Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels

Toxicon 55 (2010) 1425–1433

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Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels Yuzhe Yuan a,1, Lan Luo a, 2, Steve Peigneur b, 2, Jan Tytgat b, Shunyi Zhu a, * a

Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insect & Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R. China b Laboratory of Toxicology, University of Leuven, O&N 2, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 October 2009 Received in revised form 16 February 2010 Accepted 18 February 2010 Available online 26 February 2010

The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: BmKITc BmKITc2 Prokaryotic expression DRG neurons DmNav1/tipE

1. Introduction Voltage-gated sodium channels (VGSCs) are complex membrane proteins that regulate and control electrical excitability of insect and mammalian muscles and nerves

Abbreviations: CD, circular dichroism; CSab, cysteine-stabilized a-helix/ b-sheet; DMEM, Dulbecco’s modified Eagle’s medium; DRG, dorsal root ganglia; EK, enterokinase; GST, glutathione-S-transferase; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MBP, maltosebinding protein; RP-HPLC, reverse phase high performance liquid chromatography; TFA, trifluoroacetic acid; VGSC, voltage-gated sodium channel; TTX-S, tetrodotoxin-sensitive; TTX-R, tetrodotoxin-resistant. * Corresponding author. Fax: þ86 010 64807099. E-mail address: [email protected] (S. Zhu). 1 Present address: Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, Sichuan Province, China. 2 These authors equally contributed to this work. 0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2010.02.019

(Catterall et al., 2007). Modification of the pharmacological activities of these channels by toxins from various venomous animals causes rapid immobilization of their preys. Scorpion venom-derived depressant toxins comprise a subfamily of bioactive polypeptides that contain 61–65 amino acids stabilized by four disulfide bridges (Possani et al., 1999; Rodriguez de la Vega and Possani, 2005; Gurevitz et al., 2007). These b-toxins bind to the receptor site 4 of the VGSCs and affect channel activation in a manner by shifting the voltage dependence of activation in the hyperpolarizing direction. In a recent work, Bosmans et al. have found that btoxins can interact with multiple Na channel paddle motifs from domains II, III or IV in rNav1.2, but only with domain II paddle in rNav1.4 (Bosmans et al., 2008). Scorpion depressant toxins exhibit preferential ability in induction of a transient contraction paralysis of insect larvae followed by a progressive flaccid paralysis, making them attractive

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candidates for construction of transgenic plants or viral vectors to control pests. In fact, by combination of viral promoters, LqhIT2 and Lqh-dprIT3 have shown strong insecticidal efficacy (Gurevitz et al., 2007). So far, more than 24 depressant toxins have been functionally characterized (Fig. 1). Although most of them show high preference for insect VGSCs and modulate their activation (Bosmans et al., 2005; Karbat et al., 2007), some members are active on mammals, e.g. (1) BmKIM, a recombinant peptide toxic to insects and mammals, could inhibit Naþ currents in rat DRG neurons and ventricular myocytes (Peng et al., 2002); (2) BmKdITAP3 was reported to have a dual bioactivity, a depressant toxicity on insects and an analgesic effect on mice (Guan et al., 2001); (3) BmKAEP had little toxicity on mice and insects but was found to have an anti-epilepsy effect in rats (Wang et al., 2001); (4) LqhIT2 was demonstrated to bind and affect rat skeletal muscle channels (Cohen et al., 2007). In this study, we recombinantly produced two new depressant toxins (BmKITc and BmKITc2) in E. coli and characterized their structural and functional features. BmKITc and BmKITc2 differ by only one amino acid subsitution at position 52 (Thr52Lys) and therefore constitute an interesting starting point of investigation. As described below, we have found that this single-residue change resulted in functional diversification of these two toxins, making BmKITc rather than BmKITc2 a weak inhibitor of the TTX-R and TTX-S Naþ channels in rat DRG neurons. This observation highlights the crucial role of lysine 52 in interacting with mammalian VGSCs. In addition, although lacking sequence conservation in the equivalent positions corresponding to the ‘pharmacophore’ of scorpion b-toxins (Karbat et al., 2007), these two peptides were able to produce a significant change on the slow phase of inactivation of the Drosophila DmNav1/tipE channels expressed in Xenopus oocytes. 2. Materials and methods 2.1. Materials Male Sprague-Dawley rats (180–200 g) were purchased from the Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All primers used in this study were synthesized by SBS Genetech (Beijing, China): Bm-IT-F (50 -ATGGATCCGATGACGATGACAAGGATGGATATATAAGAGGAAGT-30 ); Bm-IT-R (50 -ATGTCGACTTAGCTACCGCATGTATTACTTTC-30 ); Bm-IT-RRn (50 -TTTCATTATCAGGAAGGCCTTCACACCA-30 ) and Bm-IT-FRn (50 -AATGGAAATATGAAAGTAATACATGCGGT-30 ), in which Bam HI and Sal I sites are underlined once and codons for enterokinase (EK) is italized. Reagent sources: Glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech Inc.); Enterokinase (Sinobio Biotech Co. Ltd., Shanghai, China); Amylose affinity resin (New England Biolabs, Ipswich, USA); Trypsin (Ameresco); Collagenase and trypsin inhibitor (Sigma). 2.2. Construction of pGEX-6P-1-BmKITc2 expression vector and its expression in E. coli Isolation of the BmKITc2 clone has been described previously (Tian et al., 2008) and its ligation into pGEX-6P-

1 was performed according to the published method (Yuan et al., 2007). In order to remove the carrier glutathione-Stransferase (GST) to exactly obtain the peptide, we introduced an EK cleavage site at the Bam HI downstream (Fig. S1, provided as supplementary data). The recombinant plasmid was transformed into E. coli DH5a and positive clones were confirmed by DNA sequencing. Expression of the fusion protein GST-BmKITc2 in E. coli BL21(DE3) was induced by 0.5 mM IPTG at 25  C when an OD600 reached 0.7. The fusion protein obtained from a sonication supernatant by affinity chromatography with glutathione-Sepharose 4B beads was digested with EK at 22  C for 5 hr. The released BmKITc2 was separated from GST using reverse-phase HPLC on C8 column (Agilent Zorbax, Eclipse XDB-C8, 4.6 mm  150 mm, 5 mm). Elution was carried out using a linear gradient of 10–70% acetonitrile in 0.1% trifluoroacetic acid (TFA) in watere (v/v) within 40 min with a flow rate of 1 ml/min. 2.3. Construction of pMAL-p2X-BmKITc expression vector and its expression in E.coli For constructing pGEX-6P-1-BmKITc, we mutated site 52 by inverse PCR to amplify the template pGEX-6P-1BmKITc2 by two phosphated primers (Bm-IT-FRn and Bm-IT-RRn) (Fig. S1). The PCR condition is as follows: 35 cycles of 1 min at 94  C, 45 sec at 62  C, and 8 min at 72  C. Self-circularization of the PCR product was carried out by DNA ligase and then the circularized product was transformed into E. coli DH5a. Positive clones were confirmed by DNA sequencing. Subsequently, we cut the coding region of BmKITc by Bam HI and Sal I and ligated it into the downstream malE gene, which encodes maltose-binding protein (MBP), of the pMAL-p2X expression vector (Fig. S1). The constructed vector was transformed into E. coli DH5a for protein expression. Expression of fusion proteins was induced with 0.2 mM IPTG at 37  C until an OD600 reached 0.5. Cells were harvested 5 hr after addition of IPTG by centrifugation and the pellet was resuspended in the column buffer (20 mM Tris–HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA) for sonication. The supernatant was then loaded to an amylose affinity resin for 2 h, followed by washing unbounded proteins using the column buffer containing 10 mM b-mercaptoethanol. Fusion proteins bound on amylose resin were eluted using the column buffer containing 10 mM maltose and then were digested with EK at 22  C for 24 h until about 80% BmKITc was released. The recombinant product was purified using RP-HPLC on C18 column (Agilent Zorbax 300SBC18, 4.6 mm  150 mm, 5 mm) as described previously. 2.4. Characterization of recombinant protein CD spectra of recombinant toxins were recorded on a JASCO J-715 spectropolarimeter (Tokyo) at a protein concentration of 0.2 mg/ml dissolved in 10 mM sodium phosphate buffer, pH 6.8. Spectra were measured at 25  C from 250 nm to 190 nm using a quartz cell of 1.0 mm length. Data were collected at 0.2 nm intervals with a scan rate of 200 nm/min. CD spectra measurement was performed by averaging three scans. Secondary structure

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contents were estimated by the standard JASCO CD analysis. Molecular weights of peptides were determined by MALDI-TOF mass spectra on a Kratos PC Axima CFR plus (Shimadzu Co. Ltd., Kyoto). 2.5. Preparation of rat dorsal root ganglia neurons Dorsal root ganglia (DRG) neurons were isolated by the method described by Xiao et al. (2005). A rat was killed by decapitation without anesthetization and its DRG was removed quickly from the spinal cord and transferred into 2 ml Dulbecco’s modified Eagle’s medium (DMEM) containing trypsin (0.5 mg) and collagenase (2.0 mg, type IA). After enzymolysis at 34  C for 20-30 min, trypsin inhibitor (1.0 mg, type II-S) was added to terminate the reaction. After centrifugation and resuspension, cells were distributed on coverslip in a 35-mm dish and incubated at 37  C (5% CO2, 95% air) for 30–60 min. And then 2 ml DMEM containing 10% fetal calf serum and 100 u/ml penicillin– streptomycin were added into the dish. Cells were cultured in a CO2 incubator at 37  C (5% CO2, 95% air) for 3 h before patch clamp. 2.6. Whole cell patch clamp recording Whole-cell patch clamp technique was used to record Naþ currents in DRG neurons (Xiao et al., 2005). The internal solution contains (in mM): CsF, 135; NaCl, 10; HEPES, 5 (pH 7.0 with 1 M CsOH), and the external bathing solution contains (in mM): NaCl, 30; KCl, 5; CsCl, 5; Dglucose, 25; MgCl2, 1; CaCl2, 1.8; HEPES, 5; tetramethylammonium chloride, 90 (pH 7.4). After establishing wholecell recording configuration, the resting potential was held at 80 mV for 2–3 min to allow adequate equilibration between the micropipette solution and the cell interior. Current traces were evoked by 50 ms þ10 mV depolarization step from a holding potential of 80 mV. Membrane currents were measured using an AxoClamp 2B amplifier (Axon Inc.) and DIGIDATA 1322A (Axon Inc.). Pulse stimulation and data acquisition were controlled by Clampex 9.0 software (Axon Inc.). Tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) currents were separated by 200 nM TTX. Statistical analyses were performed with SPSS17.0 (SPSS Inc.).

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2.8. Two-electrode voltage-clamp recordings Two-electrode voltage-clamp recordings were performed at room temperature (18–22  C) using a Geneclamp 500 amplifier (Axon Instruments, USA) controlled by a pClamp data acquisition system (Axon Instruments, USA). Whole cell currents from oocytes were recorded 4–5 days after injection. Bath solution composition was (in mM): NaCl, 96; KCl, 2; CaCl2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4). Voltage and current electrodes were filled with 3 M KCl. Resistances of both electrodes were kept between 0.7 and 1.5 MU. The elicited currents were filtered at 2 kHz and sampled at 10 kHz using a four-pole low-pass Bessel filter. Leak subtraction was performed using a P/4 protocol. Representative whole-cell currents were elicited by a 100 ms voltage pulse to 0 mV, from a holding potential of 90 mV. For activation protocols, 100 ms depolarizations ranging from 90 to 65 mV were applied from a holding potential of 90 mV in 5 mV increments. Voltagedependent steady state inactivation was determined by means of a double-pulse protocol in witch a conditioning pulse was applied from a holding potential of 90 mV to a range of potentials from 120 mV to 20 mV in 5 mV increments for 100 ms, immediately followed by a 100 ms test pulse to the voltage of maximal activation of the VGSC in control conditions. Each experiment was performed at least 5 times (n  5). All data are presented as mean  standard error of the mean. 2.9. Homology modeling Multiple sequence alignment was carried out by the program CLUSTAL X (http://bips.u-strasbg.fr/fr/ Documentation/ClustalX/).The experimental structure of LqhIT2 (pdb entry 2I61) was used as a template for modeling structures of BmKITc and BmKITc2 by programs TITO and MODELLER (http://bioser.cbs.cnrs.fr/). Models were evaluated by verify3D and PROSA. Structural superimposition, root mean square deviation (RMSD) and surface potential calculation were performed using SwissPdb Viewer software (http://swissmodel.expasy.org/ spdbv). 3. Results 3.1. Sequence and structural analysis of BmKITc2 and BmKITc

2.7. Expression of DmNav1/tipE in X. oocytes For the expression in X. oocytes, the cDNA encoding DmNav1 was subcloned into vector pGH19-13-5 and the tipE cDNA was subcloned into pGH19 vector. These vectors were linearized with Not I and transcribed with the T7 mMESSAGE-mMACHINE transcription kit (Ambion). The harvesting of stage V–VI oocytes from anaesthetized female Xenopus laevis frog was described previously (Liu et al., 2005). Oocytes were injected with 50 nl of cRNA at a concentration of 1 ng/nl using a micro-injector (Drummond Scientific, USA). The oocytes were incubated in a solution containing (in mM): NaCl, 96; KCl, 2; CaCl2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4), supplemented with 50 mg/l gentamycin sulfate and 180 mg/l theophylline.

BmKITc and BmKITc2 are two functionally unknown scorpion toxin-like peptides with only one amino acid substitution at position 52 (Thr for Lys). They both were predicted from the nucleotide sequences (GenBank accession numbers: AAD41648 and EF469252). At the protein level, BmKITc and BmKITc2 were assigned into the depressant toxin group due to sharing 56–79% sequence similarity to some characterized depressant toxins (Fig. 1A). Despite the high sequence conservation, BmKITc and BmKITc2 show some remarkable features, such as: (1) Unlike many depressant toxins whose C-termini are amidated due to the presence of the residues GKK (Fig. 1A), BmKITc and BmKITc2 are not amidated because of a mutation from GKK to SKK at their C-termini; (2) Sites

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characterized as the ‘pharmacophore’ and crucial to ‘‘voltage-sensor trapping’’ of scorpion b-toxins (Karbat et al., 2007) are completely different between LqhIT2 and BmKITc (Fig. 1B). In LqhIT2, the ‘pharmacophore’ includes Y28, E24 and I16 while in BmKITc and BmKITc2, the corresponding residues are S28, V24 and W16. The residue A13 involved in the ‘‘voltage-sensor trapping’’ of LqhIT2 is replaced by S13 in BmKITc and BmKITc2. In fact, a Ser at this position is conserved in the most depressant toxins (Fig. 1A). Whether these two new toxins still retain a similar function to LqhIT2 is a major purpose of this study. 3.2. Production of recombinant toxins Scorpion depressant toxins possess a highly dense structure core stabilized by four disulfides, not facilitating correct folding of peptides in an E. coli environment. In previous studies, several depressant toxins (e.g. LqhIT2 and its mutants, Lqh-dprIT(3) and its natural variants) have been successfully expressed in E. coli by refolding denaturated proteins from inclusion bodies (Turkov et al., 1997; Gurevitz et al., 2007), however, all these recombinant products carry an extra Met residue in their N-termini. On the contrary, the Mesobuthus martensii toxin BmKIM was expressed in E. coli in a soluble form (Peng et al., 2002), in which fusing an Nterminal GST resulted in the recovery of intact functional peptides. We therefore tried this system firstly to express BmKITc2. As expected, the fusion protein of GST-DDDDKBmKITc2 (about 33 KDa) was expressed in a soluble form (Fig. S1) and thus could directly be purified by GST affinity chromatography. The purified fusion protein released a product of 6.9 KDa after EK digestion, as identified by SDSPAGE. One major HPLC peak eluted at 18.5 min was collected for MALDI-TOF analysis (Fig. 2). The experimental MW detected is 6883.13 Da, 8.5 Da less than the calculated MW (6891.63 Da) from its reduced primary sequence, suggesting that eight hydrogen atoms in the cysteines were removed to form four disulfide bridges. The final yield of the recombinant peptide is approximately 0.1 mg/L E. coli culture. For BmKITc, we initially used the same method described above to prepare its recombinant peptide but failed due to its instability when the fusion protein was digested by EK. Instead, the pMAL-p2X vector containing MBP as the carrier protein was chosen. In this expression system, the fusion protein (MBP-DDDDK-BmKITc (53 KDa)) was also expressed in a soluble form and thus was directly purified by using amylose affinity chromatography. The fusion protein was cleaved by EK and the resultant product was separated by RP-HPLC. Recombinant BmKITc was eluted at 22.5 min and its experimental MW detected is

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6910.83 Da, 7.87 Da less than the calculated MW (6918.70 Da) from its reduced primary sequence, suggesting that four disulfide bridges have been formed. The final yield of BmKITc is approximately 0.1 mg/L E. coli culture. 3.3. Structural features of recombinant peptides Comparative modeling using the experimental structure of LqhIT2 as a template confirmed that BmKITc and BmKITc2 can adopt highly similar structures with their template, suggesting that they both maintain the conserved CSab folding. This is further verified by their CD spectra recorded from 190 nm to 250 nm, in which a typical structural feature similar to scorpion depressant toxins (e.g. BmKIM and LqhIT2) (Peng et al., 2002; Karbat et al., 2007) was observed, as characterized by a minimum at 207 nm and a maximum at 190 nm (Fig. 3A). Estimation of secondary structure contents from their CD data using the standard JASCO CD analysis revealed that these two recombinant peptides have similar values to BmKIM and LqhIT2. Overall, BmKITc2 has a 2–4% higher helical and sheet content than BmKITc (Fig. 3B). 3.4. Functional analysis of recombinant toxins First, we evaluated the activity of BmKITc and BmKITc2 on Drosophila DmNav1/tipE channels expressed in X. oocytes (Fig. 4). The results show a small increase on DmNav1/tipE in the presence of the recombinant peptides. Moreover, they both produced a small but significant change on the slow phase of inactivation of DmNav1/tipE. In fact, no complete inactivation is reached any more, such that one can postulate an overload by Naþ ions which indeed causes or explains the toxicity of depressant toxins on insects. From the effect of BmKITc2 on the steady-state inactivation curve, incomplete inactivation can also be seen as compared with control conditions. Such incomplete inactivation of insect sodium channels has previously been observed in other depressant toxins, such as LqhIT2, BotIT2, BmKITb, LqqIT2 and Lqh-dprIT3-e (Borchani et al., 1996; Benkhalifa et al., 1997; Valdez-Cruz et al., 2004; Ferrat et al., 2005; Strugatsky et al., 2005; Bosmans et al., 2005; Wang et al., 2003) and is possibly associated with the inward movement of DII/S4 and the outward movement of DIV/S4 of the channel (Gurevitz et al., 2007). Interestingly, no typical b-effect was observed, confirming our findings on steady-state activation process (see Fig. 4B). Subsequently, we tested the effects of BmKITc and BmKITc2 on rat DRG sodium channels by using the whole cell patch clamp technique. As shown in Fig. 5, the TTX-R

Fig. 1. Sequence and structural comparison of BmKITc2 and BmKITc with functionally characterized depressant toxins. (A) Multiple sequence alignment. Cysteines involved in disulfide bridge formation are shadowed in yellow and identical residues to BmKITc in grey. Secondary structures are extracted from the published structure of LqhIT2 (pdb entry 2I61), in which four disulfide bridgs are indicated by lines. Funcitonal residues in LqhIT2 are highlighed in color: the pharmacophore (red); important for ‘‘voltage-sensor trapping’’ (green); conferring toxin specificity (blue). Sequence sources: BmKITc (AAD41648); BmKITc2 (EF469252); LqqIT2 (Zlotkin et al., 1985); LqhIT2 (Zlotkin et al., 1991); BotIT2 (Borchani et al., 1996); BotIT4 and BotIT5 (Borchani et al., 1997); BaIT2 (Ceste`le et al., 1997); AaIT5 (Nakagawa et al., 1997); LqhIT5 (Moskowitz et al., 1998); OsI1 (Kozlov et al., 2000); BmKIT2 (Li and Ji, 2000); Bs-dprIT1-4 (Ali et al., 2001); BmKIT4 (Jiang et al., 2001); BmKAEP (Wang et al., 2001); BmKIM (Peng et al., 2002); BotIT6 (Mejri et al., 2003); BmKITa and BmKITb (Wang et al., 2003); Lqh-dprIT3, LqhdprIT3a and Lqh-dprIT3e (Strugatsky et al., 2005); Bsaul1 (Nikkhah et al., 2006). {, Two toxins from different origins have identical amino acid sequences. *, This toxin is also named BmKdITAP3. a, Amidation at the C-terminus due to the presence of GKK. (B) Structural mapping of functional residues of LqhIT2 and of the equivalent amino acids in the BmKITc model. Two structures are shown in identical orientation. (C) Superimposition of the side-chain atoms indicated in Fig. 1B between LqhIT2 and BmKITc, in which different side-chains in the pharmacophore are highlighted by a dotted circle and the residue involved in ‘‘voltage-sensor trapping’’ by a solid circle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Recombinant production of BmKITc2 and BmKITc. (A) RP-HPLC profile of recombinant peptides. Inset: SDS-PAGE showing the HPLC-purified peptides. (B) The determination of MWs of the recombinant peptides by MALDI-TOF.

currents were reduced by 20.0  10.0% (n ¼ 7) and 4.0  2.2% (n ¼ 5) at 5 mM BmKITc and 5 mM BmKITc2, respectively. However, only the effect caused by BmKITc is statistically significant in the paired sample t-test (P ¼ 0.011, two-tailed), while BmKITc2 did not significantly affect TTX-R currents (P ¼ 0.183, two-tailed). Moreover, at the same concentration, BmKITc was significantly more potent on the TTX-R currents than BmKITc2 (unpaired samples two-tailed t-test, P ¼ 0.021) (Fig. 5). A similar phenomenon was also observed in TTX-S currents affected by these two recombinant peptides, in which only BmKITc significantly inhibited TTX-S currents at 10 mM (Fig. 5). The dose-response curve revealed that the inhibitory effect of BmKITc on TTX-R is concentration-dependent, despite being incomplete even at a concentration of 30 mM. No effect (i.e. no shift) on the voltage dependence of channel

activation in the hyperpolarizing direction was observed for these toxins (data not shown, but confirmatory with the results observed in oocytes, see Fig. 4B). 4. Discussion In this work, we describe the prokaryotic expression, structural and functional characterization of two new scorpion depressant toxins in M. martensii. We found that recombinant BmKITc and BmKITc2 induce a maintained current at the last phase of inactivation of insect Naþ channels and they both displayed differential potency against rat DRG neuron Naþ currents. Although an inhibitory effect on total Naþ currents of rat DRG neurons has been recorded in BmKIM (Peng et al., 2002) and BmKIT2 (Li et al., 2000), this is to the best of our knowledge the first

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Fig. 3. Structural features of recombinant peptides. (A) CD spectra. (B) Comparison of secondary structure contents of different depressant toxins. BmKITc, BmKITc2 and BmKIM: calculated from their CD data; LqhIT2: extracted from its structural coordinates (pdb entry 1612I61).

report that BmKITc blocks TTX-R currents. Considering TTX-R Naþ channels are the next target for analgesic drugs, this finding is particularly attractive. Moreover, our observation also provides a possible molecular basis to explain analgesic effect of several M. martensii venom-derived depressant toxins on mammals, in which DRG TTX-R

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sodium channels could be their targets (Wang et al., 2000; Guan et al., 2001). Similar activity on insect DmNav1/tipE but different potency towards rat DRG sodium channels between BmKITc and BmKITc2 highlights a crucial mammalian-selective role of the residue K52 in BmKITc. By contrast, no significant activity alteration on DmNav1/tipE observed between BmKITc and BmKITc2 suggests site 52 could not be involved in the interaction with the insect channel, consistent with a previous computer model of the LqhIT2- DmNav1 channel complex, in which the site is far away from the interface (Tian et al., 2008). The model structures show that K52 renders BmKITc a positive charged surface as compared to BmKITc2 (Fig. 5H), which might have modified the activity of this toxin by charge interaction with mammalian DRG Naþ channels. In fact, a single charged residue substitution resulted in a dramatic functional change has been also observed in the scorpion toxins ikitoxin and birtoxin (Inceoglu et al., 2002), in which a substitution at position 23 from Glu of birtoxin to Gly of ikitoxin renders the latter much less effective. These two examples strengthened the importance of charged residues located on molecular surface of toxins in interaction with Naþ channels. It is worthy of mentioning that some depressant toxins (e.g. BmKIM, BmKdITAP3, BmKAEP and LqhIT2) (Fig. 1A) are active on mammals but their site 52 is occupied by a Thr as in BmKITc2. An explanation for this is that site 51 of these toxins provides a positively-charged residue (Lys) to interact with the mammalian sodium channels. In BmKITc2 this site is occupied by an acidic residue (E51).

Fig. 4. Effects of BmKITc2 and BmKITc on Drosophila para/tipE. (A) Representative traces of Drosophila para/tipE when the peptides added. (B) The normalized activation curves. 3 mM BmKITc2: V1/2 ¼ 30.61  0.07 mV; control: V1/2 ¼ 28.57  0.08 mV. 3 mM BmKITc: V1/2 ¼ 29.87  0.14 mV. Control: V1/2 ¼ 28.08  0.16 mV. (C) The normalized inactivation curve: 3 mM BmKITc2: V1/2 ¼ 72.03  0.16 mV; control: V1/2 ¼ 73.26  0.20 mV. 3 mM BmKITc: V1/2 ¼ 79.81  0.44 mV. Control: V1/2 ¼ 76.4  0.24 mV.

Fig. 5. Differential potency and structural basis of BmKITc2 and BmKITc on peak sodium currents in rat DRG neurons. (A, C) Representative traces of TTX-S currents when the peptides added. (B, D) Representative traces of TTX-R currents when the peptides added. (E) Statistics of the effects of peptides on TTX-S peak sodium currents: 5 mM BmKITc2: I ¼ 0.9296  0.003; 10 mM BmKITc: I ¼ 0.8888  0.0438. Control: I ¼ 1. (F) Statistics of the effects of peptides on TTX-R peak sodium currents: 5 mM BmKITc2: I ¼ 0.9604  0.0224; 5 mM BmKITc: I ¼ 0.7819  0.0338. Control: I ¼ 1. (G) Dose–response curve of BmKITc against TTXR currents. (H) Electrostatic potential surfaces of BmKITc and BmKITc2 calculated by Spd-Viewer. Differential surface charges between the two peptides are highlighted in a red dotted circle. Colors ranged from red (negative potential) to white (zero potential) to blue (positive potential).

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Although several mutations occurring at positions previously characterized as the ‘‘hot spot’’ of b-toxins, it appears these substitutions do not completely diminish the activity of BmKITc and BmKITc2 on DmNav1/tipE. Thus, more work is needed to clarify the functional surface of these two new toxins associated with their dual bioactivity. Rational design of novel molecules based on the BmKITc sequence with improved TTX-R activity is under way. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors are grateful to M.S. Williamson for sharing the DmNaV1 and tipE clones. This work was supported by the following grants: (1) The National Natural Science Foundation of China (30730015 and 30621003); (2) The 973 Program from the Ministry of Science and Technology of China (2010CB945304); (3) Bilateral Cooperation for the 16th Session of the Sino-Belgian S&T Mixed Commission to S. Z; and (4) G.0330.06 and G.0257.08 (F.W.O. Vlaanderen), OT05-64 (K.U.Leuven), P6/31 (Interuniversity attraction Poles Program- Belgian State- Belgian Science Policy) and BIL 07/ 10 (China) to J.T. Appendix. Supplementary material Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.toxicon.2010. 02.019. References Benkhalifa, R., Stankiewicz, M., Lapied, B., Turkov, M., Zilberberg, N., Gurevitz, M., Pelhate, M., 1997. Refined electrophysiological analysis suggests that a depressant toxin is a sodium channel opener rather than a blocker. Life Sci. 61, 819–830. Borchani, L., Mansuelle, P., Stankiewicz, M., Grolleau, F., Ceste`le, S., Karoui, H., Lapied, B., Rochat, H., Pelhate, M., el Ayeb, M., 1996. A new scorpion venom toxin paralytic to insects that affects Naþ channel activation. Purification, structure, antigenicity and mode of action. Eur. J. Biochem. 241, 525–532. Bosmans, F., Martin-Eauclaire, M.F., Swartz, K.J., 2008. Deconstructing voltage sensor function and pharmacology in sodium channels. Nature 456, 202–208. Bosmans, F., Martin-Eauclaire, M.F., Tytgat, J., 2005. The depressant scorpion neurotoxin LqqIT2 selectively modulates the insect voltagegated sodium channel. Toxicon 45, 501–507. Catterall, W.A., Ceste`le, S., Yarov-Yarooy, V., Yu, F.H., Konoki, K., Scheuer, T., 2007. Voltage-gated ion channels and gating modifier toxins. Toxicon 49, 124–141. Cohen, L., Troub, Y., Turkov, M., Gilles, N., Ilan, N., Benveniste, M., Gordon, D., Gurevitz, M., 2007. Mammalian skeletal muscle

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