OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae

ARTICLE IN PRESS Toxicon 51 (2008) 1424– 1430

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OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae$ Yousra Abdel-Mottaleb a, Thomas Vandendriessche a, Elke Clynen b, Bart Landuyt b, Amir Jalali c, Hossein Vatanpour d, Liliane Schoofs b, Jan Tytgat a, a

Laboratory of Toxicology, University of Leuven, Onderwijs and Navorsing II, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium Research Group Functional Genomics and Proteomics, University of Leuven, Naamsestraat 59, 3000 Leuven, Belgium Department of Pharmacology and Toxicology, Jundishapur University of Medical Science, Ahvaz, Iran d Department of Pharmacology and Toxicology, Shaheed Beheshti University of Medical Science, Tehran, Iran b c

a r t i c l e i n f o

abstract

Article history: Received 14 June 2007 Received in revised form 17 March 2008 Accepted 18 March 2008 Available online 29 March 2008

The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically. OdK2 consists of 38 amino acids, including six conserved cysteine and a C-terminal lysine residue, as revealed by the unique use of a quadrupole ion cyclotron resonance Fourier-transform mass spectrometer. Based on multiple sequence alignments, OdK2 was classified as a-KTX3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned K+ channels (vertebrate Kv1.1–Kv1.6, Shaker IR and hERG) expressed in Xenopus laevis oocytes. Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.272.7 nM. & 2008 Elsevier Ltd. All rights reserved.

Keywords: Odonthobuthus doriae Voltage-gated potassium channels OdK2 a-KTX3.11 Quadrupole ion cyclotron resonance Fourier-transform mass spectrometry (Q ICR FT MS) Multiple sclerosis

1. Introduction The Iranian yellow scorpion Odonthobuthus doriae is a member of the Buthidae family and is especially found in the central and southern part of Iran (Jalali et al., 2005). Its stings cause various effects ranging from local pain, inflammation and necrosis to muscle paralysis. A study was carried out to identify the compounds or toxins in the venom of O. doriae that modulate physiological processes at the cellular level for the following reasons (Huys et al.,

$ Ethical statement: The authors declare that this work has not been published elsewhere and that the guidelines for animal welfare have been followed.  Corresponding author. Tel.: +32 16323404; fax: +32 16323405. E-mail address: [email protected] (J. Tytgat). URL: http://www.toxicology.be (J. Tytgat).

0041-0101/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.03.027

2002; Jalali et al., 2005; Abdel-Mottaleb et al., 2006a; Maertens et al., 2006): (a) very little is known about the bioactive substances in the venom of this scorpion, (b) the discovery of new toxins can be the key to gain insight into the molecular mechanisms of scorpionism, (c) selective toxins can be used for purifying channels from native tissue, determining their subunit composition and elucidating the pharmacological and physiological roles of voltage-dependent ion channels in target tissues and (d) selective toxins can be used as lead compounds for therapeutic drugs. Earlier studies of O. doriae venom revealed the presence of an a-like Na+ channel toxin, OD1, which affects the insect channel para/tipE (Jalali et al., 2005). The same toxin was later found to potently modulate Nav1.7 (Maertens et al., 2006). Moreover, only one K+ channel toxin was identified from the venom of O. doriae, OdK1, which selectively blocks Kv1.2 channels and

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belongs to subfamily a-KTx8 (Abdel-Mottaleb et al., 2006a). Toxic peptides have played a fundamental role for studying many structural and functional features of K+ channels. Scorpion toxins that target K+ channels are compact peptides that typically contain 23–43 amino acid residues and three or four disulfide bridges (Rodriguez de la Vega and Possani, 2004). Moreover, almost all of these toxins adopt a highly conserved secondary structure, the cysteine-stabilized a/b scaffold. These toxins have been classified into four large families: a-, b-, g- and k-KTx (Rodriguez de la Vega and Possani, 2004). A systematic nomenclature was proposed for a-KTx’s based on 13 independent subfamilies (Tytgat et al., 1999), which were later increased to 20 (Rodriguez de la Vega and Possani, 2004; Abdel-Mottaleb et al. 2006b). K+ channels are a ubiquitous family of membrane proteins that play a critical role in a wide variety of physiological processes, including the regulation of heart rate, muscle contraction, neurotransmitter release, neuronal excitability, insulin secretion, epithelial electrolyte transport, cell volume regulation and cell proliferation. Based on that critical role, K+ channels have been recognized as potential targets for therapeutic drugs for many years (Wickenden, 2002). For instance, voltagegated Kv1.3 channels have been proposed as a therapeutic target for immunomodulation of autoreactive effector memory TEM cells, which play a major role in the pathogenesis of autoimmune diseases (Beeton et al., 2005). In proof of this hypothesis, the Kv1.3-blocking peptides such as the sea anemone toxin ShK have been isolated and tested in animal models (Beeton et al., 2001). More potent derivatives of ShK such as ShK-Dap (Kalman et al., 1998) and ShK-F6CA (Beeton et al., 2003) have also been developed. In addition to peptide blockers of Kv1.3, small molecules such as Psora-4 (Vennekamp et al., 2004) and PAP-1 (Schmitz et al., 2005) have also been identified as potent blockers of Kv1.3 and have been found to suppress the proliferation of human TEM cells. Here we describe the very first example of a Kv1.3 channel-blocking toxin from the venom of the scorpion O. doriae, OdK2 (trivial name abbreviated from the genus and species first letter followed by K relating to K+ channels and the number 2 indicating the second K+ channel toxin from the venom of O. doriae; Abdel-Mottaleb et al., 2006a).

For enzymatic cleavage, 100 ml of the sample eluting between 23.7 and 24.7 min was incubated with 0.2 mg of the endoprotease Asp-N (Roche) in 10 mM Tris (pH 8) for 24 h at 37 1C and subsequently with 50 mM dithiothreitol (DTT) for 2 h at 37 1C. The resulting peptide fragments were separated on a Waters Symmetry C18 column (4.6  250 mm, 5 mm) using a gradient from 1–50% CH3CN with 0.1% TFA in 40 min (flow rate 1 ml/min). Peaks were collected manually. The fraction eluting between 23.7 and 24.7 min of the original fractionation, as well as the fractions resulting from the AspN digest, were analyzed by MALDI-TOF MS on a Reflex IV (Bruker daltonics GmbH): 1 ml of the sample solution was mixed with 1 ml of a saturated solution of acyano-4-hydroxycinnamic acid in acetone on a steel target and air dried. A quadratic calibration (o10 ppm) was performed using a standard peptide mixture (Bruker daltonics GmbH). Positive ion spectra were recorded in the reflectron mode from m/z 500–3500. In order to distinguish between K and Q, an accurate mass measurement was performed. For this, the venom peptide and Substance P (Bruker daltonics GmbH) were dissolved in 50% CH3CN/ 0.5% HCOOH to a final concentration of approximately 500 fmol/ml and were injected at a flow rate of 180 ml/min by the use of a syringe pump that was connected to the ESI source of an quadrupole ion cyclotron resonance Fourier-transform (Q ICR FT) mass spectrometer (Apex Qe, Bruker daltonics GmbH). The instrument was first externally calibrated with the fragmentation spectrum of the double charged ion of Substance P that was fragmented in the quadrupole by collision-induced dissociation (CID). Calibration was repeated until a mass accuracy of less than 1 ppm was reached. The accurate masses of several multiple charged peptide ions (3–7) were then determined in the infinity ICR cell of the Q ICR FT mass spectrometer. The accurate monoisotopic mass of the intact venom peptide was finally inferred from these multiple charged mass values by deconvolution. For amino acid sequencing, 100 ml of the fraction was spotted on a precycled Biobrene Plus TM-coated micro TFA filter and sequenced by Edman degradation on an automated Procise protein sequencer (Applied Biosystems).

2. Materials and methods

The amino acid sequence of OdK2 was analyzed by BLAST (Altschul et al., 1997) and FASTA (Pearson and Lipman, 1988) searches.

2.1. Purification and sequence determination of Odk2 HPLC analyses were performed on a Gilson liquid chromatograph. One milligram of venom was dissolved in 1 ml 0.1% TFA in water, filtered (Millex-HV, 0.45 mm, Millipore) and separated on a Waters Symmetry C18 column (4.6  250 mm, 5 mm) using a linear gradient from 10–25% CH3CN with 0.1% TFA in 60 min, followed by a gradient to 50% CH3CN with 0.1% TFA in 5 min with a constant flow rate of 1 ml/min. Fractions were collected manually.

2.2. Sequence comparison analysis

2.3. Electrophysiological experiments 2.3.1. Expression in oocytes Kv1.1, 1.2, 1.3, 1.4, 1.5, 1.6 and Kv1.1 mutant as well as hERG and Shaker IR channels were prepared as previously described (Stuhmer et al., 1988; Tytgat et al., 1995; Huys et al., 2004). For the Kv1.1 mutant, three residues critical for the binding of a-dendrotoxin on Kv channels were mutated (A352P, E353S and Y379H; Tytgat et al., 1995).

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Stage-V and -VI Xenopus laevis oocytes were harvested by partial ovariectomy under anaesthesia (3-aminobenzoic acid ethyl ester methanesulfonate salt, 0.5 g/l, Sigma). Anaesthetized animals were kept on ice during dissection. The oocytes were defolliculated by treatment with 2 mg/ ml collagenase (Sigma) in Ca2+-free ND-96 solution (in mM: NaCl 96, KCl 2, MgCl2 1, HEPES 5, adjusted to pH 7.5). Between 1 and 24 h after defolliculation, oocytes were injected with 50 nl of 50–100 mg/ml cRNA. The oocytes were then incubated in ND-96 solution (supplemented with 50 mg/l gentamycin sulfate and 1.8 mM CaCl2) at 16 1C for 1–5 days. 2.3.2. Electrophysiological measurements Two-electrode voltage-clamp recordings were performed at room temperature using a GeneClamp 500 amplifier (Molecular Devices) controlled by a pClamp data acquisition system (Molecular Devices). Whole-cell currents from oocytes were recorded 1–5 days after injection. Voltage and current electrodes were filled with 3 M KCl. Resistances of both electrodes were kept as low as possible (o0.5 MO). The current records were sampled at 0.5 ms intervals and filtered using a four-pole Bessel filter at 1 kHz. Linear components of capacity and leak currents were not subtracted for hERG channels. For Kv1.1–1.6 and Shaker IR, the leak currents were subtracted using a -P/4 protocol. 3. Results and discussion 3.1. Purification and primary sequence determination MALDI-TOF MS of the fraction eluting between 23.7 and 24.7 min of the gradient (Fig. 1) showed a doubly charged ion at m/z 2037.31 (monoisotopic mass) corre-

sponding to a peptide with Mr 4072.62 Da. Using Edman degradation based sequencing, the first 36 N-terminal amino acids were determined as GVPTDVKCRGSPQCIQP CKDAGMRFGKCMNGKCHCT. This sequence shows similarity to members of subfamily 3 of the short-chain -KTx, especially to Bs6 with which it differs in only three amino acids. When we assume that the last two C-terminal amino acids are also conserved between Bs6 and our toxin, then the complete sequence GVPTDVKCRGSPQ CIQPCKDAGMRFGKCMNGKCHCTPQamide has a calculated monoisotopic mass (containing three disulfide bridges) of 4072.8013 Da. The peptide was also cleaved using AspN, which resulted in two peaks on HPLC with a monoisotopic mass of 1660.70 and 2081.86 Da on MALDI-TOF MS. The first observed mass corresponds to the sequence DVKCRGSPQCIQPCK and the second indeed matches to DAGMRFGKCMNGKCHCTPQamide. However, as the amino acids Gln (Q) and Lys (K) differ by only 0.0364 Da, additional evidence was needed to unequivocally prove the identity of the last C-terminal residue. Therefore, an accurate mass measurement was performed on a Q ICR FT mass spectrometer, which reaches a sub-ppm mass accuracy. The mean monoisotopic mass of the intact venom peptide (4072.8381) was inferred from five multiple charged mass values by deconvolution, and shows that the last C-terminal residue corresponds to Lys (Mr 4072.8377) instead of Gln (Mr 4072.8013). Although FT ICR MS is not brand new, it is not often implemented by life science people yet. Here, we provide a nice example of how the extremely high resolution and mass accuracy of Q ICR FT can be exploited for the analysis of ‘‘difficult’’ biochemical questions. 3.2. Comparative sequence analysis The amino acid sequence of OdK2 was aligned using BLAST (Altschul et al., 1997), which retrieved a-KTx belonging to subfamily a-KTx3 with high similarity scores (Fig. 2A). Based on the criteria published by Tytgat et al. (1999), OdK2 was classified as a-KTx3.11. Therefore, it is reasonable to assume that the peptide will adopt the wellknown cysteine-stabilized a/b scaffold and therefore contains the typical disulfide pairing of other a-KTx subfamilies (Mouhat et al., 2004). 3.3. Electrophysiological studies

Fig. 1. Purification of OdK2 from the venom of Odonthobuthus doriae. (A) Soluble venom (1 mg) was dissolved in 1 ml 0.1% TFA, filtered (Millex-HV, 0.45 mm, Millipore) and separated on a Waters Symmetry C18 column using a linear gradient from 10–25% CH3CN with 0.1% TFA in 60 min, followed by a gradient to 50% CH3CN with 0.1% TFA in 5 min with a constant flow rate of 1 ml/min. The fraction containing OdK2 is indicated by an arrow.

We studied the pharmacological profile of OdK2 on an extensive panel of eight K+ channels, of which seven belong to the Shaker type (Kv1.x). Fig. 3 illustrates that OdK2 is a potent Kv channel blocker. The most remarkable features of OdK2 are: (a) its selectivity to Kv1.3 (5000 fold the IC50 value of Kv1.3 failed to inhibit seven other Kv channels: Kv1.1, Kv1.2, Kv1.4–Kv1.6, Shaker IR or hERG channels) and (b) its potency to block Kv1.3 channel at nanomolar concentrations (the IC50 value was 7.272.7 nM), which is the same range as ChTx and Pi toxins (Miller et al., 1985; Sands et al., 1989; Peter et al., 2000). Interestingly, the other members of subfamily aKTx 3 seem to be more potent than OdK2 on Kv1.3 (IC50

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Fig. 2. (A) Sequence alignment of OdK2 with other a-KTx3 subfamily members (Crest et al., 1992; Garcia et al., 1994; Laraba-Djebari et al., 1994; Jaravine et al., 1997; Meki et al., 2000; Kozminsky-Atias et al., 2007). %ID is the identity score of the peptide compared to OdK2. Among the other members of subfamily a-KTx 3, OdK2 amino acid sequence bears the highest similarity to Bs6 (Ali et al., 1998). (B) Sequence alignment of the pore region of Kv1.1–Kv1.6 channels and Kv1.1 mutant. The most important amino acid residues for the channel–toxin interaction are highlighted in red (Gly375 and His399, Kv1.3 numbering) while the common residues between Kv1.3 and the other Kv1.x channels in the figure are highlighted in blue.

value of OSK1 is 14 pM; Mouhat et al., 2005); yet, unlike OdK2, the selectivity to a single Kv1.x family member is lacking. Among the other members of subfamily a-KTx3, OdK2 amino acid sequence bears the highest similarity to Bs6 (Ali et al., 1998) for which the biological target has not yet been identified. Earlier studies report key motifs that play a crucial role in the interaction between a-KTx3 and Kv1.3 channels, namely, residues Lys27 and Phe25 which form the functional dyad and are conserved throughout the aKTx3 subfamily, including OdK2 (Dauplais et al., 1997; Gilquin et al., 2005). Additionally, the residues Met29, Asn30 and Ser11 contribute in the primary binding cores of scorpion toxins of the a-KTx3 and are conserved in OdK2 (Dauplais et al., 1997; Gilquin et al., 2005). Interestingly, OdK2 lacks Lys16, which is replaced by a Gln16 (Fig. 4). The impact of Lys16 on the potency of the toxin has been demonstrated in the double mutant named OSK1-Lys16Asp20, which generated the most potent Kv1.3 peptide blocker known so far (IC50 is 3 pM; Mouhat et al., 2005). To investigate the reason behind the selectivity of OdK2 to Kv1.3 channels, a sequence alignment of the pore regions of Kv1.1–Kv1.6 was made (Li and Gallin, 2004; Fig. 2B). Earlier studies on the interaction between a-KTx3 and Kv1.x channels have indicated some amino acid residues in the pore region as targets for the toxin-channel recognition. These residues are Gly375, Asp381, Tyr395, Asp397 and His399 (Yu et al., 2004). Since Asp381, Tyr395 and Asp397 exist in all six channels while Gly375 and His399 exist only in Kv1.3, both amino acid residues

Gly375 and His399 could account for the selectivity of OdK2 to Kv1.3 channels; however, the importance of those residues remains to be proven by mutagenesis. To further determine the footprint of OdK2 on Kv1.3 channels, we took advantage of a Kv1.1 triple mutant (Tytgat et al., 1995) in which a Y379H mutation was made to match His399 of Kv1.3 channels (Somodi et al., 2004) as well as two other mutations A352P and E353S. The Kv1.1 mutant was originally made to study the footprint of a–dendrotoxin on Kv channels. Interestingly, OdK2 was able to induce a block of 21.773% (mean7SEM, n ¼ 4) in the mutant channel at a concentration of 35 nM. Higher concentrations of OdK2 (up to 35 mM) however did not induce a significant increase in the block of the channel. Qualitatively, we proved that the mutant is affected by OdK2 but quantitatively the effect was not equal to the one seen on Kv1.3. An explanation could be that the toxin interacts with a His399, causing the partial block and that alternative contact points are required to block the channel. The precise footprint of OdK2 on Kv1.3 channels remains to be elucidated, ideally via co-crystallization, knowing that the subtle difference in the amino acid sequence of the pore region causes dramatic effects on the selectivity of the toxin to the channels. Kv1.3 channels together with Ca2+-activated K+ channels (IKCa1) regulate the membrane potential and Ca2+signaling in T lymphocytes. Autoreactive memory T lymphocytes have been implicated in the pathogenesis of multiple sclerosis (MS), type-1 diabetes mellitus and psoriasis (for a review see Wulff et al., 2003). In MS, myelin-reactive lymphocytes exhibit a memory pheno-

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Fig. 3. (A–H) Selected current traces showing the effect of OdK2 on Kv1.1–Kv1.6, Shaker IR and hERG channels at a concentration of 35 nM. The toxin blocked the current through Kv1.3 by 95% while no effect was seen on the other channels. For Kv1.1–Kv1.6 and Shaker IR, currents were evoked by a depolarization from a holding potential of 90 mV to a test potential of 0 mV and followed by a test potential of 50 mV. hERG currents were induced by a depolarization to 0 mV from a holding potential of 90 mV followed by a test potential of 70 mV. (I) Concentration dependence of toxin OdK2. IC50 value was 7.272.7 nM for Kv1.3 while no effect was observed on Kv1.1, Kv1.2, Kv1.4, Kv1.5 or hERG channels. The data for Kv1.6 and Shaker IR are not shown. For the dose–response curves the percentage of unblocked current was plotted as a function of increasing toxin concentrations. Each point shown is the mean7SEM for three to five experiments.

type and are believed to contribute to the inflammatory attack on the central nervous system because of their ability to induce experimental autoimmune encephalomyelitis (EAE) in animals. Strategies designed to specifically suppress the function of chronically activated memory T cells without impairing the function of naive T cells might therefore have value in the treatment of autoimmune diseases (Wulff et al., 2003). In this respect, selective toxins such as OdK2, blocking Kv1.3 at low nanomolar concentration could present a potential lead compound to study and explore the channels in native tissues for the determination of potential drugs in pathogenic conditions.

Acknowledgments

Fig. 4. Model of AgTx2 (PDB ID: 1AGT) showing the important residues involved in the interaction of the toxin with Kv1.3 channels (Yu et al., 2004). The model was constructed by SWISS-MODEL (Kopp and Schwede, 2004) and visualized using MOLMOL program (Koradi et al., 1996).

We thank Prof. O. Pongs for providing the cDNA for the Kv1.2, 1.4, 1.5 and 1.6 channels. The Kv1.3 clone was kindly provided by Prof. M.L. Garcia and the Shaker IR clone was kindly provided by Prof. G. Yellen. The authors would like to acknowledge the skilful technical assistance of

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Mr. L. Vanden Bosch and Ms. S. Debaveye. The authors would also like to thank Dr. Elke Vermassen for the constructive comments on the manuscript. T. Vandendriessche and E. Clynen are, respectively, a research assistant and a postdoctoral fellow of the Fund for Scientific Research (FWO)-Flanders. B. Landuyt is a postdoctoral fellow of the Institute for the Promotion of Innovation through Science and Technology (IWT)-Flanders. The authors would like to thank the Flemish Government for the mass spectrometry facility ProMeta. This work was also supported in part by Grants OT/05064, G.330.06 from the Fund for Scientific Research (FWO)-Flanders and P6/31 (Interuniversity Attraction Poles Program-Belgian State, Belgian Science Policy). References Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006a. The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae. FEBS Lett. 580 (26), 6254–6258. Abdel-Mottaleb, Y., Coronas, F.V., de Roodt, A.R., Possani, L.D., Tytgat, J., 2006b. A novel toxin from the venom of the scorpion Tityus trivittatus, is the first member of a new alpha-KTX subfamily. FEBS Lett. 580 (2), 592–596. Ali, S.A., Stoeva, S., Schutz, J., Kayed, R., Abassi, A., Zaidi, Z.H., Voelter, W., 1998. Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 121 (4), 323–332. Altschul, S.F., Madden, T.L., Schuffer, A.A., Zhang, J., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25, 3389–3402. Beeton, C., Barbaria, J., Giraud, P., Devaux, J., Benoliel, A.M., Gola, M., Sabatier, J.M., Bernard, D., Crest, M., Beraud, E., 2001. Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation. J. Immunol. 166 (2), 936–944. Beeton, C., Wulff, H., Singh, S., Botsko, S., Crossley, G., Gutman, G.A., Cahalan, M.D., Pennington, M., Chandy, K.G., 2003. A novel fluorescent toxin to detect and investigate Kv1.3 channel upregulation in chronically activated T lymphocytes. J. Biol. Chem. 278 (11), 9928–9937. Beeton, C., Pennington, M.W., Wulff, H., Singh, S., Nugent, D., Crossley, G., Khaytin, I., Calabresi, P.A., Chen, C.Y., Gutman, G.A., Chandy, K.G., 2005. Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases. Mol. Pharmacol. 67 (4), 1369–1381. Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., Martin-Eauclaire, M.F., 1992. Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom. J. Biol. Chem. 267 (3), 1640–1647. Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, B., Roumestand, C., Vita, C., de Medeiros, C.L., Rowan, E.G., Harvey, A.L., Menez, A., 1997. On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channelblocking toxins with unrelated structures. J. Biol. Chem. 272 (7), 4302–4309. Garcia, M.L., Garcia-Calvo, M., Hidalgo, P., Lee, A., MacKinnon, R., 1994. Purification and characterization of three inhibitors of voltagedependent K+ channels from Leiurus quinquestriatus var. hebraeus venom. Biochemistry 33 (22), 6834–6839. Gilquin, B., Braud, S., Eriksson, M.A., Roux, B., Bailey, T.D., Priest, B.T., Garcia, M.L., Menez, A., Gasparini, S., 2005. A variable residue in the pore of Kv1 channels is critical for the high affinity of blockers from sea anemones and scorpions. J. Biol. Chem. 280 (29), 27093–27102. Huys, I., Dyason, K., Waelkens, E., Verdonck, F., van Zyl, J., du Plessis, J., Muller, G.J., van der Walt, J., Clynen, E., Schoofs, L., Tytgat, J., 2002. Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom. Eur. J. Biochem. 269 (7), 1854–1865. Huys, I., Xu, C.Q., Wang, C.Z., Vacher, H., Martin-Eauclaire, M.F., Chi, C.W., Tytgat, J., 2004. BmTx3, a scorpion toxin with two putative functional

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