Phaiodotoxin, a novel structural class of insect-toxin isolated from
the venom of the Mexican scorpion
Anuroctonus phaiodactylus
Norma A. Valdez-Cruz
1
, Cesar V. F. Batista
1
, Fernando Z. Zamudio
1
, Frank Bosmans
2
, Jan Tytgat
2
and
Lourival D. Possani
1
1
Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico,
Cuernavaca, Mexico;
2
Laboratory of Toxicology, University of Leuven, Leuven, Belgium
A peptide called phaiodotoxin was isolated f rom the venom
of the scorpion Anuroctonus phaiodactylus. It is lethal to
crickets, but non toxic to mice at the doses assayed. It has 7 2
amino acid residues, with a molecular mass of 7971 atomic
mass un its. Its covalent structure was determined by Edman
degradation and mass spectrometry; it contains four disul-
fide-bridges, of w hich one of the pairs is formed between
cysteine-7 and cysteine-8 (positions Cys63–Cys71). The
other three pairs a re formed between Cys13–Cys38, Cys23–
Cys50 and Cys27–Cys52. Comparative sequence analysis

channels [1], K
+
channels [2,3], Cl
–
channels [4] a nd Ca
2+
channels [5,6]. The scorpion Anuroctonus phaiodactylus
belongs to t he family Iuridae. Human accidents with these
scorpions have not been reported to cause symptoms of
intoxication. However, they are toxic to insects and other
arthropods from which they prey on. Scorpion toxins
affecting Na
+
channels are polypeptides with 61–76 amino
acid residues long, showing two basic different pharma-
cological a ctivities, either a or b according to their mode
of action and binding properties [7–9]. The a-scorpion
toxins (a-ScTxs) slow Na
+
current inactivation in v arious
excitable preparations, upon their binding to site 3, but
they show vast differences in p reference f or insect and
mammalian Na
+
channels. Accordingly, they are divided
into classical a-toxins that are highly active in mammalian
brain, a-toxins that are very active in insects and a-like
toxins that are active in both the mammalian and the insect
central nervous system [10]. b-Toxins shift the activation
voltage of sodium channels to more negative membrane

Mexico. Fax: +52 777 3172388, Tel.: + 52 777 3171209,
E-mail:
Abbreviations: a.m.u., atomic mass unit; CD-immobilon, cationic,
hydrophilic, charged polyvinylidene fluoride membrane; COS7,
monkey kidney cell line 7; CNBr, cyanogen bromide; GH3, rat
pituitary cell line; ScTX, scorpion toxin; TE671, human cerebellar
medulloblastoma cell line 671.
(Received 13 August 2004, accepted 14 Octo ber 2004)
Eur. J. Biochem. 271, 4753–4761 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04439.x
at the C -tail section of the toxins [14–16]. The authors
propose that evolutionary events occurred at the C-terminal
region, which plays an important role in determining
functional d iversification and constitute an important site
for Na
+
-channel recognition [16,17].
Here we describe the isolation and characterization of an
insect specific toxin from the scorpion Anuroctonus phaiod-
actylus, collected in Baja California, Mexico. We have
isolated and chemically and functionally characterized th is
peptide. The gene that codes for the toxin and several
isoforms were obtained. The three major characteristics of
phaiodotoxin are: its lethal effect on crickets, but non toxic
to mice; its different arrangement of the disulfide bridges,
and its pharmacological effect on para/tipE Na
+
channel
expressed on Xenopus laevis oocytes, where it causes an
important increment on the window of Na
+

saline; 0.15 m
M
NaCl in 0.1 m
M
sodium phosphate buffer,
pH 7.4) were injected intraperitoneally. These assays were
conducted using a minimum number of animals required to
validate t he experimental data, according to the guidelines
for animal usage of our Institute (the protocols were
approved by the Institutional Committee for Animal
Welfare). U sually, injection on two or three animals i s
considered enough to see if there is a visible effect on mice.
Lethality t ests on crickets weighing approximately 100 mg
were performed injecting 3 lL of variable amounts of
venom and/or fractions at the intersegments of the right leg.
Phaiodotoxin in amounts of 0.2, 0.5, 0.8 and 1.0 lgof
peptide per animal were injected, using two crickets at a time
and repeating the same procedure four times. The main
symptoms of intoxication were: flaccidity, impairment of
movements, paralysis and death.
Primary structure determination of phaiodotoxin
The amino acid sequence o f the N-terminal portion of
phaiodotoxin was obtained by Edman degradation carried
out with an automatic apparatus Beckman LF 3000 Pro-
tein Sequencer (Palo Alto, CA, USA), using the peptide
adsorbed on CD Inmmobilon m embranes (Beckman part
number 290110). A sample of the toxin was also sequenced
from its N-terminal region, after reduction and alkylation
in situ with acrylamide by the method described in [18]. In
order to c omplete the full sequence several fragments of the

formed. Initially, 100 lg o f phaidoto xin was digested with
lysine-C endopeptidase (Lys-C). Subsequently, another
sample was treated with two enzymes chymotrypsin and
aspartic-N (Asp-N), all from Boehringer (Mannheim,
Germany), using the conditions described by the manu-
facturer. In order to confirm the disulfide pairs found, an
independent sample was processed using CNBr cleavage
[19]. T he products were s eparated by HPLC and directly
sequenced.
Sequence analysis
Nucleotide sequence similarities were searched with the
BLAST
program using the databases of GenBank (National
Center for Biotechnology Information). The sequences
obtained were edited and aligned using
CLUSTAL
-
X
[21].
Gene cloning of phaiodotoxin
Total R NA was isolated from venomous glands situated at
the last postabdominal segment (telson) of one Anuroctonus
phaiodatylus scorpion, by the method of Chirgwin et al.
[22]. Total RNA (500 n g) was u sed as template t o gener-
ate cDNA using the oligonucleotide poliT22NN
[23]. For gene amplification two primers were used:
4754 N. A. Valdez-Cruz et al.(Eur. J. Biochem. 271) Ó FEBS 2004
5¢-AARTTYATHCGRCAYAAG-3¢ and poliT22NN. We
cloned t he product o f the amplification in EcoRV site of
phagemid p KS(–) ( Stratagene, L a J olla, C A, USA). This

): NaCl,
96;KCl,2;CaCl
2
,1.8;MgCl
2
,2andHepes,5(pH7.4),
supplemented with 50 mgÆL
)1
gentamycin sulfate.
Electrophysiological recordings in
Xenopus
oocytes
Two-electrode vo ltage-clamp recordings were performed at
room temperature (18–22 °C) using a GeneClamp 500
amplifier (Axon Instruments, Union City, CA, USA)
controlled by a pClamp data acquisition s ystem (Axon
Instruments). Whole-cell currents from oocytes were recor-
ded 4 days after injection. Voltage and currents electrodes
were filled with 3
M
KCl. Resistances of both electrodes
were kept as low as possible ( < 0.5 MX). Bath solution
composition was (in m
M
): NaCl, 9 6; KCl, 2; CaCl
2
,1.8;
MgCl
2
, 2 and Hepes, 5 (pH 7.4). Using a four-pole low-pass

bioassays were repeated four times with phaiodotoxin, given
identical results. T his is s imilar to w hat was described by
Zlotkin et al. [29] for the insect toxin LqhIT2 of the
scorpion Leirus quinquestriatus hebraeus.
Despite the fact that phaiodotoxin was not toxic to mice,
using in vivo experiments at high doses (100 lg per mouse),
several cell l ines in culture (see Materials and methods) were
tested for possible electrophysiological effects on mamma-
lian Na
+
channels. It is w orth mentioning that scorpion
toxins such as Cn2 (toxin 2 from the scorpion Centruroides
noxius), specific for m ammals, have LD
50
values in the
range of 0.25 lg per 20 g m ouse bodyweight [30]. T hus,
mice injected with 400-fold more phaiodotoxin than that
required by other scorpion toxins, did not show any toxicity
symptoms, from which we assumed this peptide is not t oxic
to mice. E lectrophysiological tests conducted with micro-
molar concentrations of phaiodotoxin in the cell culture
systems mentioned (COS7, TE671, GH3 and cerebellum
granular cells) showed no effect (data not shown), from
which we surmised that this peptide was rather specific for
insects.
The primary structure of phaiodotoxin was obtained by a
combination of direct Edman degradation and mass
spectrometry analysis, as shown in Fig. 1C. Alkylated toxin
permitted to identify the first 39 residues (underlined with
the word ÔdirectÕ in the fi gure). Two subsequent p eptides

for PhTx3 is Asn instead of Glu. The sequences are deposited into GenBank, accession numbers AY781122–AY781124.
Fig. 1. Phaiodotoxin purification. (A) Soluble venom (30 mg o f protein) was separated by Sephadex G-50 column. Frac tion s of 1.0 mL ea ch were
collected. Fraction II was toxic to insects and was further separated. (B) This fraction was applied to a semipreparative C18 reverse-phase column of
the H PLC system an d eluted with a linear gradient f rom solvent A (0.12% t rifluoroacetic acid in water) t o B (0.10% TFA in acetonitrile), run
during 60 min. The major component (asterisk) is the one with toxic activity. The inset shows the second HPLC separation of this component using
an analytical C18 column, eluted with similar gradient (pure to xin i ndicated b y aste risk). (C ) Full a mino acid sequence of phaiodotoxin a s describe d
in text. The numbers on top of th e s equenc e indic ate po sit ion o f the residu es. U nde rlined a mino acids with the word di rect me an s direct s equ ence b y
Edman degradation; those with CNBr were determined from peptides obtained by cyanogen bromide cleavage and those underlined by MS/MS
were determined by mass spectrometry fragmentation (some are overlapping sequence s). The pep tide G40–Y51 was obtained after chymotryptic
cleavage. This sequence is deposited into the SwissProt databank, accession number P84207.
4756 N. A. Valdez-Cruz et al.(Eur. J. Biochem. 271) Ó FEBS 2004
deduced amino acid sequences of two additional clones,
corresponding to putative isoforms of the toxin, labeled
PhTx2 and PhTx3. In these two peptides there is only one
amino acid change in each (L15S and D15N, respectively).
The s ignal p eptide is rich in hydrophobic residues, as
expected, and the amino acid length is similar to other
insect-toxin gene cloned [31–33].
Determination of the disulfide bridges
The digestion of native phaiodotoxin with endopeptid ase
Lys-C produced five peptides ( data not shown). The one
eluting at 27.05 min was sequenced and allowed the
identification of the heterodimeric peptide correspondent
to the C -terminal region o f the toxin (residues M62 to A72).
The automatic sequencer showed Met for amino acid o f
position 1; the Cys71 was not seeing, because it was bond to
Cys63. The amino acids in posit ion 2 were Ala72 and
cystine, confirming that the d isulfide bridge was b etween
Cys63-Cys71. The molecular mass found was 1175 atomic
mass units The expected theoretical value was 1159.39

odotoxin with representative examples of a-andb-ScTXs,
Fig. 3. Amino acid sequence comparison. T his figure shows the alignment of selected amino acid sequence of toxins and t heir disulfide bridge
arrangements. Phaiodotoxin is shown in the first line (PhTx) and t wo additional groups of sequences are shown thereafter. The first group
(11 sequences) is from the a-ScTXs, t he second is from the b-ScTXs. Birtoxin is the sho rtest. The de pressant and the long-chain ex citatory are in the
last two lines. T he right columns indicate percentage of similarities ( S) and identities (I). The brackets indicate h ow the disulfide patterns are
arranged. Solid lines indicate the disulfide bridges common to all of them, whereas broken lines are special disulfide pairin g. Dashes (–) were
introduced to increase similarities. Toxins sequences were obtained from data bank and the abbreviations stand for: AaH, Androctonus australis
Hector; Amm, Androctonus mauretanicus mauretanicus;Bj,Buthotus judaicus;Bot,Buthus occitanus tunetanus;Cn,Centruroides noxius;Lqh,
Leiurus quinq ues tria tus hebra eus ;Lqq,L. q. quinquestriatus;Me,Mesobuthus eupeus;Bo,Buthus occitanus;Bm,Buthus martensi Karsch; Ts, Tityus
serrulatus. The alignments were obtained with the program
CLUSTAL
-
X
, with best scores. Similarities and identities were calculated using the
pairwaise alignment algorithms by EMBOSS (www.ebi.a c.uk/emboss/align/).
Ó FEBS 2004 Phaiodotoxin, an insect-toxin (Eur. J. Biochem. 271) 4757
Fig. 4. Electrophysiological effects of phaiodotoxin on para/tipE expressed in Xenopus oocytes. In all panels, h represents control conditions a nd
n represents the effect of 2 l
M
phaiodotoxin after an application of 2 min . (A) Current traces were evoked from a n oocyte expressing para/tipE by
a 25 ms depolarization to )10 mV fro m a holdin g poten tial of )90 mV. On the left, an averaged trace (n ¼ 5) is shown before and afte r add ition of
2 l
M
phaiodotoxin (indicated). On th e right, a curren t–voltage relationship of p ara/tipE expressed in oocytes is shown before and after addition of
2 l
M
phaiodotoxin (n ¼ 5). A small increase i n current is noticed and changes in the activation process are presen t. Current traces w ere evoked by
10 mV depolarization steps from a holding poten tial of )90 mV. E ach point represents the mean ± SEM. (B) Phaiodotoxin shifts the voltage
dependence of activation of para/tipE. The left figure represents the normalized conducta nce/ voltag e relatio nship of para/tipE in th e absence
(h,V

as mentioned earlier is a toxin that causes flaccidity and/or
paralysis when injected into insects, rather than excitation.
All these toxins have a conserved core of three disulfide
bridges a s shown in F ig. 3 . However, t he fourth disulfide
pair of the excitatory toxins shown in this figure has a
distinct disulfide pattern. Thus, phaiodotoxin is a novel,
third different type of arrangement for the fourth disulfide
bridge. Exceptions to all of them are birtoxin and ikitoxin,
which have only three disulfide bridges [13,37], and are the
shortest ones.
The data reported here for phaiodotoxin supports the
proposition of Froy and Gurevitz [38], that the C-terminal
tail of the S cTXs are playing an important role in the
biological activity of these toxins, and should constitute an
important point of diversification of the interacting surfaces
with Na
+
channels [16,17].
Phaiodotoxin affects voltage-gated Na
+
channels
of insects
The activity o f the phaiodotoxin was electrophysiologically
tested on the cloned insect voltage-gated Na
+
channel,
para, coexpressed i n Xenopus l aevis oocytes with the in sect
Na
+
channel subunit, tipE. Current traces were evoked

gated sodium channel mutations which resulted in a gain-
of-function defect lead to either enhanced excitability
(myotonia) or inexcitability (periodic paralysis) in heart,
skeletal muscle or brain [ 39]. Most often this phenomenon is
caused by a partial impairm ent of inactivation or shifted
voltage dependence. Moreover, Cannon [39] showed that
even a subtle disruptio n of inactivation (on average, about
2% of channels fail to inactivate) is sufficient to cause
myotonia. If an increase in the window current can result in
action potential prolongation, a reduced window current
will contribute to shortening of the action potential. A 60%
reduction in window current is reported to be responsible
for ventricular arrhythmias in Brugada syndrome [40].
These results highlight the importance of the window
current.
For the first time, we describe a toxin that causes an
alteration of window current in insects. As phaiodotoxin
causes an increase in window current of about 225% in
insect voltage-gated sodium chan nels, i t i s most probable
that this will have drastic effects on the insect itself (as
shown in the bioassays).
Phylogenetic considerations on phaiodotoxin
As phaiodotoxin is the first Na
+
channel-specific t oxic
peptide ever isolated from a scorpion of the family Iuridae,
it was tempting to analyze possible e volutionary aspects of
this peptide in the context of other known examples. The
great majority of known Na
+

Science and Technology (CONACyT), Mexican Government, and
IN206003-3 from D ireccio
´
n G eneral de Asuntos del Personal Acad-
emico (DGAPA), UNAM to L.D.P. The authors are grateful to
Dr Martin S. Williamson, IACR- Rothamsted, UK, for sharing the
para and tipE clone; C. Maertens and R. Rodriguez de la Vega for the
discussions and Dr Alexei Licea for helping with the capture of
Ó FEBS 2004 Phaiodotoxin, an insect-toxin (Eur. J. Biochem. 271) 4759
scorpions. Experiments with COS7 and TE671 cells were kindly
performed by P rofessor Enzo Wanke and Rita R estano-Cassulini, from
the University of M ilano at Biccoc a, Italy, and those w ith GH3 and
cerebellum g ranular cells were performed by Dr Gianfranco Prestipino
from the I nstitute of Cybernetics and Biophysics, C.N.R. in Genova,
Italy. N.A.V C. was a recipient of a scholarship from CONACyT and
DGAPA-UNAM.
References
1. Catterall, W.A. (1980) Neurotoxins that act on voltage sensitive
sodium channels in excitable membranes. Annu.Rev.Pharmacol.
Toxicol. 20, 15–43.
2.Carbone,E.,Wanke,E.,Prestipino,G.,Possani,L.D.&
Maelicke, A. (1982) Se lective blockage of v oltag e-depen dent
K
+
channels by a novel scorpion toxin. Nature 296, 90–91.
3. Possani, L.D., Ma rtin, B . & Svendsen, I. ( 1982) T he pr imary
structure o f Noxiustoxin: a K
+
channel blocking peptide from the
venom o f the scorp ion Centruroides noxius Hoffmann. Carlsberg

Scorpion toxins specific for Na
+
-channels. Eur. J . Biochem. 264,
287–300.
10. Gordon, D., Gilles, N., Bertrand, D., Molgo, J., Nicholson, G.M.,
Sauviat, M.P., Benoit, E., Shichor, I., Lotan, I., Gurevitz, M.,
Kallen, R.G. & He inemann, S.H. (2002) Scorpion toxins differ-
entiating among neuronal sodium channel subtypes: nature’s
guide for design of selective drugs. In Perspectives in Molecular
Toxinology (Menez, A., ed.) pp. 215–238. Wiley, Chichester, UK.
11. Catterall, W.A. (1992) Cellular and molecular biology of voltage-
gated sodium channels. Physiol. Rev. 72, S15–S48.
12. Kobayashi, Y., Takashima, H., Tam aoki, H., K iogoku, Y.,
Lambert, P., Kuroda, H., Chino, N., Watanabe, T.X., Kimura,
T., Sakakibara, S. & Moroder, L. (1991) The cystine-stabilized
alpha-helix: a common structural m otif of ion-channel blocking
neurotoxic peptides. Biopolymers 31, 1213–1220.
13. Inceoglu, B., Lango, J ., Wu, J., H awkins, P., So uthern, J. &
Hammock, B.D. (2001) Isolation and c haracterization of a novel
type of neurotoxic peptide from the venom of the South African
scorpion Par abuthus transvaalicus (But hidae). Eur. J. Biochem.
268, 5407–5413.
14. Zilberberg, N ., Froy, O ., Loret, E., Cestele, S., Arad, D., Gordon,
D. & Gurevitz, M. (1997) Identification of structural elements of a
scorpion alpha-neuro toxin importa nt for r ecepto r site r ecogn ition.
J. Biol. Chem. 272, 14810–14816.
15. Froy, O., Zi lberberg, N., Gordon, D., Turkov, M., Gilles, N.,
Stankiewicz, M., Pe lhate, M ., Loret, E., Oren, D.A., Shaanan, B.
& Gurevitz, M. (1999) The putative bioactive surface of
insect-selective scorpion e xc itatory ne urotoxin s. J. Biol. C hem.

enriched in ribonuclease. Biochemistry 18, 5294–5299.
23. Corona, M., Valdez-Cruz, N.A., Merino, E., Zurita, M. &
Possani, L.D. (2001) Genes and peptides from the scorpion
Centruroides sculpturatus Ewing, that re cognize N a
+
-channels.
Toxicon 39, 1893–1898.
24. Warmke, J.W., Reenan, R.A.G., Wang, P., Qian, S., Arena, J.P.,
Wang, J ., Wunderler, D., Liu, K., Kaczorowski, J.P., Van Der
Ploeg, L. H.T., Ganetzky, B. & C ohen, C.J. (1997) Functional
expression of Drosophila para sod ium channels. Modulation by
the membrane protein tipE and toxin pharmacology. J. Gen.
Physiol. 110, 119–133.
25. Feng, G., Dea
´
k, P., Chopra, M. & Hall, L. (1995) Cloning an d
functional analysis of tipE, a novel membrane protein that
enhances Drosophila para so dium channel f unction. Cell 82,
1001–1011.
26. Liman, E.R., Tytgat, J. & Hess, P. (1992) Subunit s toichiometry of
a mammalian K
+
channel d etermi ned b y construction of m ulti-
meric cDNAs. Neuron 9, 861–871.
27. Attwell,D.,Cohen,I.,Eisner,D.,Ohba,M.&Ojeda,C.(1979)
The steady state TTX-sensitive (ÔwindowÕ) s odium current in car-
diac Purkinje fibres. Pfl u
¨
gers Arch. 37 9 , 137–142.
28. Pisciotta, M., Coronas, F.I., Bloch, C., Prestipino, G. & Possani,

variations at the interacting surface. Toxicon 41, 125–128.
35. Froy, O., Amit, E., Kleinberger-Doron, N., Gurevitz, M. &
Shaanan, B. (1998) An excitatory scorpion toxin with a distinctive
feature: an additional alpha helix at the C terminus and its
implications for interaction with insect sodium channels. Structure
6, 1095–1103.
36. Loret, E.P., Mansuelle, P., Rochat, H. & Granier, C. (1990)
Neurotoxins active on i nsects: amino acid sequences, c he mical
modifications, and secondary structure estimation by circular
dichroism o f toxins from the scorpion Androctonus a ustralis
Hector. Biochemistry 29 , 1492–1501.
37. Inceoglu, A.B., Hayashida, Y., Lango, 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, 5 369–5376.
38. Froy, O. & Gurevitz, M. (2003) New insight on scorpion diver-
gence inferred from c omparative analysis of tox in structure,
pharmacology and distribution. Toxicon 42 , 549–555.
39. Cannon, S.C. (2001) Volta ge-gated ion channelopathies of t he
nervous system. Clin.Neurosc.Res.1, 104–117.
40. Mok, N.S., Priori, S.G., Napolitano, C., Chan, N.Y., Chahine, M.
& Baroudi, G. (2003) A newly characterized SCN5A mutation
underlying Brugada syndrome unmasked by hyperthermia.
J. Cardiovasc. Electrophysiol. 14, 4 07–411.
Ó FEBS 2004 Phaiodotoxin, an insect-toxin (Eur. J. Biochem. 271) 4761