatory effect of these venoms resulted from a presynaptic action. This was expressed in the increase of quantal content and in the repeated contraction of the muscle which was correlated to the repetitive firing of the nerve. Both the axonal excitation and conduction block were assumed to result from the axonal membrane depolarization. There is an apparent resemblance between the effects of scorpion venom on arthropods and those on mammals. This is expressed in the excitatory symptoms of envenomation and the action of scorpion venom on the neuromuscular preparations of an insect, crustacean (PARNAS and RUSSELL, 1967; PARNAS et al., 1970) and mammal (KAtZ and EDWARDS, 1972; Brazil et al., 1973; ZLOTKIN et al., 1978). In all the above preparations, scorpion venom caused muscular stimulatory effects due to presynaptic excitation at the level of the exposed nerve endings resulting in the release of the corresponding transmitter. This similarity in action could have led to the assumption that the effect of scorpion venoms on mammals and arthropods is due to the same chemical factors in the crude venom. By the aid of the assay of the contraction paralysis of blowfly larvae (Fig. la), it has been found that there was no correlation between larvae paralysis and mice lethal potencies of 16 different Buthidae venoms (ZLOTKIN et al., 1971a, Table 1), suggesting that the two toxic activities may result from different factors. This possibility was supported by the finding that the potent toxins 1 and 2 of A. australis, which are strongly lethal to mammals and isolated according to the criterion of mice lethality (ROCHAT et al., 1967; MIRANDA et al., 1970), were completely inactive on larvae (ZLOTKIN et al., 1971b). The final proof of the diversity between factors affecting mammals and those paralyzing insects in scorpion venoms, was

obtained by starch gel electrophoretical separation of the venom of A. austrulis (ZLotKIN et al., 1971b; Fig. 2A), and six other Buthinae v e n o m s {ZLOTKIN et al., 1972b', Fig 2B, C). It has been found that three of the above venoms contain more than one fraction which cause contraction paralysis of larvae. These fractions were lethal to fly larvae and readily inactivated by trypsin, thus demonstrating their protein nature (ZLOTKIN et al., 1972b}. The above early findings led to the detection and isolation of the scorpion venom insect toxins (see below}. The existence of an additional group of toxins, the crustacean toxins, was initially demonstrated by the fact that in contrast to the crude venom of A. australis, its derived insect toxin (IT, see below) and M T 1 and 2 were unable to affect an isopod (terrestrial crustacean) or a scorpion suggesting the presence of discrete neurotoxins (ZLOTKIN et al., 1972a) specifically affecting these arthropods. Using a bioassay of isopods paralysis coupled with gel filtration and ion exchange column chromatography, a protein specifically toxic to isopods (crustacean toxin, AaCT) was isolated and purified from the venom of A. australis (ZLOTKIr~ et al., 1975; Table 2). Several crustacean toxins have been recently isolated from the venom of a chactoid scorpion Scorpio maurus palmatus (LazArovicl, 1980; Table 2}.

3. I N S E C T T O X I N S Insect toxins derived fi'om the vemml qf dw Androctonus scorpions

The isolation and purification of the AaIT was monitored by the above contraction-paralysis assay of fly larvae {ZLOTKIN et al., 1971a: Fig. la) using the same technical procedure previously employed for the purification of the scorpion venom mammal toxins (MIRANDA et al., 1970; ZLOTKIN et al.. 1978). Following a sequence of steps composed of water extraction

Fig. 1. Responses of SarcophagaJalculata blowfly larvae to the injection of various scorpion venoms and their derived insect toxins. A. Typical contraction paralysis as induced by various buthid scorpion venoms (Table 1) as well as the AaIT, AmIT and BjITI insect toxins. B. Typical flacid extended paralysis as induced by the crude venom of S. m. palmatus and its derived insect toxins as well as the BjIT2. Bar corresponds to 3.8 mm.

o

0

@,

U

•~ . , . , >~r,

..- ~ '='-0 ;> 0 ~ "5 o ~ . " . C

= ~"~

~

~

=~

~ .E

'E

N

,=,,~ b

£~o o0~

I

g.~ ~_~ ~. ~

i

--~

I

<

~

.

x ~ - -

I

g oS

222

[

An approach to insect neuropharmacology

223

..2

=

'**

"5 '7,< O t-

+4" -H-

,<

O

..j

"7.

"O

O

I...

<

~5 ~ "~

O O r~

"O

O

< < ["o

~N o

224

Et, IAHU ZLOTKIN

//1

D

15 E c ~Oea I0'

c

6

7 EFFLUENT(liter)

8 ac~


04

05

06

or

o8

09

io

Efflueni' Vol (I)

°'!I B

00+

t'5 50

~0 ,5O ~00 25O Efflueni Volume (ml)

004

_

100

200

/

N (3

F

FRACTION NUMBIER( 3r.I )

~c~ o.z-

DEXTRAN/'~

O.t- ~UE

~l/

!;I '~

i', ~.2

C G

f". Y

• \

~

/'V.%

o}.

6.6

Vo=~42

"

~

0~

~FRACTIOI~

/

~

,' " ' , (o

~ 1.z

"1 l~

"~ .

is.

EFFLUENT VOLUME(titers/

Eq,].

Gr

'

0.8 ..//

g (~4

<

//./ 0.4

1O

4,,/

08

,.02 " "Y

100 200 FRACTION NUMBER ( 3 ml )

~

I I

MT

EFFLUENT VOLUME (ml)

Fig. 3, Isolation and puritication of insect toxins derived from the venoms of ,4. a~L~lrali.~ (A ('t B. judaicus {D, E) and S. maurus (F, G) scorpions. A, Recycling gel filtration on Sephadcx G. 50: Four columns of 3,2 x 100 cm in series in 0.1 M a m m o n i u m acetate buffer pH K5 K6; tlow rate 60 m l h r . The mixture submitted to fractionation is the water extract of 2 g of crude venom, vertical arro\~s and numbers correspond to the beginning of the consecutive cycles. Fractions of the elation curves indicated by the full line are collected. The material marked by the dotted line is recycled. Toxicity' to mice is located in fraction RI and R2 (Mmg:,aDA et at., 1970), toxicity to fly larvae is located in fraction IT and toxicity to isopods is located in fractions E and R2 (taken from ZLOTKIN et al.. 1972b), B. Puritication ol the insect toxin: C h r o m a t o g r a p h y on DEAE Sephadex A-50 of the fly larvae toxic fraction IITI obtained in A from 0.5 g of crude venom. Column 2 x 200 cm in 0.1 M acconium acetate buffer pH S.50: flow rate 12 ml/hr. The horizontal arrow indicates the fraction toxic to fly larvae. C, Chromatography on Amberlite CG 50 of the toxic fraction obtained in B from 0.5 g of venom. Column 2 x 200 cm in 0.2 M a m m o n i u m acetate buffer pH. 6.30; flow rate 12ml:hr. The horizontal arrow indicates the Iinall} purified insect toxin fraction (taken from ZLOTKIX et ul.. 1971C). D, Gel filtration on Sephadex GY0 linc of 2 0 0 m g of Lowry protein of the water extract of the crude venom of B. judaicus. Two columns, 202 x 1.94 cm in series. A m m o n i u m acetate buffer 0.1 M pH 8.5. Flow rate 15 ml/hr. The vertical arr(m indicates elation volume of Dextran blue (DB = void volume). IT corresponds to the region of separ ation where toxicity was found for fly larvae (according to the contraction paralysis assay) and locusts (according to paralysis or excitation). CT represents the region of crustacean toxicity (according to paralysis of isopods). Fraction IL which contained about 80°;. of toxicity to fly larvae and locusts, ~as employed for further purification. E. Cation-exchange chromatography in CM Cellulose C M 5 2 IWha~man) of 30 mg of Sephadex G 50 F II, using a m m o n i u m acetate buffer. Equilibrium conditions: buffer 0.05M pH 6.9. Free flow rate 75ml/hr. First gradient: 0.05 (I.25 M pH 6.9 buffer. 2nd gradient: 0.25 1.0 M. pH 6.9 buffer. Gradients flow rate 5 ml/hr. Fraction lla represents the immediale insect

OA~

An approach to insect neuropharmacology dialysis, recycling Sephadex G50 Gel filtration and ion exchange equilibrium chromatography by DEAESephadex A-50 followed by Amberlite CG-50 (Fig. 3A-C), a final product, 267-fold purified and with a yield of 95Vo based on toxicity, was obtained. The AaIT is a polypeptide composed of about 70 amino acids including eight half cystines. Amino acid analysis of the alkylated compared with the reduced-alkylated peptide have clearly indicated that all the cyteic groups are involved in four disulphide bridges (ZLOTKINet al., 1971c). On the basis of its composition the AaIT resembles the mammal toxins derived from the same venom as judged by its total number of residues, the presence of four disulphide bridges the low amount of phenylalanine, histidine, arginine, tryptophan and the absence of methonine (Table 2). For comparative purposes an additional insect toxin was isolated from the venom of a related species of scorpion, that of Androctonus mauratanicus (AmIT, ZLOTKIN et al., 1979). The purified toxin is about 330 times more toxic than is the crude venom in its larval contraction-paralysis activity, and is composed of 67 amino acids with an estimated mol. wt of 8129 (Table 2). It strongly resembles the AalT in terms of its toxicity, amino acid composition, mol wt and selective binding affinity to insect nervous tissue (see next section). Like A. australis venom, the crude venom of A. m. mauretanicus also contains toxins specifically affecting crustaceans (ZLOTKIN et al., 1979) and mammals (ZLOTKIN et al., 1978). Buthotus judaicus insect toxins Against the contrasting background of the diverse forms of the scorpion venom mammal toxins (ZLOTKIY et al., 1978; ROCHAT et al., 1979) it was decided to investigate further the apparently well-defined chemical and pharmacological consistency demonstrated by the AaIT and AmIT by varying the venom source. The venom of the Israeli black scorpion Buthotus judaicus (Buthinae) apart from belonging to a different genus and being relatively limited in the geographical distribution, is characterized by a very low toxicity to mammals (0.5-1.0mg/mouse, LESTER, unpublished) and the induction of mixed, excitatory and depressory symptoms in insects (LESTER et al., 1982). Two toxic proteins BjIT 1 and 2, selectively paralytic and lethal

225

to insects, were purified using gel permeation and ion exchange chromatography (LESTER et al., 1982; Fig. 3D, E). Their chemical purity was assessed by column chromatography, disc electrophoresis, isoelectrophocusing (Fig. 4) and amino acid analyses. Their biological specificity and toxicity were determined by a series of paralysis and lethality assays employing the larvae of the blowfly Sarcophaga falculata, and the locust, Locusta migratoria. BjlT 1 is approx. 40 times more toxic than the crude venom (according to its fly larvae paralysing activity), is composed of 67 amino acids including six half cystines (Table 2) and has an estimated mol. wt of 7532 and a pH~ value of 8.20. It causes an immediate contraction paralysis of fly larvae (Fig. la) and a quick excitatory "knock-down" effect on locusts. BjlT 2 is about 36 times more toxic than the crude venom according to the paralytic potency in fly larvae. It is composed of 69 amino acids including six half cystines and has an estimated mol wt of 7894, a unique amino acid composition (Table 2) and pH~ value of 8.30: BjlT 2 causes a flaccid paralysis of fly larvae (Fig. l b) and a slow progressive paralysis and eventually death of locusts. Scorpio maurus palmatus insect toxins According to numerous morphological (VACHON, 1952) as well as some biochemical characteristics (Govrrorq and KOVOOR, 1978) scorpions are divided into two groups: buthoids and chactoids. The buthoids, which include the dangerous, medically important scorpions, comprise 4 0 ~ of the known species and consist of a single family, the Buthidae. The chactoids consist of five families and are, in general, medically insignificant. The vast majority of the chemical and pharmacological studies of scorpion venoms were performed with buthoid venoms (GoYFrOy and KovooR, 1978; ZLOTKIN et al., 1978), including their employment as pharmacological tools for the study of excitability in biological systems (CATTERALL, 1980) and as sources for the above mentioned animal group specific toxins. It appeared to us that the so far poorly investigated chactoid venoms may serve as an additional source of interesting pharmacologic substances. Preliminary data (ZLoa-KIN et al., 1972c, 1973) have indicated that the venom of the chactoid scorpion Scorpio maurus palmatus possesses an extremely

paralytic factor (as assayed on locusts) which was finally purified by two additional steps of chromatography: DEAE-Cellulose and CM-Sephadex columns. Fraction IIB represents the slow paralytic factor to fly larvae and to locusts which was finally purified by two additional column chromatographical manipulations both with CM--Cellulose columns (taken from ZLOTKINet al., 1980). F. The different pharmacologically active fractions isolated from the venom of the scorpion Scorpio maurus palmatus (Scorpionidae). Sephadex G-50 column chromatography. 680ODzs0 units of water extracts were charged on two columns of 198 x 1.7 cm connected in series and eluted by ammonium acetate 0.1 M pH 8.5 buffer. Flow rate of 15ml/hr was applied and fractions of 10ml were collected. Absorbance at 280nm was automatically recorded (unbroken line). Fractions collected are indicated by horizontal double headed arrows. Fractions I IV contain: high mol. wt enzymes such as hyaluronidase and phosphatase (I), phospholipases (It), direct lyric factors (III) and neurotoxic poly-peptides (IV). Fractions IVa-VI are non-proteinaceous substances. G. CM-Sephadex G-25 cation-exchange chromatography of the Sephadex 6-50 toxic fraction IV. Column: 20 x l cm; buffer; ammonium acetate. At equilibrium conditions (Eq): 0.1 M pH 8.5. Linear gradient of molarity up to 2.0 M, pH 8.5. Flow rate of 4 ml/hr was applied and fractions of 1.25 ml were collected. 40 OD280 units were charged on the column. Full line absorbance at 280 nm; dotted line, linear gradient of buffer concentration IP = insect immediate and reversible paralysis. IT = insect toxicity, CT = crustacean toxicity, MT = mammal toxicity. The insect toxic (IT) fraction was further treated on a column of CM-Sephadex C-25 resulting in the purification of the two insect toxins (Table i). (Taken from ZLOTKINet al., 1980).

226

ELIAHU ZLOTKIN

low toxicity to mammals and an unusual symptomatology to insects, In contrast to the immediate contraction paralysis of larvae (Fig. la) induced by the Buthidae scorpion venoms (ZLoTKIy et al., 1971a} this venom induced a slow progressive flacidity (Fig. l b). This has directed our attention to the insect toxic factors in this scorpio venom, resulting in the isolation and chemical-pharmacological characterization of several insect toxic components. It has been shown (LAZAROVICIet al., t9821 that the toxicity of the crude venom to insects is due to three separate groups of substances, the so-called cytotoxins, phospholipases and neurotoxins (Fig. 3F). The neurotoxic fraction contains two factors: the fast reversibly paralytic (IP) and the slow lethal (IT) (Fig. 3G). The slow lethal factor is composed of two toxins (SmIT 1 and 2), which were obtained by an additional step of column chromatography and the purity of which was assessed by column chromatography, disc electrophoresis, isoelectrophocusing, analytical ultracentrifugation and amino acid analyses. SmIT 1 and 2 are two polypeptides possessing unique amino acid compositions with mol. wts of 3232 and 3963 pH~ 8,8 and 9.2 and supposed to contain two and three disulphide bridges, respectively (Table 2). Each of the two separated toxins has demonstrated only a slight increase in its specific toxicity and a very low recovery (o) ~00 t

5O

i11

i

/.7

/

/

(b)

I

[

I

l

I

I

I

I

,5

I

t

I

I

I

I0

i~g/ Sorcopho9o

fly

I00

!U

I"2 "IT,

IT, (2)

So

J

o

_~--/ I0

i

I

L

L

l

1

50

,u.g/ LocastO

[

L

tOO Locust

Fig. 5. Synergistic interactions in lethality among the insect toxic components of the venom of the scorpion S. palmatus. (a) The interaction between CM-Sephadex C25 fractions (Fig. 3G) IT and IP, on blowfly lethality. (b/ The interaction between the two insect toxins IT1 and IT2 (Table 2) in locust lethality. Circles, mortality curves of each substance separately. Triangles, Mortality curves of mixture of the substances. Numbers in parentheses indicate the ratio in wt between the combined substances (taken from LAZAROV1CIet al., 19821.

of toxicity when compared to the crude venom. A clear cooperative interaction was demonstrated between the fast paralytic and lethal (IP and IT respectively, Fig. 3G) fractions as well as between the two insect toxins (Fig. 5) resulting in an evident recovery of the original toxicity to insects of the crude venom.

4. N E U R O T O X I C I T Y Neuromuscular ~f~]bcts

Simple considerations concerning the symptomatology, doses and speed of response pointed that the above insect toxins substantially affect the nervous system. Neurophysiologic studies were performed mainly with the different toxins derived from the venom of the scorpion A. australis (Table 2). Some early evidence was obtained, suggesting that the specificity or selectivity in the action of these toxins is based on specific affinity to nervous systems of the corresponding groups of animals. It has been found that an isopod toxic fraction obtained by Sephadex G50 chromatography (ZLOTKIN et al., 1972a) was able to mimic the excitatory and blocking action of the crude venom on the crayfish stretch receptor organ, in contrast to the insect and mammal toxins which were inactive PANSA et al., 19731. Similarly, the insect toxin was able to mimic the action of the crude venom in performing an excitatory block of the induced afferent transynaptic response of the sixth abdominal ganglion of the cockroach P e r i p h m e t a americana, in contrast to the complete inactivity of high doses of the A a M T 2 (D'AJELLO et al., 19721. The amount of IT, however, employed for the above CNS blockage was at least two orders of magnitude greater than used for the quick paralysis of the whole animal. Furthermore ventral nerve cords dissected from the AaIT-paralyzed insects possessed normal electrophysiological properties [D'AJELLO et al., 19721. This together with the resistance and impermeability of intact nerves (PARNAS et al., 1970; ZLOTKI~ et al., 19701 to scorpion venom have pointed to the neuromuscular junctions as the true targets for the paralytic action of scorpion venoms and their toxins. The action of Aa-IT, CT and MT 1 and 2 were studied in a series of neuromuscular preparations including those of a mammal (TINTPULVER et al., 19761, a crustacean (RATHMAYER et al.. 1977) an arachnid (RUHLAND et al., 19771 and an insect (WALTHER et al.. 1976, Fig. 6). It was concluded that the muscular stimulatory effects of the different toxins on the different preparations were due to an excitatory presynaptic action or the motor axons. The crustacean and the mammal toxins have demonstrated a relative specificity to the corresponding neuromuscular preparations (Table 3). The pharmacological versatility of these substances may follow either from a multiplicity of active sites or from differential affinities to identical receptor sites in different organisms. It is noteworthy that the presence of a component such as the crustacean toxin, which by itself is non-lethal to mammals but is able to perform an excitatory action m mammalian systems, may possess certain pharmaceutical advantages. The discrepancy between the relative capacity of Aa-CT and A a M T 2 to affect the locust

Fig. 4. Polyacrylamide gel disc electrophoresis (a-d) and analytical isoelectric focusing (e-f) of the two insect toxins derived from the venom of B..[udaicus. All quantities refer to Lowry protein, a-d. The gels were run from anode to cathode in fl-alanine buffer, pH 4.5 at 3 mA/gel for 40 rain. Staining was with 1% Amido Black in 730 glacial acetic acid. a b, 15 and ll0/xg of IT-I respectively. ~ d , 20 and 100pg of IT°I1 respectively, e-f, The gel contained Ampholites in the range of pH 3-10 and the run from anode to cathode lasted for 3.5 hr at a constant voltage of 200V. e. 25pg of IT-I f. 20#g of IT-II. (Taken from LESTER et al., 1982.)

227

An approach to insect neuropharmacology

A -J

it It

IIIqqilqqqqqlqtl

,I

llll. il!iiiliii

'wliJlllljJlll

:

r

11 •

•

•

•

•

•

•

•

•

•

•

•

•

•

B

229

1976; RATHMAYER et al., 1978). The axonal effect of the AaIT was mainly expressed in the induction of repetitive firing (Fig. 6) without affecting the timecourse of the action potentials thus contrasting the crustacean and mammal toxins (WALTHER et al., 1976). It was also shown that the various peripheral branches of the motor nerve serve as the primary target of the insect toxins (WALTHER e t al., 1976). Finally, these results (Table 3) suggested that only the insect toxin is selective in its activity and that the way it affects the axon might be quite different from that previously reported for scorpion venoms and toxins (WALTHER et al., 1976 and see below). Bindin9 studies

Fig. 6. A, Neuromuscular effect of the AalT on the locust hind leg extensor tibias nerve muscle preparation. A, start of two trains of slow ejps synchronously recorded from two distant locations on the muscle 50 min after application of 0.15 ~g/ml of insect toxin. Calibration: l0 mV, 100 msec. B, Spontaneous neuromuscular activity caused by insect toxin. Synchronous recording from the "slow" motor axon (upper trace) and a fibre in the main part of the extensor tibiae muscle (lower trace) 65 min after application of approx. 0.5/lg/ml of toxin. Calibration: 50msec; upper 0.5 mV, lower 10mV. These data (A, B) clearly indicate a presynaptic excitatory action on the motor nerve. (Taken from WALTHER e t al., 1976). nerve muscle preparation and their extremely low ability to paralyze the whole animal (Table 3) may suggest that the specificity of the above toxins to the respective groups of animals may be, at least in part, attributed to their resistance to inactivation processes in the body. It is also noteworthy that there exists a close resemblance between the response of the crayfish and the spider nerve-muscle preparation to the different toxins (Table 3). This may indicate certain structural and functional similarities between the neuromuscular systems of these two classes of arthropods, distinguishing them from insects. The AaIT, however, was the most potent and active only on the insect nerve muscle preparation (WALTHER e t al.,

The selectively of AalT and A m l T was further demonstrated in a series of binding experiments with the radio-iodinated toxins to nervous and non-innervated tissue preparations of a locust and nervous tissues of a crustacean (TEITELBAUMet al., 1979; ZLOTKIN et al., 1979) and a mammal (TE]TELaAUM, unpublished). Specific binding of the insect toxins was obtained only with the insect nervous tissue (Table 4). Analyses of assays of competitive displacement of the [125I]AalT (TEITELBAUM,unpublished) and [125I]AmlT (ZLOTK1N et al., 1979; Fig. 7) by the native toxins in locust nervous tissue preparations have indicated the presence of two classes of non-interacting binding sites (in contrast to a single class suggested in preliminary assays--TEITELBAUM et al. (1979). The first class of binding sites is characterized by its high affinity and low capacity, and the second class possesses slow affinity and high capacity (Fig. 7). This phenomenon of multiple classes of binding sites, which contrasts the single class of binding sites shown with the scorpion venom mammal toxins in mammalian nervous tissue preparations (CATTERALL, 1980), deserves further attention (see also Concluding Remarks). A recent preparation of neuronal plasma membrane vesicles derived from synaptosomes of the locusts CNS may serve, in the future as a more suitable medium for the study of the interaction of the insect toxins with insect neuronal tissues. The pharmacological functionality

Table 3. The relative activity of different toxins derived from the venom of the scorpion A. australis in several nerve muscle preparations* and insect paralysis Toxic material* Crude venom Insect toxin Crustacean toxin Mammal toxin I Mammal toxin II

Insect,

Crustacean§

Arachnidll

Mammal¶

1 115 18 7 2

1 No effect 127 27 4

1 No effect 67 11 5

1 No effect 0.7 1 25

Insects** paralysis 1 25 0.7 5.7 0.2

* Based on a comparison of the average minimal doses causing an evoked repetitive muscular response. Numbers express the activity relative to that of the crude venom. * The different toxins were purified from the crude venom of the scorpion Androctonus australis Hector (MIRANDAet al., 1970; ZLOTKINet al., 1971a, 1975). ;~ Locust leg extensor tibiae preparation (WALTHERet al., 1976). §The crayfish walking leg dactylus opener preparation (RATHMAYERet al., 1977). J[The claw closer muscle preparation in the leg of a mygalomorph spider (RUHLANDet al., 1977. ¶ The guinea pig ileum smooth muscle preparation (Tintpulver et aL, 1976). ** Determined by injection into the body cavity of second and third instar Locusta migratoria.

230

ELIAHU ZLOTKIN Table 4. Specific binding of A. ausu'alis [~2Sl]qT to locust and crayfish nervous tissue and non-innervated locust tissues*

Unhtbelled IT+ (l~g)

Preparation Locust nervous tissue

0 20 0 20 0 20 0 20

Locust Malpighian tubules Locust fat body Crayfish nervous tissue

['eSl]-lT bound { n g m g protein) 0.47 0.27 0.32 0.29 (I.49 0.49 O. 12 0.1 I

± ± ± + ± -+ +

0.013 0.010 0.020 0.015 0.010 0.016 0.007 0.005

Specifically bound [12~I]-lT I n g m g protein) 0.20 + 0.023 0.(/3 + 0.035 o.(10 + 0.026 0.01 + 0.012

* Taken from TEITELBAUMet al., 1979. For each tissue preparation 4 6 determinations ~erc performed. The results are expressed as the mean _+ SD. + Doses of unlabelled IT present in the incubation mixture. The binding obtained in absence of unlabelled IT represents total binding while that obtained in the presence of 20/~g represents non-specific binding. ++Calculated as the difference between total and non-specific binding measured for each tissue preparation.

of this preparation was demonstrated by its capacity to actively transport 7-aminobutyric acid in a sodium and chloride dependent manner (GORDON et al., 1982). Studies on isolated insect axo;~s. The selectivity of AaIT was determined by a series of different lethality assays, neurophysiological studies and, the above, binding studies indicating a selective affinity to the insect nervous system. It was concluded that the axonal membranes serve as the primary target for the action of the insect toxins. It was therefore of interest to test the effects of the different insect toxins on an insect axonal preparation (PELHArE and ZLOTKIN, 1981, 1982, LESTER et al., 1982; LAZAROVlCl et at., 1982). The isolated giant axon from the central ner-

vous system of the cockroach PeripIaneta americana, in both, current and voltage clamp experiments using a double oil-gap, single fibre technique (P~CHON and BOISTEL, 1967), was employed. Figure 8 presents the data concerning the AaIT and AaMT 2. The latter is the most potent scorpion toxin to mammals (ZLOTK~Net aL, 1978) and was previously shown to be practically inactive to insects (ZLoTKIX et al., 1971b, 1972a; WALTHER et aL, 1976 and Table 4). In current-clamp a prolongation of the evoked action potential was induced by AaMT 2 (Fig. 8a). Such response was previously shown to occur in the giant squid axon, ( N A R A H A S H 1 e t al., 1972), in frog nerve fibre (KOPPENH6FER and SCHMn)T, 1968) treated by

._.q

ID Q.

15o -

Ioo

rE E

50

rn O ~O°

l io~

I iOz ONabelled

A mm-IT,

k iO3

iO4

nM

Fig. 7. Specific binding of Am IT to locust CNS homogenate. Incubation mixtures of 0.7 ml contained 0,158 mg of tissue protein, 016 p-mole of [~251]-Am IT (100,000 counts/min) and varying concentrations of unlabelled Am IT. N o n specific binding was determined in the presence of 7.6 ~tM of unlabelled Am IT. Each point represents the mean of three experiments and the horizontal bars indicate the range of the determinations. Analysis (KLOTZ and HUNSTON, 1971; DAtlLQUIST, 1978) of these data has resulted in biphasic Scatchard plot and a linear Hill plot with a slope close to 1. This indicates two classes of non-interacting binding sites. The first class is characterized by its high affinity (K• = 2.4 riM) and low capacity (300 f-mole/mg protein} the second is of a low affinity (K D = 9.5 itM) and a high capacity (93 p-mole per mg protein}. Similar data were obtained with AalT (TEITELBAUM unpublishedl, (Taken from ZLOTK1N et al.. 19791.

An approach to insect neuropharmacology

(0 ) MTII

231

(b) IT 4 0 mV 4 ms in (o) 4 0 ms in (b)

Membrane voltage (mY)

( c ) MTII

-40

-6O

-20

0

e) 'E u E

-I

--

o (d)

~

ZT

~

2ms i

I mAcm

-2

o E ID

j

Ip MTII Ip IT

-3

Fig. 8. The effects of AaIT and AaMT2 on the action potentials and sodium currents recorded from the isolated axon of the cockroach CNS. a-b, Current-clamp experiments. (a) 3.5 #M of AaMT2 (b) 1.3 #M of AaIT. c-d, Voltage clamp experiments. The preparation was pretreated by 4-aminopyridine thus suppressing the potassium currents. (c) and (d) sodium currents following the treatment by 3.5 and 1.3 #M of AaMT2 and IT respectively corresponding to pulses from Eh = -60 mV to Em= l0 mV in 10mV steps. (e) Current voltage relations of the data obtained in (c) and (d). Ip-Na + peak current II-sodium late, steady state current. (Taken from PELHATEand ZLOTKIN, 1981).

crude scorpion venoms or in crustacean axonal prep- aration in current- and voltage-clamp conditions arations treated by purified scorpion toxins (ROMEY et (LESTER et al., 1982). In current clamp BjIT1 induces al., 1975; RATHMAYERet al., 1977). In the presence of repetitive activities (Fig. 9b, c) and BJIT2, a block of AaIT, however repetitive firing of action potentials the evoked action potentials (Fig. 9e-g). These two (Fig. 8b) accompanied a 2-7 mV depolarization of the opposite effects may be explained by their different axonal membrane (see also Fig. 6B), was obtained. In effects as sodium permeabilities. Both toxins increase contrast to the AaMT 2 the effect of the AaIT could the sodium resting permeability (IT2 markedly more) not be reversed by washing and could be reached with resulting in a progressive depolarization of the axonal concentrations of one order of magnitude lower than membrane. Concerning the activatable sodium perwith AaMT2. Voltage clamp experiments were per- meability, both toxins slightly slow the turning off of formed in the presence of 2 × 10-4M 4-aminopyr- sodium transient inward current (Fig. 9i, j). The peak idine thus selectively blocking the potassium current sodium current, however, is increased by BjITI and (PELHATE and PtCHON, 1974). Under voltage clamp, decreased by BjIT2 (Fig. 9i, j). This essential difference AaMT2 slowed the Na + current turn off irrespective may, at least partially, account for the contrasting of the membrane potential, with no effect on the peak symptoms the two toxins induce in the whole insect. sodium current (Fig. 8c, e). By contrast Aa-IT slowed As in the case of the AaIT the Bj insect toxins were the Na + current turnoff most effectively at low irreversible in their axonal action (Fig. 9g). negative values of applied voltage and increased by Finally it may be concluded that the two insect 18% (S.D. _+ 3, n = 16) the peak sodium current toxins, which elicit contrasting symptoms at the level (Fig. 8d, e). It was concluded (PELHATEand ZLOTKIN of the whole animal, differ mainly in their opposite 1981, 1982) that the repetitive activity induced by effects on the activatable sodium permeability in the AaIT results from the voltage dependent modulation axonal preparations. A complementary interpretation of sodium inactivation coupled with an increase in may be based on the assumption that BjIT2 increases sodium permeability. The voltage clamp studies have the proportion of the open Na + channels by possibly also indicated that in contrast to the partial suppres- "locking" these channels in their open conformation sory effect on the potassium current induced by the A. and therefore it induces a sustained depolarization australis mammal and crustacean toxins, the AaIt had which finally blocks the axon. BJITI on the other no effect on this current (PELHATE and ZLOTKIN, hand, seems to modify the "kinetics" of the channel 1982). finally inducing excitatory phenomena. It is, however, The actions of the BJIT-1 and -2 inducing the fast premature to draw conclusions concerning the pharexcitatory-spastic and the slow depressory flaccid macology of these two insect toxins before performing paralyses of fly larvae, respectively, were also investi- a complementary study of their effects on insect gated on the above in vitro cockroach axonal prep- neuromuscular preparations. Possible postsynaptic

ELIAHU ZLOTKIN

232

i

4140mV Ims(a&b) 400ms ~ )

~40mV 2ms

o 1,

12

13

Iso.v h

i

2mI

------

i~lJ A

Fig. 9. The effects of BjlT1 and 2 on the action potentials and sodium currents recorded from the cockroach axon. (a c) Action of IT1 (65 #g per ml. 85 #M): (a) control action potentials; (b) after 8 rain of superfusion with IT1 the three superimposed records represent: (1) a series of several sweeps demonstrating a slow depolarization of about 5 mV; (2) a sweep indicating repetitive activity which resulted in a 15 20mV depolarization and (3) a partial block of impulses: (c) 5 rain later, the same axon being superfused with normal saline and repolarized to the control resting potential (by passing a constant hyperpolarizing current). A stimulation of 100 msec 10 nA has induced a burst of repetitive activity of action potentials. (d g) Action of IT2 (120l~g/ml, 15#M: (d) control action potential (e) 2rain of superfusion with the toxin resulted in 5 mV depolarization accompanied by a progressive reduction in the amplitude of the action potential: (fl 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 IT2 (applied at arrow II. 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). (h j) Voltage clamp experiments: (h) control: transient sodium current recorded after the suppression of the potassium current with 4-aminopyridine in voltage pulse to - 10 mV and of 7 ms duration; (i) sodium current recorded after 3 rain of exposure to ITI (26,ug/mt, 3.5 #M) indicating an increase in the peak current and the persistence of a small maintained current; (j) sodium current recorded after 5 min of exposure to IT-If (90 #g/ml, 11/~M) indicating an evident decrease in the peak current and the persistence of a small maintained current. (Taken from LESTER et ~11., 19821.

muscular action which, in addition, may also account for the opposite symptoms produced by the two insect toxins, should be experimentally excluded. The synergic interaction between the SmIT1 and 2 previously demonstrated in assays of lethality (LAZAROV]Cl et al., 1982) (Fig. 5B) was also expressed in the above axonal preparation. When assayed on the isolated cockroach axon under voltage clamp conditions, the c o m b i n a t i o n of the SmITI and 2 caused a reversible blockage of both the sodium and potassium currents (Fig. 10). This may explain the specific symptomatology and the mechanism of the paralysis of these toxins. The fact that the blockage of both ions is the consequence of cooperative interaction may suggest certain structural associations a n d / o r similarity betweeen the sodium and potassium channels in insect axons. Such a possibility deserves further experimentation. 5. T H E C O V A L E N T S T R U C T U R E O F

THE Aa-IT On the basis of the information reviewed so far, it appears that the AaIT selectively interacts with components associated to sodium conductance in insect

axonal membranes. As such it may be employed in the future as a tool for the chemical characterization of these c o m p o n e n t s thus suggesting the study of its structure function relationships. These considerations have motivated the most recent determination of the complete covalent structure of AaIT (DARBON et al., 1982). The amino acid sequence of AaIT was established by means of extensive phenylisothiocyanate degradation in a liquid sequencer p r o g r a m m e d either with a "quadrol p r o g r a m m e " for native or modified protein or "'dimethyl benzylamine/parvalbumin p r o g r a m m e " for peptides. Due to the low solubility of the S-alkylated toxin in quadrol buffer the sequence was finally established through peptide degradation. The primary structure of the AaIT and the main steps of the enzymatic and sequential degradation are presented in Fig. 11. The t r y p t o p h a n residue, which was, initially, spectrophotometrically determined in the native toxin was not found in the sequence determination. The molar extinction coefficient (at 277 nm) of the carboxymethylated derivative is in a perfect accordance with the six tyrosine residues present. The unusual spectral properties of the native toxin can be attributed either to a special conformation of the native toxin or to the

233

An approach to insect neuropharmacology

*

::L~)

....................................................................................................................................................................................... I (,D IT I

IT2

I T I~-IT2

Time

(200/.~g/ml )

(314/.,z.g/ml I

(133 + 66/..~/rot )

(Stain)

Fig. 10. The action of Sm ITI and I2 on the isolated axon of the cockroach. Current clamp data a Control action potential evoked by application of 500 ,usec duration 10 nA intensity current pulse every 2.5 sec. b and c. Similar action potentials were recorded after 5 min of superfusion of the axon with 200,ug/ml of each of the two insect toxins, d and e, A mixture of 133 and 66 #g/ml of SmITI and 2 respectively induced a progressive decrease in the amplitude of the action potential (as shown by the superimposed recording--d), resulting in a complete block 3 rain after the application (e). Voltage clamp data f, Ionic currents recorded by applications of a twin pulse to - 10 mV and +40 mV from a holding potential of - 6 0 m V in an axon superfused with a solution of 2% BSA in saline g and h: Axon superfused with a mixture of the two insect toxins (133 and 66 #g/ml of IT 1 and 2 respectively) indicating a continuous decrease in the peak inward current (*) and the late outward current ( + ) (g), resulting in a substantial blockage of ionic currents after 5 rain of application (h). The lower graphs are the plot vs time of I peak and I late currents for three different experiments including a separate and combined application of the two insect toxins, as shown. Horizontal bars indicate the duration of application of the toxins. (Taken from LAZAROVICIet al., 1982). presence of a non-covalently bound chromophore in acetic acid and differentiation of the paired amino the native toxin which is lost upon denaturation, acids by determination on tryptic peptides, of the The position of the four disulphide bridges was acspecific radioactivities of the S[14C]-carboxymethycurately deduced by analysis of proteolytic peptides ~ lated phenylthiohydantoin cystines. Of the four disulbefore and after performic oxidation, and by partial phide bridges present in both the A m M T 2 (KoPEYAN labelling of the half cystine residues with [14C].iodo. et al., 1974) and AmIT (Fig. 12) three are in homolo-

t NH z -- L y s - L y s - A s n -

Gly- Tyr-AlO-Vol-

~I=

IO & s p - S e r - $ e r - G l y - L y s ~b, l o - P r o -

*3

20 Glu- Cys- Leu- Leu- Ser-Asr~- Tyr- Cys-Asn-

=L

T4

30 I~ 40 Asn -- Gin - Cys -- T h r - L y s - V o l - - His -- Tyr - A l o - - A s p - L y s - - Oly - T y r - C y s - C y s - L e u - Leu - - S e t - - C y s - T y r - Cy.s - P h e - G I . y -

601 -

T6

k

5O L e u --6,sn - A S p

-- ~ - s p - L y s - L y s

c9

7O -- V o l - L e u -- O l u -- I l e - S e r

=1=

-- A s p - T h r - -

Arg-Lys

C,o

--5er -Tyr

- Cys--Asp

~I~

-Thr

- - T h r -- Ile -- ILe -- A s n -- C O O H

c,,

Fig. 11. The primary structure of the AaIT: positions determined by automatic degradation of carboxymethylated protein. Peptides-"T": obtained after tryptic digestion. Peptides "C': obtained after chymotryptic digestion.

,~

234

ELIAHU ZLOTK1N

I OH

7©

Fig. 12. Comparison of the location of the disulphide bridges on the AalT and the AaMT2 (MTI.

gous positions. The fourth differs in that of one of the two half-cystines involved, Cys 12 in A m M T 2 shifts to Cys 38 in the insect toxin. On the basis of several considerations (DARBON et al., 1982) concerning the primary sequence of scorpion venom toxins it is likely that the position of the disulphide bridges is the same for all scorpion neurotoxins active on mammals. 6. C O N C L U D I N G

REMARKS

Scorpion venoms contain discrete groups of neurotoxic polypeptides specifically affecting different groups of animals. The fact that these substances are able to discriminate between phylogenetically and taxonomically related groups of arthropods such as insects and crustaceans in curious. According to common knowledge these two groups of organisms share identical nervous systems from an anatomical, physiological, pharmacological and chemical points of view. The distinction between insect and crustacean nervous systems was strongly emphasized by binding studies with the AaIT (Table 4) and AmlT. These studies have also demonstrated the existence of the two classes (Fig. 7) of binding sites for the Aa and Am insect toxins. Three different considerations indicate that the neurotoxic action of these toxins is associated with the high affinity and low capacity rather than the low affinity and high capacity binding sites. Firstly there is an accordance between the K D of the high affinity sites and the threshold concentrations of the AalT in the neuromuscular (5 20 nM, WALTHr~Ret al., 1976) and axonal (about 100 nM, PELHAHi et al., 1982) preparations. Secondly there is, also, an accordance between the K D and density of the high affinity sites for the insect toxins (Fig. 7) with those which were measured for the single class of binding sites of the scorpion venom mammal toxins in mammalian nervous systems (JOVER et al., 1978: COURAUI) ff a[.. 1978: CATTFRALL, 1977; RAY et al., 1978). The third consideration is based on a rough estimation of order of magnitudes of mol wts of membrane bound proteins, including those which are supposed to represent the Na + channels in mammalian systems (TAMKUN and CATTERALL, 1981). The extremely high density of the low affinity IT binding site (Fig. 7) makes it highly improbable that these sites are directly associated with membranal components involved in ionic conductance. Six different insect toxins were examined in this review. These substances share some basic qualities such as being polypeptides, possessing a selective neurotoxicity to insects and affecting the ionic con-

ductance in insect axons. They strongly differ, however in, their amino acid composition. In spite of the temptation to associate the apparent resemblance and consistency in the chemical compositions of the AaIT and AmIT (Table 2) with their unique and selective mode of action, such a conclusion should be avoided. The BjIT1 possesses an entirely different amino acid composition (Table 2) but is able to mimic their pharmacologic action of AaIT and AmlT. This may indicate the need for a comparative study of the primary structure of these toxins. Possible identities in their sequences may correspond to the 'active sites' of these molecules. When comparing the compositions of the different groups of toxins presented in Table 1, it appears that the resemblance in the amino acid compositions is associated with the biological source of the substances rather than with their pharmacological action. This element is emphasized when considering, for example, the amino acid compositions of the insect toxins of S. m. palmatus venom, which clearly resemble the crustacean and mammal toxins derived from the same venom and strongly differs from the insect toxins derived from the venom of the other three scorpion species (Table 2). In addition to the relative resemblance in the composition of different toxins derived from the same venom it is noteworthy that AalT and AaMT2 also share the identical localization of three out of their four disulphide bridges (Fig. 12). The shift of one half cysteine residue in the insect toxin may induce a major conformational change in the structure ot this protein which in turn, may account for the pharmacologic specificity of this toxin. The scorpion venom insect toxins also differ in their axonal effects and they can be divided into the following three categories:- (1)The AalT, AmlT and BJIT1 increase in an irreversible manner the actionpotential (activatable) sodium permeability and slo~ its turning off. (2) The BJIT2 irreversibly increases the resting sodium permeability and decreases the "activable" sodium permeability. (3)The SmlTI and 2 con> prise a third category and differ markedly from thc above buthoid insect toxins by their cooperative interaction, simultaneous suppression of the conductance of the sodium and potassium ions and a clear reversibility in their action. The chemical and pharmacological diversity of the insect and other scorpion venom toxins is interesting from different aspects. From the evolutionary point of view scorpions are believed to represent one of thc most ancient groups of terrestrial animals. Their basic form, body organization and anatom}, has nol

An approach to insect neuropharmacology changed, during the last 350 millions of years (KJELLESVIG-WAER1N6, 1966). These "living fossils", however, demonstrate a high degree of flexibility and diversity in the composition and function of their venoms. It would appear that the "selective pressure" was mainly directed to and " a b s o r b e d " by the venom gland (which is an essential instrument for the survival of the scorpions) thus enabling their morphological conservatism. F r o m the neuropharmacological point of view the very existence of these selective and diverse forms of insect toxins indicate a certain structural functional uniqueness, complexity and versatility of the ion conducting components in insect axonal membranes. These toxins may be, thus employed as pharmacological tools for the chemical characterization of insect axonal structural elements directly involved in excitability and ion conductance. This approach is strongly supported by some recent developments in m a m m a l i a n neurochemistry. Using neuroblastoma and synaptosomal preparations coupled with a scorpion v e n o m m a m m a l toxin, it was shown that the toxin binds specifically to receptor sites associated with voltage-sensitive sodium channels and its binding was sensitive to the conformational state of the sodium channel as modulated by voltage (CATTERALL et al., 1979; CATTERALL, 1980). This information raises additional interest in the neuropharmacological significance of the scorpion venom insect toxins. Acknowledgements--The studies presented in this review were supported by a grant from the Israel Academy of Sciences (1972/74) and grants nbs 730 and 2461/81 from the U.S.-lsrael Binational Science Foundation (BSF), Jerusalem.

REFERENCES ADAM K. R. and WEISS C. (1959) Actions of scorpion venom on skeletal muscle Br. J. Pharmac. 14, 334-339. BEARG W. J. (1961) Scorpions biology and effect of their venom. Bull. Univ. Kanass agric. Exp. Stn. 649. BRAZIL O. V., NEDER A. C. and CORRADO A. P. (1973) Effects and mechanism of action of Tityus serrulatus venom on skeletal muscle. Pharmac. Res. Commun. 5, 137 150. CATTERALL W. A. (1977) Membrane potential dependent binding of scorpion toxin to the action potential Na + ionophor. J. biol. Chem. 2512, 8660-8668. CATTERALL W. A. (1980) Neurotoxins that act on voltage sensitive sodium channels in excitable membranes. A. Rev. Pharmac. Toxic. 20, 15 43. CATTERALL W. A., MORROW C. S. and HARTSHORNE R, P. (1979) Neurotoxin binding to receptor sites associated with voltage sensitive sodium channels in intact, lysed and detergent-solubilized brain membranes. J. biol. Chem. 254, 11379-I 1387. COURAUD F., ROCHAT H. and LISSITZKY S. (1978) Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na + ionophore in neuroblastoma cells. Biochem. biophys. Res. Commun. 83, 1525-1530. D'AJELLO W., ZLOTKIN E., MIRANDA F., L1SSITZKY S. and BETTINI S. (1972) The effect of scorpion venom and pure toxin on the cockroach central nervous system. Toxicon. 10, 399-404. DAHLQUIST F. W. (1978) The meaning of Scatchard and Hill plots. Meth. Enzym. 48, 270-299. DARBON H., ZLOTKIN E., KOPEYAN C., VAN RIETSCHOTEN J.

235

and ROCHAT H. (1982) Covalent structure of the insect toxin of the North African scorpion Androetonus australis Hector. Int. J. Peptide Protein Res. 20, 320-330. DEL POZO E. C. and ANGUIANO L. G. (1974) Acciones del venero de alacran sobre la actividad motora de musculo estriado. Rev. hist. Salubr. Ep!ferm. trop. (Mex) 8, 231-263. GORDON D., ZLOTKIN E. and KANNER B. (1982) Functional membrane vesicles from the nervous system of insects. I sodium and chloride dependent 7-aminobutyric acid transport. Biochem. Biophys. Acta 688, 229-236. GOYFFON M. and KOVOOR J. (1978) Chactoid venoms. In Arthropod Venoms (Ed. by BETTINI S.), pp. 395-418. Springer, Berlin, Heidelberg. HOUSSAV B. A. (1919) Action physiologique du venin des scorpions (Buthus quinquestriatus and Tityus bahiensis). J. Physiol. Path. g~n. 18, 305-317. JOVER E., MARTIN-MOUTOT N., COURAUD F. and ROCHAT H. (1978) Scorpion toxins: specific binding to rat synaptosomes. Bioehem. Biophys. Res. Commun. 85, 377 382. KAMON E. and SHULOV A. (1963) Estimation of locust resistance to scorpion venom. J. Insect Path. 5, 206-214. KATZ N. L. and EDWARDS CH. (1972) The effect of scorpion venom on the neuromuscular junction of the frog. Toxicon 10, 133-137. KJELLESVIG-WEARING E. N. (1966) Silurian scorpions of New York. J. Paleom. 40, 359 375. KLOTZ I. M. and HUNSTON D. H. (1971) Properties of graphical representations of multiple classes of binding sites. Bioehemistry 10, 3065-3069. KOPEYAN C., MARTINEZ G., LISSITZKY S., MIRANDA F. and ROCHAT H. (1974) Disulfide bonds of toxin II of the scorpion Androctonus australis Hector. Eur. J. Biochenr 47, 483-489. KOPPENHOFER E. and SCHMIDT H. (1968) Incomplete sodium inactivation in nodes of Ranvier treated with scorpion venom. Experientia 24, 41 42. LA GRANGE R. G. and RUSSELLF. (1971) Effects of Centrurbides sculpturatus and C. gertschi venom on the mammalian nerve-muscle preparation: a possible mechanism of action. Proc. West Pharmae. Soc. 14, 163-165. LAZAROVlCI P. (1980) Studies on the composition and action of the venom of the scorpion Scorpio maurus palmatus (Scorpionidae). Ph.D. thesis Hewbrew University of Jerusalem (in Hebrew). LAZAROVICI P., YANAI P., PELHATE M. and ZLOTKIN E. (1982) Insect toxic components from the venom of a chactoid scorpion Scorpio maurus pahnatus (Scorpionidae). J. biol. Chem. 257, 8397 8404. LAZAROVlCl P. and ZLOTKIN E. (1982) A mammal toxin derived from the venom of a chactoid scorpion. Comp. Biochem. Physiol. 71 C, 177-18 I. LESTER D., LAZAROVICI P., PELHATE M. and ZLOTKIN E. (1982) Two insect toxins from the venom of the scorpion Buthotus judaicus: purification characterization and action. Biochem. Biophys. Acta. 701,370-381. MIRANDA F., KOPEYAN C., ROCHAT C., ROCHAT H. and LlSSlTZKY S. (1970) Purification of animal neurotoxins. Isolation and characterization of eleven neurotoxins from the venom of the scorpions Androctanus australis Hector, Buthus oceitanus tunetatus and Leiurus quinquestriatus. Eur. J. Biochem. 16, 514 523. NARAHASHI T., SHAPIRO B. I., DEGUSHI T., SCUKA M. and WANG CH. M. (1972) Effects of scorpion venom on squid axon membranes. Am. J. Physiol. 282, 85(~857. PANSA M. C., MIGLIORI-NATALIZIG. and BETTINI S. (1973) Effect of scorpion venom and its fractions on the crayfish stretch receptor organ. Toxicon II, 283 286. PARNAS I., AVGAR O. and SHULOV A. (1970) Physiological effects of venom of Leiurus quinquestriatus on neuromuscular systems of locust and crab. Toxicon 8, 67-79. PARNAS I. and RUSSELL F. E. (1967) Effects of venoms on nerve, muscle and neuromuscular junction. In Animal

236

ELIAHU ZLOTKIN

Toxins (Ed. by RUSSELL F. E. and SAUNDERS P. R.k pp. 401 427. Pergamon, Oxford, New York. PELHATE M. and PWHON Y. (19741 Selective inhibition of potassium current in the giant axon of the cockroach. J. Physiol., Lond. 242, 90--91. PELHATE M. and ZLOTKIN E. (1981) Voltage dependent slowing of the turn off of Na + current in the cockroach giant axon induced by the scorpion venom "'insect toxin". J. Physiol., Lond. 319, 30 31. PELHATE M. and ZLDTKIN E. (1982) Actions of insect toxin and other toxins derived from the venom of the scorpion Androctonus australis in isolated giant axons of the cockroach (Periplaneta americana). J. exp. Biol. 97, 67 77. PICHON Y. and BOISTEL J. (1967) Current voltage relations in the isolated giant axon of the cockroach under w)ltageclamp conditions. J. e.,:p. Biol. 47, 343 355. RATHMAYER W., WALTHER Crt. and ZLDFKIN E. (19771 The effect of different toxins from scorpion venom on neuromuscular transmission and nerve action potentials in the crayfish. Comp. Biochem. Physiol. 56c, 35 38. RATHMAYER W.. RUtILAND M.. TINTPULVER M., WAr.litER CH. and ZLOTKIN E. (1978) The effect of toxins derived from the venom of the scorpion Androctom~s austrulis Hector on neuromuscular transmission. In Toxins: Animal. Plant and Microbial. (Ed. by ROSliNBER(; P.), pp. 629-637. Pergamon, Oxford. RAY R., MDRROW C. S. and CATTERAI,L W. A. {1978} Binding of scorpion toxin to receptor situs associated with voltage sensitive sodium channels in synaptic nerve ending particles. J. biol. Chem. 253, 7307 7313. ROMEY G., CtnCHEPORTICHE R., LAZI)UNSKI M., RO('HAT H., MIRANDA F. and LISSITZKY S. (1975) Scorpion neurotoxin: a presynaptic toxin which affects both Na + and K ~ channels in axons. Biochem. biophys. Res. Cnmmllll. 64, 115 121. ROCHAT C., ROCHAT H., MIRANDA F. and LISSIrZKY S. (1967) Purification and some properties of the neurotoxins of Amh'octomcs australis Hector. Biochemistry 6, 578 588. RO('HAT H., BERNARD P. and CDURAUD F. (1979) Scorpion toxins: Chemistry and mode of action. Adv. in Cytophurmac. 3, 325 333. RUHLAND M., ZLOTKIN E. and RATHMAYER W. (1977) The effect of toxins from the venom of the scorpion Androctom~s australis on an spider nerve-muscle preparation. Toxicon 15, 157 160. STAHNKE H. L. (1966) Some aspects of scorpion behavior. Bull. 5th Cal(ll Acad. Sci. 65, 65 80. TAMKUN M. M. and CATTERAI,L W. A. (1981) Reconstitutions of the voltage sensitive sodium channel of rat brain from solubilized components. .I. biol. Chem. 256, 11457-11463. TEITELBALTM Z., LAZAROVICI P. and ZLOTKIN E. (1979) Selective binding of the scorpion venom insect toxin to insect nervous tissue. Insect Biochenl. 9, 3 4 3 346. TINTPULVER M., ZERACHIA T. and ZLOTKIN E, (1976) The

action of toxins derived from scorpion venom on the ileal smooth muscle preparation. Toxicon 14, 371 377. VAC'H()N M. (1952) Etudes sur les Scorpions. Institute Pasteur de Algerie. W,',L'IHIiR C.. ZLOTKIN E. and RArlIMAYER W. (1976) Action of different toxins from the scorpion Androctomus australis on a locust nerve muscle preparation. J. Insect. Physiol. 22, 1187 1194. YAIIEL-NIv A. and ZLOTKIN E. (1979) ('omparative studies on venom obtained from individual scorpions by natural stings. 7b.x-icon 17, 435 446. ZI()TKIN E., Bt,~INDHEIM S. A. and SHt;LOV A. (1970) Effect of the venom of the scorpion Leiurus quinquestriutus on the tympanic nerve of the locust Locusta mioratoria migratorioides. Toxicon 8, 47 49. ZLOTKIN E., FRAENKIL G., MIRANDA E. and hSSHZkV S. (1971a) The effect of scorpion venom on blowfly larvae: a new method for the evaluation of scorpion venom potency. 7bvicon 9, 1 s l ZLOrKIx E.. LEBOVITS N. and SHVLOV A. (1972c)Toxic effects of the venom of the scorpion Scorpio muurus pulmatu,s (Scorpionidae). Rir. Parassit. 33, 237 243. ZLOTKIN E., LEBOVITS N. and SHULDV A. (1973) Hemolytic action of the venom of the scorpion Scorpio maurus palmatus (Scorpionidae}. In Animal and Plant Toxins (Ed. by KAISER E.I, pp. 67 72. Goldman, Munchen. ZLOTKIN E.. MARTINEZ O., RO('HAT H. and MIRANDA F. {19751 A protein toxic to crustacea from the venom of the scorpion Androctom~s austrulis. Insect Biochem. 5, 243 250. ZLO['KIN E., MIRANI)A F.. Kt~P~YAN G. and LIssprzK~ S. (1971b) A new toxic protein in the venom of the scorpion Androclomts australis Hector. To.xicon 9, 9 13. ZLO'rKIN E., MIRANDA F, and LISSITZKV S. (1972a) A factor toxic to crustacean in the venom of the scorpion Androctomts australis Hector. ['osicon 10, 211 216. ZLOrKIN E.. MmAYDA F. and Lisslrz~z¥ S. (1972b) Proteins in Scorpion venoms toxic to m a m m a l s and insects. To\icon 10,207 210. ZI.OIKIN E., MmANDA F. and ROCHA! H. (1978) Chemistry and pharmacology of buthinae scorpion venoms. In Arthropod Venoms (Ed. by BEXXrNI S.). pp. 317 369. Springer, Berlin, Heidelberg. ZLOTKIN E., RO(;HAT H., KUPEYAN ('., MIRANDA F. and LISSIrZKY S. (1971c) Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector. Biochimie (Paris) 53, 1073 1078. ZL()TKIN E., TEITELBAUMZ , ROCHAT H. and MIRANDA F. 11979) The insect toxin from the venom of the scorpion Amh'octonus muuretanicus: Purification, characterization and specificity. Insect Biochem. 9, 347- 354. Zt.OTKIN E., TErrELBAt!M Z.. LESTER [). and LAZAROVI('I P. (1980) Toxins selective to insects derived from scorprion venoms. In Natural Toy)us (Ed. by EAKER D. and WADOSTR6M T.), pp. 637 646. Pergamon Press. Oxford, New York.