Báo cáo khoa học: An ‘Old World’ scorpion b-toxin that recognizes both insect and mammalian sodium channels A possible link towards diversification of b-toxins ppt

An ‘Old World’ scorpion b-toxin that recognizes both insect
and mammalian sodium channels
A possible link towards diversification of b-toxins
Dalia Gordon
1
, Nitza Ilan
1,6
, Noam Zilberberg
2
, Nicolas Gilles
3
, Daniel Urbach
1
, Lior Cohen
1
, Izhar Karbat
1
,
Oren Froy
1
, Ariel Gaathon
4
, Roland G. Kallen
5
, Morris Benveniste
6
and Michael Gurevitz
1
1
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel;
2

Department of Life Sciences,
Ben-Gurion University, Israel;
3
CEA, De
´
partment d’Inge
´
nie
´
rie et d’Etudes des Prote
´
ines, France;
4
Bletterman Research Laboratory
for Macromolecules, The Hebrew University-Hadassah, Medical School Jerusalem, Israel;
5
Department of Biochemistry and
Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA;
6
Department of Physiology and Pharmacology,
Sackler School of Medicine, Tel-Aviv University, Israel
Scorpion toxins that affect sodium channel (NaCh) gating
in excitable cells are divided into a- and b-classes. Whereas
a-toxins have been found in scorpions throughout the world,
anti-mammalian b-toxins have been assigned, thus far, to
ÔNew WorldÕ scorpions while anti-insect selective b-toxins
(depressant and excitatory) have been described only in the
ÔOld WorldÕ.
2
This distribution suggested that diversification

of b-toxins into distinct pharmacological groups occurred
after the separation of the continents, 150 million years ago.
We have characterized a unique toxin, Lqhb1, from the ÔOld
WorldÕ scorpion, Leiurus quinquestriatus hebraeus,that
resembles in sequence and activity both ÔNew WorldÕ
b-toxins as well as ÔOld WorldÕ depressant toxins. Lqhb1
competes, with apparent high affinity, with anti-insect and
anti-mammalian b-toxins for binding to cockroach and rat
brain synaptosomes, respectively. Surprisingly, Lqhb1also
competes with an anti-mammalian a-toxin on binding to rat
brain NaChs. Analysis of Lqhb1 effects on rat brain and
Drosophila Para NaChs expressed in Xenopus oocytes
revealed a shift in the voltage-dependence of activation to
more negative membrane potentials and a reduction in
sodium peak currents in a manner typifying b-toxin activity.
Moreover, Lqhb1 resembles b-toxins by having a weak effect
on cardiac NaChs and a marked effect on rat brain and
skeletal muscle NaChs
3
. These multifaceted features suggest
that Lqhb1 may represent an ancestral b-toxin group in ÔOld
WorldÕ scorpions that gave rise, after the separation of the
continents, to depressant toxins in ÔOld WorldÕ scorpions and
to various b-toxin subgroups in ÔNew WorldÕ scorpions.
Keywords: scorpion toxins; sodium channel subtypes; toxin
diversification.
Scorpion Ôlong chainÕ toxins affecting voltage-gated
sodium channels (NaCh) are polypeptides of 61–76 amino
acids long that traditionally are divided between two
major classes, a and b, according to their physiological

effects on channel gating and their binding properties
[1–3]. a-Toxins slow sodium channel inactivation upon
binding to a homologous cluster of binding sites named
receptor site-3, and are subdivided into distinct groups
according to their potency for mammalian and insect
receptors and their affinity for sodium channel subtypes
[2,4–7]. a-Toxins predominate in the venom of Buthidae
scorpions of the ÔOld WorldÕ (Africa and Asia), but some
representatives have been also identified in ÔNew WorldÕ
(America) scorpions [1]. b-Toxins shift the activation
voltage of sodium channels to more negative membrane
potentials upon binding to receptor site-4 [2,7,8], and vary
greatly in their effects on various animals. Css2 and Css4
(from Centruroides suffusus suffusus) show specificity for
mammals [1]; Cll1 (from C. limpidus limpidus), Cn5, and
Cn11 (from C. noxius) are highly effective on crustaceans
[9–12]; Ts7 and Tst1 (from Tityus serrulatus and T. stig-
murus), and Tbs1 and Tbs2 (from T. bahiensis)are
highly effective on both insects and mammals [1,12–16].
b-Toxins, active on mammals, have thus far been assigned
to scorpions of the ÔNew WorldÕ (Tityus and Centruroides
species), whereas depressant and excitatory b-toxins,
which modify exclusively the activation of insect sodium
channels, have been found strictly in ÔOld WorldÕ Buthoids
[1,2,12,17,18]
4
.
In addition to their effects on sodium channel gating,
classification of toxins to either the a- or b-classrelieson
competition binding assays against the ÔOld WorldÕ toxin,

Aah2, from Androctonus australis hector,andtheÔNew
WorldÕ toxin, Css2, from Centruroides suffusus suffusus,
respectively [1,2]. Further distinction between toxins can be
made using competition binding assays utilizing LqhaIT
Correspondence to D. Gordon or M. Gurevitz, Department of Plant
Sciences, George S. Wise Faculty of Life Sciences,
Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
1
Fax: + 972 3 6406100, Tel.: + 972 3 6409844,
E-mail: or
Abbreviations:Lqhb1, Leiurus quinquestriatus hebraeus beta toxin 1;
LqhIT2, anti-insect selective depressant toxin; LqhaIT, anti-insect
a-toxin; Lqh2, anti-mammalian a-toxin; Css2, Css4, Centruroides
suffusus suffusus b-toxins 2 and 4; Ts7, Tityus serrulatus toxin 7
(also called Ts1 and c-toxin); NaCh, sodium channel.
(Received 27 March 2003, accepted 29 April 2003)
Eur. J. Biochem. 270, 2663–2670 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03643.x
(from Leiurus quinquestriatus hebraeus)forthea-anti-insect
and Lqh3 for the a-like groups [4,5]; Bj-xtrIT (from
Buthotus judaicus [19]) and AahIT [17] for the anti-insect
selective excitatory, and LqhIT2 for the depressant b-toxin
groups [2,20].
The ability of excitatory and depressant anti-insect
selective toxins to compete with ÔNew WorldÕ b-toxins, such
as Ts7, suggests that common features may exist in their
receptor sites on various insect and mammalian sodium
channels [1,2,21,22]. As b-toxins that affect mammalian
sodium channels have not been identified in the ÔOld WorldÕ,
it was assumed that diversification of anti-mammalian
b-toxins in the ÔNew WorldÕ, and excitatory and depressant

toxins in the ÔOld WorldÕ occurred from an unknown
ancestral progenitor after the separation of the continents
150 million years ago.
Yet, an ÔOld WorldÕ scorpion toxin, AahIT4, with high
affinity for the AahIT binding site on insect neuronal
membranes, could also compete with a- (Aah2) and
b- (Css2) anti-mammalian toxins for binding to rat brain
synaptosomes [23]. As AahIT4 shares little sequence
similarity with any of the known anti-mammalian scorpion
toxins [1,12], and no information was available on its mode
of action, it was considered a unique member of a new
pharmacological group of neither a- nor b-type toxins
[12,23]. More recently, two toxins have been purified from
the Asian scorpion, Buthus martensii Karsch, BmK AS and
BmK AS-1, which share 80 and 86% sequence identity
with AahIT4 [24]. These toxins are weakly toxic to insects,
are not toxic to mice and inhibit Na
+
currents in neurons
of rat dorsal root ganglia [25]. Still, no pharmacological
details that allow their classification to a- or b-toxins have
been provided.
Here we report the isolation and characterization of an
ÔOld WorldÕ toxin, Lqhb1 that probably belongs to the same
group as AahIT4 if sequence similarity and binding features
are examined
5
.Lqhb1 competes with both a- and b-toxins
for binding to rat brain synaptosomes and with excitatory
anti-insect selective toxins in insect neuronal membranes.

We show that Lqhb1 affects insect and mammalian NaCh
subtypes in a manner that typifies ÔNew WorldÕ b-toxins.
This suggests that b-toxins affecting mammalian NaChs
have existed and are still present in ÔOld WorldÕ scorpions.
The effects of Lqhb1 on various NaCh subtypes may
suggest that this toxin represents an ancient group of
b-toxins that gave rise to the anti-insect depressant toxins in
the ÔOld WorldÕ andtotheb-toxins active on mammals in
the ÔNew WorldÕ.
Experimental procedures
Biological material
Venom from Leiurus quinquestriatus hebraeus was collected
from scorpion stings to a parafilm membrane. Sarcophaga
falculata (blowfly) larvae and Periplaneta americana (cock-
roaches) were bred in the laboratory. Albino laboratory
ICR mice were purchased from the Levenstein farm in
Yokneam, Israel. As purified Lqhb1 was obtained in a
limited amount, the toxin was also purchased from Latoxan
(LTx-003; Valance, France) together with the anti-mam-
malian a-toxin, Lqh2.
Purification and analysis of Lqhb1
Toxin purification was carried out by conventional chro-
matographic procedures described previously [19,20].
Briefly, crude venom (6 mg) from 30 scorpions was
lyophilized, dissolved in 3 mL of 10 m
M
ammonium acetate
pH 6.7, and subjected to two chromatographic steps: anion-
exchange chromatography on a 0.8-mL DEAE-Sephadex
column (Sigma, USA) equilibrated in 10 m

M
ammonium
acetate pH 8.0. A linear gradient of 0.01–1.0
M
ammonium
acetate pH 6.7 at a flow rate of 0.5 mLÆmin
)1
at room
temperature was applied and 1.5 mL aliquots were collected
and lyophilized. Several fractions eluted by 275–375 m
M
ammonium acetate that were toxic to blowfly larvae were
combined and further purified via HPLC using an analytical
C
18
column (250 · 10 mm; Vydac, USA). Sample was
loaded in 0.1% trifluoroacetic acid in water (Buffer A) and
eluted with a stepwise increasing gradient of 0.1% trifluoro-
acetic acid in acetonitrile (Buffer B) at a flow rate of
1mLÆmin
)1
. The protein eluted after 19.5 min and con-
tained 163 lg pure polypeptide of 7463 Da (determined by
Electrospray Mass Spectrometry, Technion, Haifa, Israel)
and exerted depressant activity on blowfly larvae. Amino
acid sequence analysis, carried out by automated Edman
degradation using an Applied Biosystem (Foster City, CA,
USA) gas-phase sequencer (470 A) connected to its corres-
ponding PTH-analyzer (120 A) and data system (900 A)
identified the first 24 N-terminal residues. This amino acid

sequence was used to pull out the entire cDNA clone using
Ôback-to-backÕ oligonucleotide primers in a PCR technique
described by Zilberberg and Gurevitz [
6
26]. Briefly, two
degenerate oligonucleotides, designed according to the
protein sequence (Primer 1: 5¢-ANACYTTPCANCC
HGTNGC-3¢;Primer2:5¢-GGTGYGTNATHGAYGA
YGC-3¢; N stands for either A, G, T or C; Y for C or T;
PforAorG;HforA,T,orC;Fig.1A),wereusedas
primers for PCR (MJ Research thermocycler, USA) with
L. q. hebraeus cDNA library [27] as DNA template to
amplify the entire Lqhb1-cDNA. Reaction conditions were:
30 cycles of 1 min at 94 °C, 1 min 50 °Cand1minat
72 °C. The PCR product was blunt-ended, phosphorylated,
cloned into the SmaI site of pBluescript, and subjected to
sequence analysis using Sequenase II (United States
Biochemicals). The cloned gene was labeled with
32
Pand
used as a probe to pull out by colony hybridization the
original cDNA from the library (Fig. 1).
Recombinant toxin production
Bj-xtrIT and LqhIT2 were produced in Escherichia coli
strain BL21 and reconstituted by in vitro folding as was
described previously [19,28]. A synthetic gene was used to
produce Css4 b-toxin (I. Karbat, D. Gordon & M. Gurevitz,
unpublished observation)
7
following the procedure described

for LqhIT2 [28].
Toxicity assays
Five toxin concentrations were tested using four-day-old
blowfly larvae ( 100 mg body weight). Ten larvae were
injected intersegmentally at the rear side with each toxin
concentration in three independent experiments. A positive
2664 D. Gordon et al. (Eur. J. Biochem. 270) Ó FEBS 2003
result was scored when a characteristic paralysis (transient
immobilization and contraction replaced by gradually
increasing flaccidity) was obtained and lasted at least
15 min. ED
50
values were calculated as was described
previously [28].
Binding experiments
Insect synaptosomes were prepared from whole heads of
adult P. americana according to a previously described
method [19]. Mammalian brain synaptosomes were pre-
pared from adult albino Sprague–Dawley rats ( 300 g,
laboratory bred), as was described previously
8
[29]. Mem-
brane protein concentration was determined using a Bio-
Rad Protein Assay, using bovine serum albumin (BSA) as a
standard. Lqh2, LqhaIT, Bj-xtrIT and Css4 were radio-
iodinated by iodogen (Pierce, Rockford, USA) using 5 lg
toxin and 0.5 mCi carrier-free Na
125
I(Amersham,UK)and
the monoiodotoxins were purified using an analytical Vydac

RP-HPLC C
18
column, as was described previously [4,30].
The concentration of the radiolabeled toxin was determined
according to the specific activity of the
125
I corresponding to
2500–3000 d.p.m.Æfmol
)1
of monoiodotoxin, depending on
the age of the radiotoxin and by estimation of its biological
activity (usually 50–70%; see [30] for details). The compo-
sition of the medium used in the binding assays was (in m
M
):
choline Cl, 130; CaCl
2
,1.8;KCl,5;MgSO
4
,0.8,Hepes50;
Glucose 10, and 2 mgÆmL
)1
BSA, pH 7. Wash buffer
composition was (in m
M
): choline Cl, 140; CaCl
2
,1.8;KCl,
5.4; MgSO
4

,0.8;Hepes,50;5mgÆmL
)1
BSA, pH 7.5.
Binding assays were performed as was described previously
[29,30]. Nonspecific toxin binding was determined in the
presence of a high concentration of unlabeled toxin, as
specified in figure legends, and consisted typically of 5–15%
of total binding. Equilibrium competition binding assays
were performed using increasing concentrations of the
unlabeled toxins in the presence of a constant low con-
centration of [
125
I]toxins, and analyzed by the computer
program
KALEIDAGRAPH
(Synergy Software, Reading, PA,
USA) using a nonlinear fit to the Hill equation (for IC
50
determination). The K
i
were calculated by the equation
K
i
¼ IC
50
/[1 + (L*/K
d
)], where L* is the concentration of
hot toxin and K
d

is its dissociation constant. Each experiment
was performed in duplicate samples and repeated multiple
timesasindicated(n)foreachK
i
value. Bj-xtrIT excitatory
toxin, a marker of receptor site-4 in insect sodium channels
[19,21], was used in competition binding assays of LqhIT2
and Lqhb1 toxins to cockroach neuronal membranes.
Sodium channel expression and two-electrode
voltage-clamp assays using
Xenopus
oocytes
cRNAs encoding rat skeletal muscle (Na
v
1.4; rSkM1), rat
brain IIa (rNa
v
1.2a; rBIIA), human heart (hNa
v
1.5; hH1)
subtypes and insect Drosophila Para (DmNa
v
1; gift from
J. Warmke, Merck, New Jersey, USA)
9
sodium channel
a-subunits, and the auxiliary human b1 and insect TipE
subunits (gift from M. S. Williamson, IACR-Rothamsted,
10
UK), were transcribed in vitro usingT7RNApolymerase

and the mMESSAGE mMACHINE
TM
system (Ambion,
USA [31,32]); and were injected into Xenopus laevis oocytes
as described by Shichor et al. [21]. One to four days after
injection, currents were measured by two-electrode voltage
clamp using a Gene Clamp 500 amplifier (Axon Instru-
ments, Union City, CA, USA). Data were sampled at
10 kHz and filtered at 5 kHz. Data acquisition was con-
trolled by a Macintosh PPC 7100/80 computer, equipped
with ITC-16 analog/digital converter (Instrutech Corp.,
Fig. 1. Nucleotide sequence of the cDNA clone
encoding Lqhb1 and its deduced amino acid
sequence. The putative signal sequence is
underlined and a polyadenylation signal
appears in lowercase letters. The amino acid
stretch used for design of back-to-back oligo-
nucleotide primers is indicated by arrows 1
and 2 (see Experimental procedures for
sequence). The technique used for cloning
has been described previously [26].
Ó FEBS 2003 A novel ÔOld WorldÕ scorpion b-toxin (Eur. J. Biochem. 270) 2665
Port Washington, NY, USA), utilizing
SYNAPSE
(Synergistic
Systems, Sweden). Capacitance transients and leak currents
were removedbysubtracting ascaled control traceutilizing a
P/6protocol[32].Bathsolution contained (in m
M
):NaCl, 96;

KCl, 2; MgCl
2
,1;CaCl
2
,1.8;Hepes,5;pH 7.85.Toxinswere
diluted with bath solution containing 1 mgÆmL
)1
BSA.
OocyteswerewashedwithbathsolutionflowingfromaBPS-
8 perfusion system (ALA Scientific Instruments, Westbury,
NY, USA) with a positive pressure of 4 psi. Approximately
1 mL of toxin-containing solution was perfused over the
oocyte situated in a 200-lL chamber at room temperature.
Results
Purification and cloning of a functionally unique
b-toxin from
L. q. hebraeus
Lqhb1 was purified from the venom of L. q. hebraeus by
cation-exchange chromatography followed by RP-HPLC
(see Experimental procedures for details). Upon injection of
Lqhb1 to blowfly larvae, a short, transient contraction was
observed followed by a dose-dependent, long-lasting flaccid
paralysis (ED
50
¼ 102ngper100mgbodyweight).These
effects on blowfly larvae are typical of depressant toxins,
such as LqhIT2 [20,28]. The amino acid sequence deduced
from the cDNA nucleotide sequence reveals 73% sequence
identity with AahIT4 [23], and 85 and 91% with BmK AS
and BmK AS-1 [24], respectively (Fig. 2). The relative

molecular mass of Lqhb1, determined by mass spectroscopy,
is 7463 Da, which matches the calculated value of the amino
acid sequence deduced from the cDNA nucleotide sequence.
This suggests that Lqhb1 does not undergo post-trans-
lational processing as has been shown for other toxins [27].
Binding of Lqhb1 to insect and rat brain NaCh
The sequence similarity between Lqhb1 and AahIT4 [23]
could suggest similar activity and therefore we analyzed the
Fig. 2. Sequence alignment of toxin representatives of various pharmacological groups that affect sodium channels. The alignment relies on known
structures, putative models [2] and conserved cysteine residues. Dashes indicate gaps. Asterisks (*) designate alpha-amidation of C-termini. Cysteine
residues that are conserved in all scorpion toxins and form disulfide bonds (plane lines) are shaded by light grey, whereas cysteines involved in
unique disulfide bonds (dashed lines) are shaded by dark grey. Aah, Androctonus australis hector;Bj,Buthotus judaicus;BmK,Buthus martensii
Karsch; Cn, Centruroides noxius;Cll,Centruroides limpidus limpidus;CsE,Centruroides sculpturatus Ewing; Css, Centruroides suffusus suffusus;Lqh,
Leiurus quinquestriatus hebraeus; Lqq, Leiurus quinquestriatus quinquestriatus;Ts,Tityus serrulatus;Tst,Tityus stigmurus;Tb,Tityus bahiensis [12].
2666 D. Gordon et al. (Eur. J. Biochem. 270) Ó FEBS 2003
pharmacological profile of Lqhb1 in competition binding
assays utilizing well established a- and b-toxin markers.
Lqhb1 inhibited, though at high concentration, the binding
of the classical anti-mammalian a-toxin, Lqh2 [5,29], to
site-3 in rat brain synaptosomes (K
i
¼ 1.85 l
M
, n ¼ 2;
Fig. 3A). A significantly lower concentration of AahIT4
(60 n
M
) inhibited the binding of Aah2 [23]. Lqhb1also
competed with the anti-mammalian b-toxin, Css4, for
binding to site-4 in rat brain synaptosomes with very high

apparent affinity [K
i
¼ 0.51 ± 0.27 n
M
, (mean ± SD),
n ¼ 3; Fig. 3B]. In comparison, the apparent affinity of
AahIT4 for the binding site of Css2 is much lower
(IC
50
¼ 40 n
M
[23]). Although Lqhb1 competed with both
site-3 and site-4 toxins for binding to rat brain sodium
channels, it did not inhibit the binding of the a-insect toxin,
LqhaIT, to receptor site-3 on cockroach channels (not
shown). Yet, Lqhb1 inhibited the binding of the excitatory
toxin, Bj-xtrIT, to cockroach synaptosomes with high
apparent affinity (K
i
¼ 0.170 ± 0.017 n
M
, n ¼ 3; Fig. 3C).
These results demonstrate the similarity in binding capabi-
lities between Lqhb1 and AahIT4, although they vary
quantitatively in the apparent affinity for receptor sites 3
and 4 in rat brain sodium channels.
Effects of Lqhb1 on sodium channel subtypes
The effects of Lqhb1 on sodium currents were examined on
various mammalian and insect sodium channels expressed
in Xenopus oocytes using two-electrode voltage-clamp. In

the absence of toxin, a step-depolarization from a holding
potential of )80 to )40 mV elicits almost no current in
oocytes expressing the rNa
v
1.2a channels (Fig. 4A). How-
ever, in the presence of 2.5 l
M
Lqhb1, a significant peak
current is observed (Fig. 4A) that indicates a shift of
channel activation to more negative membrane potentials as
readily observed in Fig. 4B. In contrast, upon depolarization
to )20 mV, under which channel activation in the
absence of toxin was near maximal, peak currents were
inhibited 70 ± 10% (n ¼ 4) in the presence of Lqhb1
(Fig. 4A,B). The current–voltage (I–V) relationship delin-
eated in Fig. 4B indicates two clear effects imposed by
Lqhb1onrNa
v
1.2a channels. The shift in the voltage-
dependence of channel activation to more negative mem-
brane potentials, and a marked decrease in the sodium
peak-current amplitude typify the phenotypic change of
sodium currents induced by scorpion b-toxins active on
mammals, e.g. Ts7 and Css4 [8,14,33–35]. As ÔNew WorldÕ
b-toxins that affect mammals show weak activity on cardiac
sodium channels [34,36], the specificity of Lqhb1was
further examined on rat skeletal muscle (rNa
v
1.4) and
human heart (hNa

v
1.5) channels. The toxin effects on
rNa
v
1.4 were similar to those obtained with rNa
v
1.2a,
whereas no shift in channel activation and only little
decrease in peak current were observed in hNa
v
1.5
(Fig. 4C,D). These results indicate that Lqhb1 is similar
to other b-toxins in action and specificity to mammalian
sodium channel subtypes.
Lqhb1 is similar to the anti-insect depressant toxin,
LqhIT2, in its toxic symptoms induced in blowfly larvae,
and the ability to compete for the binding site of the
excitatory toxin, Bj-xtrIT, in insect NaChs [19,22]. There-
fore, the electrophysiological effects of Lqhb1 and LqhIT2
were compared on an insect NaCh. Both toxins revealed
typical b-toxin effects on oocytes expressing the Drosophila
Para NaCh and TipE. The peak sodium current, elicited
by depolarization to )10 mV, decreased 45 ± 13% and
66 ± 5% (n ¼ 3) in the presence of 2.5 l
M
Lqhb1and
LqhIT2, respectively. In addition, the I–V curves obtained
indicate that the appearance of the sodium current is shifted
to more negative potentials in the presence of either toxin
(Fig. 5A). Similar effects were observed when LqhIT2 was

applied on an isolated cockroach axon [18]. However, in
contrast to Lqhb1 (Fig. 4B), LqhIT2 in concentrations as
high as 50 l
M
did not affect the rNa
v
1.4 channel (Fig. 5B),
Fig. 3. Binding of Lqhb1 to receptor sites 3 and 4 in rat brain and cockroach sodium channels. Competition of Lqhb1 with the site-3 a-toxin, Lqh2 (A),
and with the site-4 b-toxin, Css4 (B), for binding to rat brain NaChs. Rat brain synaptosomes (65 lgproteinÆmL
)1
) were incubated 30 min at 22 °C
with 110 p
M
[
125
I]Lqh2 or 120 p
M
[
125
I]Css4 and increasing concentrations of the indicated toxins. Nonspecific binding, determined in the presence
of 200 n
M
Lqh2 or 1 l
M
Css4, respectively, was subtracted. (C) Competition of Lqhb1with[
125
I]Bj-xtrIT for binding to cockroach NaCh.
Cockroach neuronal membranes (16 lgÆmL
)1
) were incubated for 60 min at 22 °Cwith180p

M
[
125
I]Bj-xtrIT and increasing concentrations of the
indicated toxins. Nonspecific binding, determined in the presence of 1 l
M
Bj-xtrIT, was subtracted. The amount of bound [
125
I]toxin is provided as
the percentage of maximal specific binding without competitor. The competition curves were analyzed using the nonlinear fit of the Hill equation
(withaHillcoefficientof1)fordeterminingoftheIC
50
values (see Experimental procedures). The data points are means of two to three
measurements from representative experiments, of which the following K
i
values were obtained (in n
M
): Lqh2, 2.1; Lqhb1, 1900 (in A). Css4, 1.1;
Lqhb1, 0.64 (in B). Bj-xtrIT, 0.35; Lqhb1, 0.2 (in C).
Ó FEBS 2003 A novel ÔOld WorldÕ scorpion b-toxin (Eur. J. Biochem. 270) 2667
nor the rNa
v
1.2a and hNa
v
1.5 mammalian channels
(not shown), indicating high specificity for insect sodium
channels.
Discussion
Comparison of toxins found in ÔOld WorldÕ vs. ÔNew WorldÕ
scorpions may provide a hint about their diversification,

but first thorough characterization of their pharmacological
properties and genetic relations is needed. Scorpions of the
family Buthidae originated approximately 350 MA from
the Carboniferous scorpions, Neoscorpiones [37], and were
physically divided 150 MA upon the partition of the Upper
Jurassic Brazilo-Ethiopian continent (Africa–South Amer-
ica). Scorpion a-toxins, that affect NaCh inactivation and
resemble ÔOld WorldÕ classical a-toxins in their amino acid
sequences, have been described in the ÔNew WorldÕ,e.g.
CsE V, Ts IV, and TsTX V [1,12], which suggests that they
existed before the separation of the continents.
As b-toxins active on mammals have not been found thus
far in scorpions of the ÔOld WorldÕ, it could be assumed that
they have developed in Tityus and Centruroides ÔNew WorldÕ
scorpions after the separation of the continents [12]. Yet,
scorpion polypeptides that resemble ÔNew WorldÕ b-toxins
have been reported in the ÔOld WorldÕ and include the
nontoxic polypeptide, AahSTR1 [38], the glycosylated toxin,
Aah6 [39], and the anti-insect selective excitatory and
depressant toxin groups [2,12,17,20]. In addition, polypep-
tides with some b-toxin properties have been found in the
venom of the old world scorpions, Leiurus quinquestriatus
hebraeus [40] and Buthus martensii Karsch [25]. Another
peculiar toxin that competes for both a and b-toxin receptor
sites in rat brain synaptosomes, AahIT4, seems to be related
to b-toxins because it was recognized by antibodies raised
against the b-toxin, Css2, but not by antibodies against the
a-toxin, Aah2, or the excitatory toxin, AahIT [23].
The pharmacological properties of Lqhb1 describe for
the first time a typical b-toxinintheÔOld WorldÕ. The high

similarity in sequence of Lqhb1, Bmk AS, BmK AS-1, and
AahIT4 suggests that these toxins may belong to a unique
group of ÔOld WorldÕ b-toxins (Fig. 2). Lqhb1 resembles
AahIT4 in its high apparent affinity for receptor site-4 in
insect sodium channels (Fig. 3 [23]). Yet, whereas AahIT4
has moderate affinity for both receptor sites 3 and 4 on rat
brain synaptosomes [23], Lqhb1 competes with Lqh2 on
binding to receptor site-3 only at high concentrations, and
binds receptor site-4 in mammalian sodium channels with a
very high apparent affinity (Fig. 3). Although both toxins
seem to belong to one pharmacological group, Lqhb1exerts
typical b-toxin binding properties to a greater extent than
AahIT4. Lqhb1 and AahIT4 vary also in their effect on
blowfly larvae as AahIT4 induces contraction [23] and
Lqhb1 induces flaccid paralysis.
The sequence of Lqhb1 resembles those of ÔNew WorldÕ
b-toxins (41–50% identity; Fig. 2). The pharmacological
features of Lqhb1 are mostly similar to those of the b-toxin,
Ts7, with high affinity binding for insect and mammalian
NaChs (Figs 3–5 [1,2,13]), and preference for mammalian
brain and skeletal muscle NaCh subtypes (Fig. 4 [34]).
Notably, the sequence, binding, and electrophysiological
properties of Lqhb1 show substantial resemblance to those
of ÔOld WorldÕ anti-insect selective depressant toxins, such
Fig. 5. Comparison of the effects of Lqhb1 and LqhIT2 on insect sodium
channels. Curves I–V were obtained from two-electrode voltage-clamp
experiments using Xenopus oocytes that coexpress the Drosophila Para
a-subunit with the auxiliary insect b-subunit TipE (A), or the rNa
v
1.4

a-subunit with b1 (B). Peak amplitudes obtained in the absence of
toxin are designated by filled circles; and peak amplitudes measured
from traces elicited in the presence of toxin are represented by open
circles. Lqhb1 or LqhIT2 (2.5 l
M
) enabled Para channel sodium cur-
rents to be elicited at more hyperpolarized potentials, and decreased
the peak inward currents at potentials > )20mV.Incontrast,50l
M
LqhIT2 had no effect on currents mediated by rNa
v
1.4.
Fig. 4. Effects of Lqhb1 on mammalian sodium channel subtypes.
Current–voltage (I–V) curves were obtained with two-electrode
voltage-clamp experiments using Xenopus oocytes that coexpress rat
brain, rNa
v
1.2a (A, B), rat skeletal muscle, rNa
v
1.4 (C), and human
heart, hNa
v
1.5 (D) channel a-subunits together with the mammalian
auxiliary subunit b1. In A, currents elicited during a depolarizing pulse
to )40 mV (upper traces) or )20 mV (lower traces) in the absence and
presence of 2.5 l
M
Lqhb1, are shown. Note that in the presence of
Lqhb1, the current appears during the )40 mV pulse, in contrast to the
control trace. (B–D) Representative I–V curves are shown for experi-

ments in which the different channels were expressed in Xenopus
oocytes in the absence of toxin (control; d) and in the presence of
2.5 l
M
Lqhb1(s). Appearance of current occurs at more hyper-
polarized potentials in the presence of toxin for oocytes expressing
rNa
v
1.2a or rNa
v
1.4, but not rNa
v
1.5 channels.
2668 D. Gordon et al. (Eur. J. Biochem. 270) Ó FEBS 2003
as LqhIT2 (44–50% identity; Figs 2,3 and 5 [18,20]). The
similarity between Lqhb1 and LqhIT2 is further exemplified
in the effect on the Para NaCh expressed in Xenopus
oocytes. The two toxins still differ substantially in that only
Lqhb1 affects mammalian NaChs (Fig. 5). These features
may suggest that the group of toxins represented by Lqhb1
and AahIT4 evolved into the anti-insect selective depressant
toxins in the ÔOld WorldÕ,andintob-toxins presently found
in ÔNew WorldÕ scorpions, after the separation of the
continents. It seems that diversification of the b-toxins in
the ÔNew WorldÕ proceeded toward those with affinity for
mammals (e.g. Css2 and Css4 [1,12,36]), crustaceans (e.g.
Cn5, Cn11, and Cll1 [9–12]) and a group that acquired
a-like activity while maintaining the structural features of
b-toxins (CsEv1–3 [2,12,14])
11

. Tityus b-toxins, such as Ts7,
Tst1, and Tb1 are highly active on mammals and insects
[16], and thus seem to preserve ancient properties of Lqhb1
in the ÔNew WorldÕ.
Although all known Ôlong chainÕ scorpion toxins share a
similar structural core (a-helix packed against three anti-
parallel b-strands) [2,12,41], and genomic organization
[41,42], the identity of the ancestor polypeptide, from which
they had diverged is a riddle, which is further accentuated
due to difficulties to establish reliable phylogenetic relations
between the available toxin sequences (M. Gurevitz &
D. Gordon, unpublished observations)
12
. Nonetheless, the
recent description of a Ôlong chainÕ polypeptide with only
three disulfide bonds, birtoxin, found in the venom of the
South African scorpion Parabuthus transvaalicus [43,44],
together with the features of Lqhb1 may enable speculation
on a putative route for toxin diversification. Birtoxin is toxic
to mice, inhibits Na
+
currents in dissociated fish retinal cells
in a manner resembling the effect of b-toxins, and shows
substantial sequence similarity to b-toxins from various
ÔNew WorldÕ Centruroides species [43,44]. These features
may suggest that birtoxin represents an ancient group of
toxins that could have evolved into the excitatory anti-insect
selective toxins by acquiring a structurally distinctive forth
disulfide bond (Fig. 2), or, alternatively, into the group
represented by Lqhb1 by acquiring the forth conserved

disulfide bond found in all but the excitatory toxins. The
Lqhb1 group may have evolved after the separation of the
continents to depressant toxins in the ÔOld WorldÕ,andto
b-toxins in the ÔNew WorldÕ. Since the forth disulfide bond
in a and most b-toxins is spatially conserved [2,12,41,45], it
may be hypothesized that toxins with three disulfide bonds,
such as birtoxin, were the progenitors of a-toxins as well.
Acknowledgements
This research was supported in part by the United States–Israel
Binational Agricultural Research and Development grant IS-3259–01
(D. G. and M. G.); by the Israeli Science Foundation, grants 508/00
(D. G.) and 733/01 (M. G.); and by an EU grant QLK3-CT-2000–
00204 (D. G. and M. G.).
References
1. Martin-Eauclaire, M F. & Couraud, F. (1995) Scorpion neuro-
toxins: effects and mechanisms. In Handbook of Neurotoxicology
(Chang, L.W. & Dyer, R.S., eds), pp. 683–716. Marcel Dekker,
New York.
2. Gordon, D., Savarin, P., Gurevitz, M. & Zinn-Justin, S. (1998)
Functional anatomy of scorpion toxins affecting sodium channels.
J. Toxicol. Toxin Rev. 17, 131–159.
3. Gordon, D. (1997) Sodium channels as targets for neurotoxins:
mode of action and interaction of neurotoxins with receptor sites
on sodium channels. In Toxins and Signal Transduction
(Lazarowici, P. & Gutman, Y., eds), pp. 119–149. Harwood,
Amsterdam.
4. Gordon, D., Martin-Eauclaire, M.F., Ceste
`
le, S., Kopeyan, C.,
Carlier, E., Ben Khalifa, R., Pelhate, M. & Rochat, H. (1996)

Scorpion toxins affecting sodium current inactivation bind to
distinct homologous receptor sites on rat brain and insect sodium
channels. J. Biol. Chem. 271, 8034–8045.
5. Gilles, N., Blanchet, C., Shichor, I., Zaninetti, M., Lotan, I.,
Bertrand,D.&Gordon,D.(1999)Ascorpiona-like toxin that is
active in insects and mammals reveals an unexpected specificity of
distribution of sodium channel subtypes in rat brain neurons.
J. Neurosci. 19, 8730–8739.
6. Gilles, N., Chen, H., Wilson, H., Le Gall, F., Montoya, G.,
Heinemann, S.H. & Gordon, D. (2000) Scorpion a and a-like
toxins differentially interact with sodium channels in mammalian
CNS and periphery. Eur. J. Neurosci. 12, 2823–2832.
7. Catterall, W.A. (1992) Cellular and molecular biology of voltage-
gated sodium channels. Physiol. Rev. 72, S15–S48.
8. Strichartz, G., Rando, T. & Wang, G.K. (1987) An integrated
view of the molecular toxicology of sodium channel gating in
excitable cells. Annu. Rev. Neurosci. 10, 237–267.
9. Lebreton, F., Delepierre, M., Ramirez, A.N., Balderas, C. &
Possani, L.D. (1994) Primary and NMR three-dimensional
structure determination of a novel Crustacean toxin from the
venom of the scorpion Centruroides limpidus limpidus Karsch.
Biochemistry 33, 11135–11149.
10. Garcia, C., Becerril, B., Selisko, B., Delepierre, M. & Possani,
L.D. (1997) Isolation, characterization and comparison of a novel
Crustacean toxin with a mammalian toxin from the venom of the
scorpion Centruroides noxius Hoffmann. Comp. Biochem. Physiol.
116B, 315–322.
11. Ramirez-Dominguez, M.E., Olamendi-Portugal, T., Garcia, U.,
Garcia,C.,Arechiga,H.&Possani,L.D.(2002)Cn11,thefirst
example of a scorpion toxin that is a true blocker of Na

+
currents
in crayfish neurons. J. Exp. Biol. 205, 869–876.
12. Possani, L.D., Becerril, B., Delepierre, M. & Tytgat, J. (1999)
Scorpion toxins specific for Na
+
-channels. Eur. J. Biochem. 264,
287–300.
13. Barhanin, J., Giglio, J.R., Leopold, P., Schmid, A., Sampaio, S.V.
& Lazdunski, M. (1982) Tityus serrulatus venom contains two
classes of toxins. J. Biol. Chem. 257, 12553–12558.
14. Meves, H., Simard, J.M. & Watt, D.D. (1986) Interactions of
scorpion toxin with the sodium channel. Annu. NY Acad. Sci.
479, 113–131.
15. Becerril, B., Corona, M., Coronas, F.I.V., Zamudio, F., Calderon-
Aranda, E.S., Fletcher, P.L., Martin, B.M. & Possani, L.D. (1996)
Toxic peptides and genes encoding toxin c of the Brazilian
scorpions Tityus bahiensis and Tityus stigmurus. Biochem. J. 313,
753–760.
16. Becerril, B., Marangoni, S. & Possani, L.D. (1997) Toxins and
genes isolated from scorpions of the genus Tityus. Toxicon 35,
821–835.
17. Zlotkin, E. (1983) Insect selective toxins derived from scorpion
venoms: An approach to insect neuropharmacology. Insect
Biochem. 13, 219–236.
18. Ben Khalifa, R., Stankiewicz, M., Lapied, B., Turkov, M.,
Zilberberg, N., Gurevitz, M. & Pelhate, M. (1997) Refined
electrophysiological analysis suggests that a depressant toxin is a
sodium channel opener rather than a blocker. Life Sci. 61,
819–830.

Ó FEBS 2003 A novel ÔOld WorldÕ scorpion b-toxin (Eur. J. Biochem. 270) 2669
19. Froy, O., Zilberberg, N., Gordon, D., Turkov, M., Gilles, N.,
Stankiewicz, M., Pelhate, M., Loret, E., Oren, D.A., Shaanan, B.
& Gurevitz, M. (1999) The putative bioactive surface of insect-
selective scorpion excitatory neurotoxins. J. Biol. Chem. 274,
5769–5776.
20. Zlotkin. E., Gurevitz. M., Fowler. E., Moyer, M. & Adams, M.E.
(1993) Depressant insect selective neurotoxins from scorpion
venom: chemistry, action and gene cloning. Arch. Insect Biochem.
Physiol. 22, 55–73.
21. Shichor, I., Zlotkin, E., Ilan, N., Chikashvili, D., Stuhmer, W.,
Gordon,D.&Lotan,I.(2002)Domain2ofDrosophila Para
voltage-gated sodium channel confers insect properties to a rat
brain channel. J. Neurosci. 22, 4364–4371.
22. Gordon, D., Moskowitz, H., Eitan, M., Warner, C., Catterall,
W.A. & Zlotkin, E. (1992) Localization of receptor sites for insect-
selective toxins on sodium channels by site-directed antibodies.
Biochemistry 31, 7622–7628.
23. Loret, E.P., Martin-Euclaire, M.F., Mansuelle, P., Sampieri, F.,
Granier, C. & Rochat, H. (1991) An anti-insect toxin purified from
the scorpion Androctonus australis hector also acts on the a and b
sites of the mammalian sodium channel: sequence and circular
dichroism study. Biochemistry 30, 633–640.
24. Ji, Y.H., Li, Y.J., Zhang, J.W., Song, B.L., Yamaki, T.,
Mochizuki, T., Hoshino, M. & Yanaihara, N. (1999) Covalent
structure of BmK AS and BmK AS-1, two novel bioactive poly-
peptides purified from Chinese scorpion Buthus martensii Karsch.
Toxicon 37, 519–536.
25. Goudet, C., Chi, C W. & Tytgat, J. (2002) An overview of toxins
and genes from the venom of the Asian scorpion Buthus martensii

Karsch. Toxicon 40, 1239–1258.
26. Zilberberg, N. & Gurevitz, M. (1993) Rapid isolation of full length
cDNA clones by Ôinverse PCRÕ: Purification of a scorpion cDNA
family encoding a-neurotoxins. Anal. Biochem. 209, 203–205.
27. Zilberberg, N., Zlotkin, E. & Gurevitz, M. (1992) Molecular
analysis of cDNA and transcript encoding the depressant insect
neurotoxin of the scorpion. Leiurus quinquestriatus hebraeus.
Insect Biochem. Molec. Biol. 22, 199–203.
28. Turkov, M., Rashi, S., Zilberberg, N., Gordon, D., Ben Khalifa,
R.,Stankiewicz,M.,Pelhate,M.&Gurevitz,M.(1997)Invitro
folding and functional analysis of an anti-insect selective scorpion
depressant neurotoxin produced in Escherichia coli. Prot. Express.
Purific. 9, 123–131.
29. Gilles, N., Leipold, E., Chen, H., Heinemann, S.H. & Gordon, D.
(2001) Effect of depolarization on binding kinetics of scorpion
a-toxin highlights conformational changes of rat brain sodium
channels. Biochemistry 40, 14576–14584.
30. Gilles, N., Krimm, I., Bouet, F., Froy, D., Gurevitz, M., Lancelin,
J.M. & Gordon, D. (2000) Structural implications on the inter-
action of scorpion a-like toxins with the sodium channel receptor
site inferred from toxin iodination and pH-dependent binding.
J. Neurochem. 75, 1735–1745.
31. Gershon, E., Weigl, L., Lotan, I., Schreibmayer, W. & Dascal, N.
(1992) Protein kinase A reduces voltage-dependent Na
+
current in
Xenopus oocytes. J. Neurosci. 12, 3743–3752.
32. Wallner, M., Weigl, L., Meera, P. & Lotan, I. (1993) Modulation
of the skeletal muscle sodium channel a-subunit by the b
1

-subunit.
FEBS Lett. 336, 535–539.
33. Vijverberg, H.P., Pauron, D. & Lazdunski, M. (1984) The effect of
Tityus serrulatus scorpion toxin c on sodium channel in neuro-
blastoma cells. Pflugers Arch. 40, 297–303.
34. Marcotte,P.,Chen,L Q.,Kallen,R.G.&Chahine,M.(1997)
Effects of Tityus serrulatus scorpion toxin c on voltage-gated
Na
+
channels. Circ. Res. 80, 363–369.
35. Ceste
`
le, S. & Catterall, W.A. (2000) Molecular mechanisms of
neurotoxin action on voltage-gated sodium channels. Biochimie
82, 883–892.
36. Cestele, S., Qu, Y., Rogers, J.C., Rochat, H. & Catterall, W.A.
(1998) Voltage sensor-trapping: enhanced activation of sodium
channels by beta-scorpion toxin bound to the S3–S4 loop in
domain II. Neuron 21, 919–931.
37. Byalynitskii-Birula, A.A. (1917) Arthrogastric arachnids of
Caucasia.PartI.InScorpions (Nasonov, N.V., ed.). [English
translation from Russian by Israel Program for Scientific Trans-
lations, Jerusalem (1964)].
38. Blanc, E., Hassani, U., Meunier, S., Mansuelle, P., Sampieri,
F., Rochat, H. & Darbon, H. (1997)
1
H-NMR-derived sec-
ondary structure and overall fold of a natural anatoxin from
the scorpion Androctonus australis hector. Eur. J. Biochem. 247,
1181–1126.

39. Hassani, O., Loew, D., Van Dorsselaer, A., Papandreou, M.J.,
Sorokine,O.,Rochat,H.,Sampieri,F.&Mansuelle,P.(1999)
Aah VI, a novel, N-glycosylated anti-insect toxin from Androc-
tonus australis hector scorpion venom: isolation, characteri-
sation, and glycan structure determination. FEBS Lett. 443,
175–180.
40. Rack, M., Richter, D. & Rubly, N. (1987) Purification and
characterization of a b-toxinfromthevenomoftheAfrican
scorpion Leiurus quinquestriatus. FEBS Lett. 214, 163–166.
41. Gurevitz, M., Gordon, D., Ben-Natan, S., Turkov, M. & Froy, O.
(2001) Diversification of neurotoxins by C-tail ÔwigglingÕ –a
scorpion recipe for survival. FASEB J. 15, 1201–1205.
42. Froy, O., Sagiv, T., Poreh, M., Urbach, D., Zilberberg, N. &
Gurevitz, M. (1999) Dynamic diversification from a putative
common ancestor of scorpion toxins affecting sodium, potassium,
and chloride channels. J. Mol. Evol. 48, 187–196.
43. Inceoglu, B., Lango, J., Wu, J., Hawkina, P., Southern, J. &
Hammock, B.D. (2001) Isolation and characterization of a novel
type of neurotoxic polypeptide from the venom of the South
African scorpion Parabuthus transvaalicus (Buthidae). Eur. J.
Biochem. 268, 5407–5413.
44. Inceoglu,B.,Hayashida,Y.,Longo,J.,Ishida,A.T.&Hammock,
B.D. (2002) A single charged surface residue modifies the activity
of ikitoxin, a beta-type Na
+
channel toxin from Parabuthus
transvaalicus. Eur. J. Biochem. 269, 5369–5376.
45. Fontecilla-Camps, J.C. (1989) Three-dimensional model of the
insect-directed scorpion toxin from Androctonus australis hector
and its implication for the evolution of scorpion toxins in general.

J. Mol. Evol. 29, 63–67.
2670 D. Gordon et al. (Eur. J. Biochem. 270) Ó FEBS 2003