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 shows that phaiodotoxin belongs to the long-chain sub- family of scorpion peptides. S everal genes coding for t his 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 l ines (TE671, COS7, rat GH3 and cerebellum g ranular 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 ÔwindowÕ current is increased by 225%, which is th ought to be the cause of its t oxicity t oward 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 c haracterized from this family. Among the most important findings are four different groups of peptides, which specifically interact w ith ion channels: Na + 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 potentials upon binding to receptor site 4 [ 11]. This class includes two types o f 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 b3strandbytwoof the four d isulfide bonds. T he cysteine pair of the a-he lix motif is spaced by a tripeptide CXXXC (where C stands for cysteine and X for any amino a cid), whereas the pair of cysteine residues of the b3 s trand is separated by only one amino acid residue (CXC), usually linking the C3 (third cysteine of the s equence) to C6 and C4 t o C7 [12]. A t hird structurally conserved d isulfide bridge occur s between t he C2 of the N-terminal segment with C5 of the b2 stran d [ 9]. The fourth disulfide bond is established between C1 and C8, of the N- with t he C-terminal region. The excitatory insect toxins lack the equivalent position of C1, present in most scorpion toxins, a nd the fourth disulfide bridge is formed between C5¢ (contiguous to C5) w ith C 8 [ 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 w as mapped. Analysis of the t hree-dimen- sional models suggests that the functional differences reside Correspondence to L. D. Possani, Instituto de Biotecnologı ´ aUNAM 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 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 + 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 sco rpions were collected in Maneadero Baja California, Mexico. Their venom was obtained by electrical stimulation, dissolved in double d istilled water, centrifuged at 15 000 g for 15 min and the supernatant lyophilized and kept at )20 °C. The s oluble venom was applied t o a Sephadex G-50 column (0.9 · 190 cm) in 20 m M ammonium 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, H isperia, 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 c ases, a linear g radient was run f or 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 lLNaCl/P i (phosphate buffered 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 peptide w ere obtained after cleavage of phaiodotoxin with cyanogen bromide (CNBr), than ks to the presence o f two methionine residues in the molecule. An eight-fold excess of CNBr over toxin ( w/w) in 70% formic acid was used according to t he technique described by B iedermann [19]. After o vernight reactio n, the pr oducts were reduced with dithiothreitol for 30 min, at 56 °C and separated b y HPLC. The sub peptides were used for Edman degrad ation a nalysis. The molecular mass determination of pure phaiodotoxin and the additional sequencing work w as performed by mass spectrometry, using an LCQ Duo Finnigan mass spectrometer, as described previously [20]. All spectra were obtained in the positive-ion m ode. For sequence d etermin- ation, MS/MS s pectra pr oduced were analyzed manually and automatically by SEQUEST software. The acquisition and deconvolution of data were performed with the XCALI- BUR software 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 an alysis. It i s worth noting that for these sequences no reduction of the peptides was per- 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 construct was used to transform Escherichia coli DH5-a cells. Clone selection and DNA sequencing were p erformed as described by C orona et al.[23].Inordertocompletethe nucleotide sequence, the m ethod for rapid amplification of the 5¢-region (RACE 5¢) was applied, using RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends) protocol, according t o the instructions of the kit fr om Ambion (Austin, TX, USA). The cDNA mix was synthes- ized from poly(A)+ mRNA u sing M-MLV reverse tran- scriptase. The cDNA was joined with the a daptor provided by the kit (5 ¢-gcugauggcgaugaaugaacacugcguuugCUGG CUUUGAUGAAA-3¢) using T4 DNA ligase. The modi- fied cDNA was used as t emplate for PCR amplification. Two rounds of amplification with the primers from the Ambion kit were performed. Expression in Xenopus oocytes For t he expression in Xenopus oocytes, the para/pG H19- 13–5 vector [24] and tipE/pGH19 vector [25] were linearized with Not I 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 )1 using a Drum- mond microinjector (Broomal, P A, USA). The solution used for incubating the oocytes contained (in m M ): 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 Bessel filter, currents were fi ltered at 2 kHz and sampled at 10 kHz. Leak and capacitance subtraction w ere 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 e ffect of phaiodotoxin was also assayed i n several mammalian cell lines: TE671 (from human cerebellar medulloblastoma), COS7 (from monkey kidney fibro- blasts), GH3 and cerebellum granular ce lls from r at, 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 toxictomiceusingadoseupto100lg 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. 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 (corresponding to residues in positions M41 to R59 and M62 to K70) were s equenced 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. 1 C). The full sequence w as also confirmed by mass spectrometry. The molecular mass o f native phaiod- otoxin was s hown to b e 7971.0 atomic mass units, whereas the theoretical expected value based on the sequence obtained was 7970.3 atomic m ass units (within the experi- mental error). The correct overlapping segments were further aligned, after c loning the g ene that codes f or the toxin, as it will be discussed below. cDNA clone of phaiodotoxin Figure 2A shows t he nucleotide sequence obtained for the cloned g ene o f phaiodotoxin. In total 372 nucleotide pairs were identified. They code for the 72 amino acid residues of Ó FEBS 2004 Phaiodotoxin, an insect-toxin (Eur. J. Biochem. 271) 4755 the 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; w hereas at the 3¢-end, after the stop codon, 28 nucleotide bases were determined. Figure 2 B shows the Fig. 2. Nucleotide sequence of the g ene coding for phaiodotoxin. (A) The deduced amino acid sequence corres ponding to the gene of phaio dotoxin is indicated below each codon, starting from th e signal peptide (u nderlined). T he seque nce co rresponding to the mature peptide is indicated i n bold. A segment corresponding to the 5¢-untranslated region is s hown on the fi rst line (first 71 base pairs). The stop codon is indicated, followed by 28 base pairs of untranslated s equence. Numbers on the right side indicate both the nucleotide se quenc e and the amino acid sequence. (B) Two additional putative isoforms of phaiodotoxin were cloned and sequenced. The first line labelled PhTx contain s the amino a cid sequence of phaiodotoxin, the second and third lines show two isoforms: PhTx2, and PhTx3, respectively. Residue in position 1 6 for PhTx2 is Ser instead o f 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 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 (about 16 atomic mass units more than expected, due to the oxidation of t he methionine, i n this p articular preparation). These results showed that in phaiodotoxin, a new structural arrangement of disulfide pairs occurs between non expected cysteinyl residues. Because of t his fact, this experiment was repeated with another a liquot of toxin, but the final results were identical. Still another sample was analyzed (from the cyanogen bromide cleavage) a lso c onfirming this unusual disulfide pairing. From the other four peptides obtained after endopeptidase Lys-C cleavage (mentioned before), the one elutin g a t 3 3.08 min (data not shown) turned out t o 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 m in was found to cor respond t o t he segments that links the C ys13 with Cys38, i.e. disulfide pair: C 2–C5. The peptide eluted at 26.15 min allowed the identification of Cys23 with C ys50, 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 t o form the last missing disulfi de bridge. Furthermore, this is one of the c onstant disulfide pairs found in all the scorpion toxins described to data. In this way, as shown in F ig. 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-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 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 s teady-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 prese nted as a Bo ltzm ann sigm oidal fit. Each p oint rep resents the mean ± SEM of data from five e xperiments. (C) Superimposed graphics of th e activation and steady-state inactivation curves without toxin (left) and with phaiodotoxin (right). The window current of para/tipE with p haiodoto xin is 225% larger than witho ut the toxin. Th e inset below the grap hs shows the superimposed enlarged window currents without (black) and with phaiodotoxin (black + grey). 4758 N. A. Valdez-Cruz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 chosen and modified from a n earlier publication by G ordon and G urevitz [34]. The amino acid similarities of phaiod- otoxin are closer to those of a-ScTXs, showing variable scores of 30–49% similarity and only 22–3 2% of identity. The similarities and identities are even lower when com- paredtotheb-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 t wo 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 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 using 25 m s step depolarizations of 5 or 10 m V to a voltage range between )70 and 40 mV from a holding potential of )90 mV. I n Fig. 4A, an averaged trace and I–V curve (n ¼ 5) are shown before and after addition of 2 l M of phaiodotoxin. An increase in current is noticed (9 ± 0.3%) and the activation process is mildly shifted to more negative potent ials (DV 1/2 ¼ 2.6±0.9mV). In Fig. 4B (left), this shift in activation is shown more clearly (n ¼ 5). On the right, the steady-state inactivation of para/ tipE channels in the absence and presence of phaiodotoxin is shown (n ¼ 5). Here, a s hift towards more positive potentials w as seen. Current traces shown were evoked by 50 ms depolarizations of 5 mV from )120 mV t o )15 mV followed by a 50 ms pulse to )10 mV, from a h olding potential of )90 mV. When the activation and inactivation curves o f control conditions on the one hand and toxin conditions on the other han d a re superimposed, we were able to determine the window current for control conditions and toxin conditions (Fig. 4 C) using the weighing method [27]. When this is performed, it is noticeable that the window current in toxin conditions (2 l M ) is about 225% that of control conditions. It is probable that this e vent 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 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 + channels specific scorpion toxins were isolated from the Buthidae family [reviewed in 9,34,38]. As s hown i n Fig. 3, the amino acid sequence similarities of phaiodotoxin are l ower 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 a ll 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 o f t he 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-e xcitatory toxins. Unfortunately thus far, the three dimensional s tructure and the genomic sequence of phai- odotoxin are not know, w hich could add some insight concerning the evolutionary links with other peptides isolated from the B uthidae scorpions. T he only p lausible indication emerging from this analysis is that the C -terminal arrangement o f this novel toxin might be responsible for its specific novel pharmacological actions: toxic to insects, where it e nlarges the ÔwindowÕ currents of N a + 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 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 Res. Comm. 47, 285–289. 4. Debin, J.A., Maggio, J.E. & Strichartz, G.R. (1993) Purification and c haracterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am.J.Physiol.264, C361–C369. 5. Chuang, R.S., Jaffe, H., Cribbs,L.,Perez-Reyes&Swartz,K.J. (1998) Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin. Nat. Neurosci. 1, 668–674. 6. Olamendi-Portugal, T., Garcı ´ a, B.I., Lo ´ pez-Gonza ´ lez, I., Van Der Walt, J., Dyason, K., Ulens, C., Tytgat, J., Felix, R., Darzon, A . & Possani, L.D. (2002) Two new scorpion toxins that target voltage- gated Ca 2+ and Na + channels. Biochem. Biophys. Res. Commun. 299, 562–568. 7. Martin-Eauclaire, M.F. & Couraud, F. (1995) Scorpion neuro- toxins: effects and mechanisms. In Handbook on Neurotoxicology (Chang, L.W. & Dyer, R.S., eds), pp. 683–716. Marcel Dekker, New York. 8. Gordon, D., Savarin, P., Gurevitz, M. & Zinn-Justin, S. (1998) Functional anatomy of scorpion toxins affecting sodium channels. J. Toxicol. Toxin. Rev. 17 , 131–158. 9. Possani, L .D., Becerril, B., Delepierre, M. & Tytgat, J . (1999) 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. 274, 5769–5776. 16. Gurevitz, M., Gordon, D., Ben-Natan, S., Turko v, M. & Froy, O. (2001) Diversification of neurotoxins by C-tail ÔwigglingÕ:ascor- pion recipe for survival. FASEB J. 15, 1201–1205. 17. Cohen,L.,Karbat,I.,Gilles,N.,Froy,O.,Corzo,G.,Angelovici, R., Gordon, D . & Gurevitz, M. (2004) Dissection o f t he fu nctional surface of an a nti-insect excitatory toxin illuminates a putative hot spot common to all scorpion beta-toxins affectin g Na + channels. J. Biol. Chem. 279, 8206–8211. 18. Brune, D .C. (1992) Alkylation of c ysteine with a crylamide for protein sequence analysis. Anal. Biochem. 207, 285–290. 19.Biedermann,K.,Montali,U.,Martin,B.,Sevendsen,I.& Ottesen, M. (1980 ) T he amino acid s equence o f proteinase A inhibitor 3 from bakers yeast. Carlsberg Res. Commun. 45, 225– 235. 20. Batista, C.V.F., del Pozo, L., Zamudio, F.Z., Contreras, S., Becerril,B.,Wanke,E.&Possani,L.D.(2003)Proteomicsofthe venom from the Amazonian scorpion Tityus cambridgei and th e role of prolines on mass spectrometry analysis of toxins. J. Chromatogr. B 803, 55–66. 21. Thompson, J.D., G ibson , T.J., Plewniak, F ., Jeanmougin, F. & Higgins, D.G. (1997) The CLUSTAL X windows interface: flexible strategies fo r multiple s equence alignment aided by quality ana- lysis tools. Nucleic Acids Res. 25, 4876–4882. 22. Chirgwin, J.M., Przybyla, A.E., McDonald, R.J. & Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from source 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, L.D. (2000) Fast K + currents from cerebellum granular cells are completely blocked b y a peptide purified fro m Androctonus australis Garzoni scorpion veno m. Biochim. Biophys. Acta 1468, 203–212. 29. Zlotkin, E., Gurevitz, M., Fowler, E. & Adams, M.E. ( 1993) Depressant insect selective neurotoxins from scorpion venom: chemistry, action, and gene cloning. Arch. Insect Biochem . Physiol. 22, 55–73. 30. Licea, A.F., Becerril, B. & Possani, L.D. (1996) FAB fragments of the monoclonal antibody BCF2 are capable of neutralizing the whole soluble venom from t he scorpion C entruroides noxius Hoffmann. Toxicon 34, 843–847. 31. Froy, O., Zil berberg, N., Gordon, D., Turkov, M., Gilles, N., Stankiewicz, M., Pe lhate, M., L oret, E., Oren, D.A., Shaanan, B. & G urevitz, M. (1999) The putative bioactive su rface of insect- selective scorpion excitatory neurotoxins. J. Biol. Chem. 274, 5769–5776. 4760 N. A. Valdez-Cruz et al.(Eur. J. Biochem. 271) Ó FEBS 2004 32. Corona, M., Zurita, M., Possani, L.D. & Becerril, B. (1996) Cloning and characterization of the genomic region encoding toxin IV-5 from the scorpion Tityus serrulatus LutZ and Mello. Toxicon 34, 251–256. 33. Ye, J G., Chen, J., Zuo, X P. & Ji, Y H. (2001) Cloning and characterization of cDNA sequ ences en coding two novel a-like- toxin precurso rs from the Chinese scorpion Buthus martensii Karsch. Toxicon 39, 1191–1194. 34. Gordon, D . & Gurevitz, M. (2003) The selectivity of scorpion alpha-toxins for sodium channel subtypes is determined by subtle 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 . Phaiodotoxin, a novel structural class of insect-toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus Norma A. Valdez-Cruz 1 ,. 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