Scorpion toxins affecting Na+channels are polypeptides with 61–76 amino acid residues long, showing two basic different pharma-cological activities, either a or b according to their mode
Trang 1Phaiodotoxin, a novel structural class of insect-toxin isolated from
Norma A Valdez-Cruz1, Cesar V F Batista1, Fernando Z Zamudio1, Frank Bosmans2, Jan Tytgat2and Lourival D Possani1
1
Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Mexico;2Laboratory of Toxicology, University of Leuven, Leuven, Belgium
A peptide called phaiodotoxin was isolated from the venom
of the scorpion Anuroctonus phaiodactylus It is lethal to
crickets, but non toxic to mice at the doses assayed It has 72
amino acid residues, with a molecular mass of 7971 atomic
mass units Its covalent structure was determined by Edman
degradation and mass spectrometry; it contains four
disul-fide-bridges, of which one of the pairs is formed between
cysteine-7 and cysteine-8 (positions Cys63–Cys71) The
other three pairs are formed between Cys13–Cys38, Cys23–
Cys50 and Cys27–Cys52 Comparative sequence analysis
shows that phaiodotoxin belongs to the long-chain
sub-family of scorpion peptides Several genes coding for this
peptide and similar ones were cloned by PCR, using cDNA
prepared from the RNA of venomous glands of this
scor-pion Electrophysiological assays conducted with this toxin
in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ cur-rents However, it shifts the voltage dependence of activation and inactivation of insect Na+channels (para/tipE) to more negative and positive potentials, respectively Therefore, the
window current is increased by 225%, which is thought to
be the cause of its toxicity toward insects Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae
Keywords: Anuroctonus phaiodactylus; disulfide bridges; insect toxin; Na+-channel; scorpion
Most of the biochemical work performed with scorpion
venom has been reported using scorpions of the family
Buthidae, probably because they are dangerous to humans
A large number of different protein and polypeptides have
been isolated and characterized from this family Among
the most important findings are four different groups of
peptides, which specifically interact with ion channels: Na+
channels [1], K+channels [2,3], Cl–channels [4] and Ca2+
channels [5,6] The scorpion Anuroctonus phaiodactylus
belongs to the 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 activities, 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 various
excitable preparations, upon their binding to site 3, but
they show vast differences in preference for 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 potentials upon binding to receptor site 4 [11] This class includes two types of toxins, excitatory and depressant [7,8]
Na+channels specific ScTxs present a conserved core formed by a-helix and three strands of b-sheet structural motifs The helix motif is linked to the b3 strand by two of the four disulfide bonds The cysteine pair of the a-helix motif is spaced by a tripeptide CXXXC (where C stands for cysteine and X for any amino acid), whereas the pair of cysteine residues of the b3 strand is separated by only one amino acid residue (CXC), usually linking the C3 (third cysteine of the sequence) to C6 and C4 to C7 [12] A third structurally conserved disulfide bridge occurs between the C2 of the N-terminal segment with C5 of the b2 strand [9] The fourth disulfide bond is established between C1 and C8,
of the N- with the C-terminal region The excitatory insect toxins lack the equivalent position of C1, present in most scorpion toxins, and the fourth disulfide bridge is formed between C5¢ (contiguous to C5) with C8 [reviewed in 9] This last disulfide bridge is not present in birtoxin, which has only three disulfide bridges, but functionally shows a b-like activity and shares homology with the Centruroides’ b-toxins [13] Recently, the functional surface of three different toxins was mapped Analysis of the three-dimen-sional models suggests that the functional differences reside
Correspondence to L D Possani, Instituto de Biotecnologı´a UNAM
Avenida Universidad, 2001 Apartado Postal 510–3 Cuernavaca 62210
Mexico Fax: +52 777 3172388, Tel.: +52 777 3171209,
E-mail: possani@ibt.unam.mx
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 October 2004)
Trang 2at 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 diversification 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 this
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+currents It is
worth mentioning that the unusual disulfide bridge is
situated at the C-terminal tail of the molecule
Materials and methods
Venom collection and purification procedure
The scorpions were collected in Maneadero Baja California,
Mexico Their venom was obtained by electrical stimulation,
dissolved in double distilled water, centrifuged at 15 000 g
for 15 min and the supernatant lyophilized and kept at
)20 C The soluble venom was applied to a Sephadex G-50
column (0.9· 190 cm) in 20 mMammonium acetate buffer
pH 4.7, resolving six fractions The second fraction contains
the phaidotoxin which was obtained in a homogeneous
form after two independent steps of purification Initially,
the separation was performed in a semipreparative C18
reverse phase column (Vydac, Hisperia, CA, USA), using a
Waters 600E HPLC, equipped with a Photodiode Array
Detector 996 from Millipore (Milford, MA, USA) The
second HPLC was carried out in an analytical C18 reverse
column In both cases, a linear gradient was run for 60 min,
from solution A (0.12% trifluoroacetic acid in water) to
60% solution B (0.10% TFA in acetonitrile)
Lethality tests
Lethality tests were carried out on female albino mice (CD1
strain) of approximately 20 g bodyweight The various
samples dissolved in 100 lL NaCl/Pi(phosphate buffered
saline; 0.15 mMNaCl in 0.1 mMsodium phosphate buffer,
pH 7.4) were injected intraperitoneally These assays were
conducted using a minimum number of animals required to
validate the experimental data, according to the guidelines
for animal usage of our Institute (the protocols were
approved by the Institutional Committee for Animal
Welfare) Usually, injection on two or three animals is
considered enough to see if there is a visible effect on mice
Lethality tests 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 lg of
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 of 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 membranes (Beckman part number 290110) A sample of the toxin was also sequenced from its N-terminal region, after reduction and alkylation
in situwith acrylamide by the method described in [18] In order to complete the full sequence several fragments of the peptide were obtained after cleavage of phaiodotoxin with cyanogen bromide (CNBr), thanks to the presence of two methionine residues in the molecule An eight-fold excess of CNBr over toxin (w/w) in 70% formic acid was used according to the technique described by Biedermann [19] After overnight reaction, the products were reduced with dithiothreitol for 30 min, at 56C and separated by HPLC The subpeptides were used for Edman degradation analysis The molecular mass determination of pure phaiodotoxin and the additional sequencing work was performed by mass spectrometry, using an LCQDuo Finnigan mass spectrometer, as described previously [20] All spectra were obtained in the positive-ion mode For sequence determin-ation, MS/MS spectra produced were analyzed manually and automatically by SEQUEST software The acquisition and deconvolution of data were performed with the XCALI-BURsoftware on a Windows NT PC data system
Determination of disulfide bridges Native toxin was digested with several specific endo-peptidases and their products were separated by HPLC (same conditions as described above) The purified dimeric peptides were directly used for Edman degradation and mass spectrometry analysis It is worth noting that for these sequences no reduction of the peptides was per-formed Initially, 100 lg of phaidotoxin 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] The products were separated by HPLC and directly sequenced
Sequence analysis Nucleotide sequence similarities were searched with the BLASTprogram using the databases of GenBank (National Center for Biotechnology Information) The sequences obtained were edited and aligned usingCLUSTAL-X[21] Gene cloning of phaiodotoxin
Total RNA 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 ng) was used as template to gener-ate cDNA using the oligonucleotide poliT22NN [23] For gene amplification two primers were used:
Trang 35¢-AARTTYATHCGRCAYAAG-3¢ and poliT22NN We
cloned the product of the amplification in EcoRV site of
phagemid pKS(–) (Stratagene, La Jolla, CA, USA) This
construct was used to transform Escherichia coli DH5-a
cells Clone selection and DNA sequencing were performed
as described by Corona et al [23] In order to complete the
nucleotide sequence, the method for rapid amplification of
the 5¢-region (RACE 5¢) was applied, using RLM-RACE
(RNA ligase mediated rapid amplification of cDNA ends)
protocol, according to the instructions of the kit from
Ambion (Austin, TX, USA) The cDNA mix was
synthes-ized from poly(A)+ mRNA using M-MLV reverse
tran-scriptase The cDNA was joined with the adaptor provided
by the kit (5¢-gcugauggcgaugaaugaacacugcguuugCUGG
CUUUGAUGAAA-3¢) using T4 DNA ligase The
modi-fied cDNA was used as template for PCR amplification
Two rounds of amplification with the primers from the
Ambion kit were performed
Expression inXenopus oocytes
For the expression in Xenopus oocytes, the
para/pGH19-13–5 vector [24] and tipE/pGH19 vector [25] were linearized
with NotI and transcribed with the T7
mMESSAGE-mMACHINE kit (Ambion) The harvesting of oocytes
from anaesthetized female Xenopus laevis frogs was as
described previously [26] Oocytes were injected with 50 nL
of cRNA at a concentration of 1 ngÆnL)1using a
Drum-mond microinjector (Broomal, PA, USA) The solution
used for incubating the oocytes contained (in mM): NaCl,
96; KCl, 2; CaCl2, 1.8; MgCl2, 2 and Hepes, 5 (pH 7.4),
supplemented with 50 mgÆL)1gentamycin sulfate
Electrophysiological recordings inXenopus oocytes
Two-electrode voltage-clamp recordings were performed at
room temperature (18–22C) using a GeneClamp 500
amplifier (Axon Instruments, Union City, CA, USA)
controlled by a pClamp data acquisition system (Axon
Instruments) Whole-cell currents from oocytes were
recor-ded 4 days after injection Voltage and currents electrodes
were filled with 3M KCl Resistances of both electrodes
were kept as low as possible (< 0.5 MX) Bath solution
composition was (in mM): NaCl, 96; KCl, 2; CaCl2, 1.8;
MgCl2, 2 and Hepes, 5 (pH 7.4) Using a four-pole low-pass
Bessel filter, currents were filtered at 2 kHz and sampled at
10 kHz Leak and capacitance subtraction were performed
using a P/4 protocol Current traces were evoked in an
oocyte expressing the cloned sodium channels by
depolari-zation between)70–40 mV, using 10 mV increments, from
a holding potential of)90 mV
The window current was estimated following the
des-cription of Attwell et al [27] using the weighing method
Electrophysiological recordings with mammalian
cell lines
The effect of phaiodotoxin was also assayed in several
mammalian cell lines: TE671 (from human cerebellar
medulloblastoma), COS7 (from monkey kidney
fibro-blasts), GH3 and cerebellum granular cells from rat, using
the technique described [28]
Results and Discussion
Purification, bioassays and chemical characterization
of phaiodotoxin Figure 1 shows the results of the chromatographic steps used for purification of phaiodotoxin In short, a gel filtration system with Sephadex G-50 column (Fig 1A) and two additional separations on HPLC (Fig 1B) provided a homogeneous peptide Toxicity tests showed that it was non toxic to mice using a dose up to 100 lg per 20 g mouse weight, but causing flaccidity and paralysis in crickets Crickets injected with little as 0.5 lg per animal showed symptoms of intoxication such as: impairment of move-ments and mild paralysis A 0.8 lg per animal dose causes
a clear flaccid paralysis, but at 1.0 lg per animal all the crickets die, within the first 2 h after injection These bioassays were repeated four times with phaiodotoxin, given identical results This is similar to what 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 lines in culture (see Materials and methods) were tested for possible electrophysiological effects on mamma-lian Na+ channels It is worth mentioning that scorpion toxins such as Cn2 (toxin 2 from the scorpion Centruroides noxius), specific for mammals, have LD50 values in the range of 0.25 lg per 20 g mouse bodyweight [30] Thus, 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 toxic
to mice Electrophysiological 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 direct in the figure) Two subsequent peptides (corresponding to residues in positions M41 to R59 and M62 to K70) were sequenced after cyanogen bromide cleavage (underlined by CNBr) The C-terminal residues of each peptide were identified by mass spectrometry frag-mentation of the same purified subpeptides (underlined MS
in the Fig 1C) The full sequence was also confirmed by mass spectrometry The molecular mass of native phaiod-otoxin was shown to be 7971.0 atomic mass units, whereas the theoretical expected value based on the sequence obtained was 7970.3 atomic mass units (within the experi-mental error) The correct overlapping segments were further aligned, after cloning the gene that codes for the toxin, as it will be discussed below
cDNA clone of phaiodotoxin Figure 2A shows the nucleotide sequence obtained for the cloned gene of phaiodotoxin In total 372 nucleotide pairs were identified They code for the 72 amino acid residues of
Trang 4the mature toxin (capital letters below the nucleotide
codons), and for 18 amino acids of the corresponding signal
peptide (underlined sequence) At the most 5¢-untranslated
region, 71 nucleotide bases were identified, just before the signal peptide; whereas at the 3¢-end, after the stop codon,
28 nucleotide bases were determined Figure 2B shows the
Fig 2 Nucleotide sequence of the gene coding for phaiodotoxin (A) The deduced amino acid sequence corresponding to the gene of phaiodotoxin is indicated below each codon, starting from the signal peptide (underlined) The sequence corresponding to the mature peptide is indicated in bold A segment corresponding to the 5¢-untranslated region is shown on the first line (first 71 base pairs) The stop codon is indicated, followed by 28 base pairs of untranslated sequence Numbers on the right side indicate both the nucleotide sequence and the amino acid sequence (B) Two additional putative isoforms of phaiodotoxin were cloned and sequenced The first line labelled PhTx contains the amino acid sequence of phaiodotoxin, the second and third lines show two isoforms: PhTx2, and PhTx3, respectively Residue in position 16 for PhTx2 is Ser instead of Leu, and residue 25 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 of protein) was separated by Sephadex G-50 column Fractions of 1.0 mL each 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 HPLC system and eluted with a linear gradient from solvent A (0.12% trifluoroacetic acid in water) to 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 toxin indicated by asterisk) (C) Full amino acid sequence of phaiodotoxin as described
in text The numbers on top of the sequence indicate position of the residues Underlined amino acids with the word direct means direct sequence by 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 sequences) The peptide G40–Y51 was obtained after chymotryptic cleavage This sequence is deposited into the SwissProt databank, accession number P84207.
Trang 5deduced 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 signal peptide 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 endopeptidase
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 of the toxin (residues M62 to A72)
The automatic sequencer showed Met for amino acid of
position 1; the Cys71 was not seeing, because it was bond to
Cys63 The amino acids in position 2 were Ala72 and
cystine, confirming that the disulfide bridge was between
Cys63-Cys71 The molecular mass found was 1175 atomic
mass units The expected theoretical value was 1159.39
(about 16 atomic mass units more than expected, due to the
oxidation of the methionine, in this particular preparation)
These results showed that in phaiodotoxin, a new structural
arrangement of disulfide pairs occurs between non expected
cysteinyl residues Because of this fact, this experiment was
repeated with another aliquot of toxin, but the final results
were identical Still another sample was analyzed (from the cyanogen bromide cleavage) also confirming this unusual disulfide pairing From the other four peptides obtained after endopeptidase Lys-C cleavage (mentioned before), the one eluting at 33.08 min (data not shown) turned out to contain a mixture of the three remaining disulfide bridges linked all together This peptide was further digested with chymotrypsin and Asp-N The mixture was separated by HPLC (data not shown), from which a peptide eluting at 25.20 min was found to correspond to the segments that links the Cys13 with Cys38, i.e disulfide pair: C2–C5 The peptide eluted at 26.15 min allowed the identification of Cys23 with Cys50, corresponding to the pair: C3–C6 The last disulfide pair was assumed to be between Cys28 and Cys52, as the molecular mass of the native peptide was consistent with the oxidation of the corresponding thiol groups, in order to form the last missing disulfide bridge Furthermore, this is one of the constant disulfide pairs found in all the scorpion toxins described to data
In this way, as shown in Fig 3, the structural arrange-ment of the disulfide bridges of phaiodotoxin constitutes a novel example of disulfide pairing for scorpion toxins Sequence comparison with other ScTXs
Figure 3 shows a comparative sequence analysis of phai-odotoxin with representative examples of a- and b-ScTXs,
Fig 3 Amino acid sequence comparison This figure shows the alignment of selected amino acid sequence of toxins and their disulfide bridge arrangements Phaiodotoxin is shown in the first line (PhTx) and two additional groups of sequences are shown thereafter The first group (11 sequences) is from the a-ScTXs, the second is from the b-ScTXs Birtoxin is the shortest The depressant and the long-chain excitatory are in the last two lines The right columns indicate percentage of similarities (S) and identities (I) The brackets indicate how the disulfide patterns are arranged Solid lines indicate the disulfide bridges common to all of them, whereas broken lines are special disulfide pairing 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 quinquestriatus hebraeus; 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.ac.uk/emboss/align/).
Trang 6Fig 4 Electrophysiological effects of phaiodotoxin on para/tipE expressed in Xenopus oocytes In all panels, h represents control conditions and
n represents the effect of 2 l M phaiodotoxin after an application of 2 min (A) Current traces were evoked from an oocyte expressing para/tipE by
a 25 ms depolarization to )10 mV from a holding potential of )90 mV On the left, an averaged trace (n ¼ 5) is shown before and after addition of
2 l M phaiodotoxin (indicated) On the right, a current–voltage relationship of para/tipE expressed in oocytes is shown before and after addition of
2 l M phaiodotoxin (n ¼ 5) A small increase in current is noticed and changes in the activation process are present Current traces were evoked by
10 mV depolarization steps from a holding potential of )90 mV Each point represents the mean ± SEM (B) Phaiodotoxin shifts the voltage dependence of activation of para/tipE The left figure represents the normalized conductance/voltage relationship of para/tipE in the absence (h, V 1/2 ¼ )20.5 ± 0.7 mV) and in the presence (n, V 1/2 ¼ )23.1 ± 0.6 mV) of 2 l M phaiodotoxin Data are presented as a Boltzmann sigmoidal fit The right figure shows the steady-state inactivation of para/tipE channels in the absence (V 1/2 ¼ )49.6 ± 0.4 mV) and presence (V 1/2 ¼ )43.8 ± 0.4 mV) of 2 l M phaiodotoxin Data are presented as a Boltzmann sigmoidal fit Each point represents the mean ± SEM of data from five experiments (C) Superimposed graphics of the activation and steady-state inactivation curves without toxin (left) and with phaiodotoxin (right) The window current of para/tipE with phaiodotoxin is 225% larger than without the toxin The inset below the graphs shows the superimposed enlarged window currents without (black) and with phaiodotoxin (black + grey).
Trang 7chosen and modified from an earlier publication by Gordon
and Gurevitz [34] The amino acid similarities of
phaiod-otoxin are closer to those of a-ScTXs, showing variable
scores of 30–49% similarity and only 22–32% of identity
The similarities and identities are even lower when
com-pared to the b-ScTXs (21–38% and 15–28%, respectively)
The cysteine residues are all aligned, although the length of
phaiodotoxin is longer (72 amino acid residues), only
surpassed by the insect-toxin Bj’xtrIT from Butothus
judaicus[35] The insect toxin 1 from Androctonus australis
(AshIT1) has 71 amino acids [36] These two last toxins were
described as insect-excitatory toxins [34–36] Phaiodotoxin
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 as shown in Fig 3 However, the 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 ScTXs 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 of the phaiodotoxin was electrophysiologically
tested on the cloned insect voltage-gated Na+ channel,
para, coexpressed in Xenopus laevis oocytes with the insect
Na+ channel subunit, tipE Current traces were evoked
using 25 ms step depolarizations of 5 or 10 mV to a voltage
range between)70 and 40 mV from a holding potential of
)90 mV In Fig 4A, an averaged trace and I–V curve (n ¼
5) are shown before and after addition of 2 lM of
phaiodotoxin An increase in current is noticed
(9 ± 0.3%) and the activation process is mildly shifted to
more negative potentials (DV1/2¼ 2.6 ± 0.9 mV) In
Fig 4B (left), this shift in activation is shown more clearly
(n¼ 5) On the right, the steady-state inactivation of para/
tipE channels in the absence and presence of phaiodotoxin
is shown (n¼ 5) Here, a shift towards more positive
potentials was seen Current traces shown were evoked by
50 ms depolarizations of 5 mV from)120 mV to )15 mV
followed by a 50 ms pulse to )10 mV, from a holding
potential of)90 mV
When the activation and inactivation curves of control
conditions on the one hand and toxin conditions on the
other hand are superimposed, we were able to determine the
window current for control conditions and toxin conditions
(Fig 4C) using the weighing method [27] When this is
performed, it is noticeable that the window current in toxin
conditions (2 lM) is about 225% that of control conditions
It is probable that this event causes toxicity in insects For
comparison, in 2001, Cannon reported that
voltage-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 impairment of inactivation or shifted voltage dependence Moreover, Cannon [39] showed that even a subtle disruption 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 channels, it is 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 toxic peptide ever isolated from a scorpion of the family Iuridae,
it was tempting to analyze possible evolutionary aspects of this peptide in the context of other known examples The great majority of known Na+channels specific scorpion toxins were isolated from the Buthidae family [reviewed in 9,34,38] As shown in Fig 3, the amino acid sequence similarities of phaiodotoxin are lower than 49%, when compared with the a-ScTx and less than 38% when compared with the b-ScTx We have enlarged this analysis
by generating a phylogenetic tree encompassing all known scorpion toxins or genes coding for similar peptides [9,34,38], but the final results clearly indicate that it is phylogenetically closer to the a-ScTxs (data not shown) However, due to the uniqueness of its sequence, it branches independently of the other a-ScTxs Figure 3 also shows that the core of the three disulfide bridges of phaiodotoxin is conserved similarly to the others, but as discussed in [16,34,38], the fourth pair is differently positioned Actually,
it is worth noticing that it is also different from the b-excitatory toxins Unfortunately thus far, the three dimensional structure and the genomic sequence of phai-odotoxin are not know, which could add some insight concerning the evolutionary links with other peptides isolated from the Buthidae scorpions The only plausible indication emerging from this analysis is that the C-terminal arrangement of this novel toxin might be responsible for its specific novel pharmacological actions: toxic to insects, where it enlarges the window currents of Na+channels, but non toxic to mammals
Acknowledgements
Supported in part by grants 40251-Q from the National Council of Science and Technology (CONACyT), Mexican Government, and IN206003-3 from Direccio´n General 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
Trang 8scorpions Experiments with COS7 and TE671 cells were kindly
performed by Professor Enzo Wanke and Rita Restano-Cassulini, from
the University of Milano at Biccoca, Italy, and those with GH3 and
cerebellum granular cells were performed by Dr Gianfranco Prestipino
from the Institute 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.
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