Expression and characterization of jingzhaotoxin-34, a novel neurotoxin from the venom of the tarantula Chilobrachys jingzhao

Peptides 30 (2009) 1042–1048

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Expression and characterization of jingzhaotoxin-34, a novel neurotoxin from the venom of the tarantula Chilobrachys jingzhao Jinjun Chen a,b,1, Yongqun Zhang a,1, Mingqiang Rong a,1, Liqun Zhao a, Liping Jiang a, Dongyi Zhang a, Meichi Wang a, Yucheng Xiao a, Songping Liang a,* a b

The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, PR China College of Biology Science and Technology, Hunan Agricultural University, Changsha 410128, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 17 January 2009 Received in revised form 23 February 2009 Accepted 26 February 2009 Available online 13 March 2009

Jingzhaotoxin-34 (JZTX-34) is a 35-residue polypeptide from the venom of Chinese tarantula Chilobrachys jingzhao. Our previous work reported its full-length cDNA sequence encoding a precursor with 87 residues. In this study we report the protein expression and biological function characterization. The toxin was efficiently expressed by the secretary pathway in yeast. Under whole-cell patch-clamp mode, the expressed JZTX-34 was able to inhibit tetrodotoxin-sensitive (TTX-S) sodium currents (IC50  85 nM) while having no significant effects on tetrodotoxin-resistant (TTX-R) sodium currents on rat dorsal root ganglion neurons. The inhibition of TTX-S sodium channels was completely reversed by strong depolarization (+120 mV). Toxin treatment altered neither channel activation and inactivation kinetics nor recovery rate from inactivation. However, it is interesting to note that in contrast to huwentoxin-IV, a recently identified receptor site-4 toxin from Ornithoctonus huwena venom, 100 nM JZTX-34 caused a negative shift of steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV. The results indicated that JZTX-34 might inhibit mammalian sensory neuronal sodium channels through a mechanism similar to HWTX-IV by trapping the IIS4 voltage sensor in the resting conformation, but their binding sites should not overlay completely. ß 2009 Elsevier Inc. All rights reserved.

Keywords: Toxin Expression Tetrodotoxin-sensitive sodium channel Whole-cell recording

1. Introduction The opening of voltage-gated sodium channels (VGSCs) is responsible for the increase in sodium permeability that initiates action potentials in electrically excitable cells. They are composed of an a subunit (260 kDa) associated with up to four different auxiliary b subunits (21–23 kDa) [3,17]. From mammals, over 10 mammalian subtypes (Nav1.1–1.9 and Navx) have been identified and characterized by electrophysiological recording, biochemical purification and cloning [15]. Most of them can express in dorsal root ganglion (DRG) neurons, except for Nav1.4 in skeletal muscles and Nav1.5 in cardiac myocytes [16]. In terms of tetrodotoxin (TTX), they can be classified into TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) types. The mechanism and physiological role of some ion channels are now better understood thanks to the discovery of animal toxins such as those from scorpions, spiders, snakes, and marine animals, which bind with high affinity and specificity to different kinds of ion channels. At

* Corresponding author. Tel.: +86 731 8861304; fax: +86 731 8861304. E-mail address: [email protected] (S. Liang). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.02.018

least six distinct receptor sites for neurotoxins on the a subunits have been identified [4]. Hitherto, many toxins acting on VGSCs were isolated from spiders and characterized, sometimes with great subtype and acting site selectivity [6,7,21,26]. These VGSCs modulating toxins from spider have been classified into two groups according to the properties: pore-blocking toxins and gating modifier toxins. HNTX-I, a pore-blocking toxin from Selenocosmia hainana, occludes the channel pore to block inward sodium currents by binding to site 1 in a manner similar to tetrodotoxin (TTX) and saxitoxin [11]. Gating modifier toxins usually modulate the movement of the voltage sensor to affect the activities of activation gate and inactivation ball by binding to neuronal sites 2–6. For example, datracotoxins from Australian funnel-web spiders compete with ascorpion toxin and slow the inactivation of sodium channel by binding to neuronal site 3 [14]. However, JZTX-III from Chilobrachys jingzhao could affect activation potential of subtype of the VGSC expressed on rat cardiac myocytes. The competitive assay by BMK1 showed that JZTX-III belonged to site-4 toxins [29]. Recently, a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4 was demonstrated. HWTX-IV from Ornithoctonus huwena was identified to inhibit activation of sodium channel by trapping the voltage

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sensor of domain II in the inward, closed configuration, which was different from scorpion b-toxins that are likely to trap the IIS4 voltage sensor in an outward configuration [26]. In the recent work, we have isolated 60 peptide toxins, compact molecules cross-linked by 3–5 disulfide bonds, from the venom of the spider C. jingzhao. The native toxin with a molecular mass of 4150.7 Da was named jingzhaotoxin-34 (JZTX-34). Because the toxin is too limited in the venom, only 12 residues were identified at N-terminal by Edman degradation sequencing [12]. Based on the partially identified amino acid sequence, we further got the fulllength cDNA encoding the toxin of interest. Here, we report its identification, expression and functional characterization. Our findings showed that JZTX-34, a 35-residue peptide, could be efficiently expressed by the secretary pathway in yeast and inhibit channel activation of TTX-S sodium channels on DRG cells. On the other hand, this toxin shares high sequence similarity with several well-characterized spider toxins that act on diverse voltage-gated ion channels. 2. Materials and methods 2.1. Strains, materials, and animals Plasmid pVT102U/a, Escherichia coli strain DH5a, and Saccharomyces cerevisiae strain S-78 (Leu2, Ura3, Rep4) were used. This plasmid pVT102U/a contains an E. coli origin of replication, a yeast 2 m origin of replication, an E. coli ampicillin resistance gene, the yeast URA3 gene encoding an orotidine-5-phosphate decarboxylase and the signal sequence of yeast prepro-a-factor [23,31]. Restriction endonucleases and T4 DNA ligase were obtained from Roche Applied Science (Mannheim Germany). Primers were synthesized by Sangon (Shanghai, China). Taq DNA polymerase was obtained from MBI. CM32-cellulose cation-exchange and Sephasil1 peptide C18 reversed-phase (12-mm ST4.6/250) columns were from Whatman and Amersham Biosciences AB (Uppsala, Sweden), respectively. All other chemicals were at least analytical grade and were purchased from Merck or Sigma. The Chinese tarantula C. jingzhao were collected in Hainan province of China. Sprague–Dawley rats were purchased from Xiangya School of Medicine, Central South University. The oocytes were collected from mature female X. laevis.

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tion enzyme sites were used for cloning purposes. The approximately 110 base pair PCR products cloned into the yeast expression vector pVT102U/a and transformed into E. coli DH5a competent cells. The recombinant plasmid pVT102U/aJZTX-34 was extracted, sequenced, and transformed into S. cerevisiae S-78 (Leu2, Ura3, Rep4) using the LiCl method [10]. After fermentation, the supernatant of the culture was adjusted to pH 4.2 with acetic acid. The sample was directly applied to a CM32-cellulose cation-exchange column, which was equilibrated with 0.1 M sodium acetate at a flow rate of 4 mL/min. Upon reaching a steady base line, the column was washed by stepwise elution with 0.1, 0.2, 0.3, and 0.5N NaCl equilibration buffer. The fractions were directly applied to a reverse-phase C18 column. Buffer A contained 0.1% trifluoroacetic acid in water; buffer B contained 0.1% trifluoroacetic acid in acetonitrile. Reversed-phase chromatography was carried out using an A¨KTA purifier chromatography system (Amersham Biosciences AB). Molecular weight information of peptides was obtained by using a MALDI-TOF–TOF mass spectrometer (UltraFlex I, Bruker Daltonics) equipped with nitrogen laser (337 nm) and operated in reflector/delay extraction mode for MALDI-TOF-peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode using the flexControl TM software. 2.4. Whole-cell patch-clamp experiments

The cDNA sequence was retrieved from a venom gland cDNA library of the Chinese tarantula C. jingzhao [5]. Venom glands of the Chinese tarantula C. jingzhao collected in Hainan province of China were harvested and put into liquid nitrogen rapidly. Total RNA was prepared by tissue homogenization in liquid nitrogen using a mortar and pestle, followed by cell lysis in the presence of TRIzol (Invitrogen). For cDNA library construction, poly A (+) RNA was purified from the total RNA on an oligo(dT)-cellulose affinity column using the mRNA Purification Kit (Promega) according to the manufacturer’s protocol. The full-length cDNA library was made by employing primer extension in accordance with the instructions provided with the CreatorTM SMARTTM cDNA Library Construction Kit (Clontech). The full protein sequence of the toxin was determined by combining Edman degradation sequencing [12] with its full cDNA sequence.

Acutely dissociated DRG cells were prepared from 4-week-old Sprague–Dawley rats and maintained in short-term primary culture using the method described by Xiao et al. [29] Briefly, the dissociated cells were suspended in essential Dulbecco’s modified Eagle’s medium containing trypsin (0.5 g/L, type III), collagenase (1.0 g/L, type IA), and DNase (0.1 g/L, type III) to incubate at 34 8C for 30 min. Trypsin inhibitor (1.5 g/L, type II-S) was used to terminate enzyme treatment. The DRG cells were transferred into 35-mm culture dishes (Corning, Sigma) containing 95% Dulbecco’s modified Eagle’s medium, 5% newborn calf serum, hypoxanthine aminopterin thymidine supplement, and penicillin– streptomycin and then incubated in the CO2 incubator (5% CO2, 95% air, 37 8C) for 1–4 h before the patch-clamp experiment. Cell currents were recorded on experimental DRG cells using whole-cell patch-clamp technique at room temperature (20– 25 8C). The patch pipettes with DC resistances of 2–3 MV were fabricated from borosilicate glass tubing (VWR micropipettes, 100 ml, VWR Company) using a two-stage vertical microelectrode puller (PC-10, Narishige, Japan) and fire-polished by a heater (Narishige, Japan). Ionic currents were filtered at 10 kHz and sampled at 3 kHz on EPC-9 patch-clamp amplifier (HEKA Electronics, German). Under the voltage clamp 70–80% series resistance compensation was applied. For sodium current recordings, the bath solution contained (in mM): 150 NaCl, 2 KCl, 5 D-glucose, 1 MgCl2, 1.5 CaCl2, and 10 HEPES at pH 7.4; the pipette internal solution contained (in mM): 105 CsF, 35 NaCl, 10 HEPES, and 10 EGTA at pH 7.4. Sodium currents were elicited at 10 mV from a holding potential of 80 mV. All experiments were completed at room temperature 22–25 8C. Experiments data were acquired and analyzed by using the program Pulsefit + Pulse 8.0 (HEKA Electronics, Germany) and Sigmaplot (Sigma). All data are presented as means  S.E., and n is the number of independent experiments.

2.3. Construction, expression, purification and identity of JZTX-34

2.5. Two-microelectrode voltage clamp experiments

The S. cerevisiae shuttle vector, pVT102U/a, was used for cloning, sequencing and transformation. The cDNA sequence coding mature peptide region of JZTX-34 amplified by PCR using synthetic oligonucleotides incorporating XbaI and HindIII restric-

Capped cRNAs encoding ion channels were synthesized after linearizing the plasmids and performing the transcription by a standard protocol (11). For in vitro transcription, the plasmids pCI containing the gene for Kv2.1 were first linearized with NotI. Using

2.2. Identification of JZTX-34

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Fig. 1. Amino acid and cDNA sequence of JZTX-34. (A) The cDNA sequence of JZTX-34. The amino acid composition of the precursor reading from the cDNA is suggested below the nucleotide sequence. The potential endoprotelytic sites are pointed out with down arrows. The sequence of mature peptide was underlined by solid line, while the polyadenylations signal, AATAAA, was double underlined. (B) Alignment of JZTX-34 with related ICK peptides from Ceratogyrus cornuatus (CcoTx3), Grammostola rosea (Omega-GsTx SIA), P. cambridgei (VaTx1, VaTx2 and VaTx3), Thrixopelma pruriens (ProTx-I), Ornithoctonus huwena (HWTX-V), H. maculata (HmTx1), Scodra griseipes (SGTx1), Grammostola spatulata (HaTx1) and Chilobrachys jingzhao (JZTX-XI, JZTX-IV) spiders. Cysteine residues characteristic of ICK peptides are highlighted in yellow and other highly conserved residues are highlighted in gray. *The stars indicate the toxins produce no or little inhibition on potassium channels Kv2.1, and the acidic residues between the forth and fifth cysteins are underlined. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

the linearized plasmids as templates, cRNAs were synthesized in vitro using the large-scale T7 mMESSAGE mMACHINE transcription kit (Ambion). Stage V–VI X. laevis oocytes were collected from mature female X. laevis under anesthesia by putting on ice. Then the oocytes were defolliculated by treatment with 1 mg/mL collagenase in calciumfree ND96 solution (pH 7.5) containing (in mM) 96 NaCl, 2 KCl, 1 MgCl2, and 10 HEPES. Between 2 and 24 h after defolliculation, oocytes were injected with 41 nL of 100–500 ng/mL cRNA using a microprocessor controlled nanoliter injector (WPI). The oocytes were then incubated in OR2 solution (pH 7.5) at 18 8C for 1–4 days. OR2 solution contains (in mM) 82.5 NaCl, 2.5 KCl, 1 CaCl2, 1 Na2HPO4, 1 MgCl2, and 5 HEPES, supplemented with 50 mg/L gentamycin sulfate (only for incubation). Whole-cell currents from oocytes were recorded using the twomicroelectrode voltage clamp (TURBO TEC-03X; NPI Electronic). Voltage and current electrodes (0.1–0.8 MV) were filled with 3 M KCl. Oocytes were studied in a 100 mL recording chamber that was perfused with an extracellular solution containing RbCl (50 mM), NaCl (50 mM), MgCl2 (1 mM), CaCl2 (0.3 mM), and HEPES (5 mM),

pH 7.5, with NaOH. Current records were sampled at 0.5 ms intervals after low-pass filtering at 2 kHz. Linear components of capacity and leak currents were not subtracted. All experiments were performed at room temperature (19–23 8C). 3. Results 3.1. Sequence analysis of JZTX-34 The amino acid sequence of JZTX-34 was identified by a combination method of N-terminal amino acid and cDNA sequencing. As shown in Fig. 1, the full length of the cDNA encoding JZTX-34 was 461 bp and the oligonucleotide sequence of the cDNA was found to comprise a 50 -untranslated region, an open reading frame, and a 30 -untranslated region. The open reading frame encoded an 87-residue peptide corresponding to the JZTX34 precursor that consisted of a signal peptide of 21 residues, a mature peptide of 35 residues, an intervening propeptide of 29 residues and 2 extra basic residues in C-terminus. The amino acid sequence of mature peptide was completely consistent with that of

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Fig. 2. Purification and identification of recombinantly produced JZTX-34. (A) Tricine/SDS-PAGE before and after purification. The protein bands were stained with Coomassie Brilliant Blue R-250. Lane 1, molecular mass markers; lane 2, fermentation supernatant of JZTX-34, lane 3, purified JZTX-34. (B) Purification of JZTX-34 from the fermentation supernatant by reversed-phase chromatography. (C) The mass spectrometry of recombinantly produced JZTX-34. The calculated molecular weight of JZTX-34 was 4156.78 Da and the measured molecular weight was 4151.094 Da.

JZTX-34 in N-terminus determined by Edman degradation [5]. An extra dipeptide (Arg-Lys) emerges at its C-terminus of the mature peptide before the stop codon. Usually, the dipeptide is a signal for amidation at C-terminal residue during the post-translational processing. After it is removed, the C-terminal residue (Val35) is amidated and six cysteine residues are involved in forming three intramolecular disulfides, the theoretical mass of mature peptide is the same as that of JZTX-34 measured by MALDI-TOF mass spectrometry. Finally, a polyadenylation signal (AATAAA) was found in the 30 -untranslated regions at position 24 upstream from the polyadenylated sequence (Fig. 1A). An online BLAST search showed that the mature peptide of JZTX-34 had considerable sequence similarity with other spider toxins (Fig. 1B). 3.2. Expression and purification Due to the limited content of naturally occurring JZTX-34 in C. jingzhao venom, we tried to express the toxin in yeast to characterize its biological functions. The sequence corresponding to mature peptide of JZTX-34 was produced by PCR. The target gene was expressed using the pVT102U/a vector [23]. Tricine/SDS-

PAGE analyses of yeast culture demonstrated that the target protein was expressed and secreted into the medium. Expressed JZTX-34 was then purified by a simple and efficient protocol. One liter of culture was harvested and initially purified by chromatography on a CM32 cation-exchange column. The next step of purification was carried out on an analytical C18 column. The elution peaks corresponding to target peptide were pooled and lyophilized. The molecular mass difference of 6 Da between measured molecular weight of JZTX-34 (4151.094 Da) and the calculated mass (4156.78 Da) was observed (Fig. 2C), indicating that three pairs of disulfide bridges were formed by six cysteines. Four milligram of target protein could be purified from 1 L of culture medium. The Tricine/SDS-polyacrylamide gel and the mass spectrometry showed a high purity of the final products (Fig. 2). Therefore, in this study, we succeeded in efficiently expressing JZTX-34 through a secretary pathway in yeast. 3.3. Effects of JZTX-34 on voltage-gated sodium channels The investigation of the biological functions of JZTX-34 was performed on adult rat DRG neurons using whole-cell patch-clamp

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Fig. 3. Effects of JZTX-34 on VGSCs. All current traces were evoked by a 50-ms step depolarization to 10 mV from a holding potential of 80 mV at every second. The currents of TTX-S were reduced significantly by 100 nM JZTX-34 (A) and completely by 10 mM (B) and 10 mM JZTX-34 (C) has no effect to TTX-R currents. (D) The concentration dependent inhibition of TTX-S sodium currents on DRG neurons. Every data point (mean  S.E.) coming from 4 to 8 cells shows current relative to control. (E) Time course for inhibition TTX-S by 1 mM JZTX-34.

technique. DRG neurons from adult animals can express two kinds of sodium channels: TTX-S and TTX-R. Here, we chose the cells with diameter of >40 or <20 mm to measure the actions of JZTX-34 on TTX-S or TTX-R sodium channels, respectively. The experimental cells were held at 80 mV for over 4 min to allow adequate equilibration between the micropipette solution and the cell interior, and then the current traces were evoked using a 50 ms step depolarization to 10 mV every second. It was found that 100 nM JZTX-34 could significantly reduce TTX-S current amplitude by 55.4 + 3.1% (n = 4) (Fig. 3A). When toxin concentration in extracellular solution increased 10-fold, almost 83% of current amplitude was depressed with a time constant (t) of 57 s (Fig. 3E). At the concentration of 10 mM, JZTX-34 was able to completely inhibit TTX-S sodium current (Fig. 3B). Because both TTX-S and TTX-R sodium channels are expressed in small DRG neurons [29], we added 200 nM TTX into extracellular solution to separate TTX-R currents from mixture currents. However, the following application of 10 mM toxin exhibited no effect on TTX-R currents (Fig. 3C). Fig. 3D shows the dose–response curve of JZTX-34 inhibiting TTX-S sodium channels. After fitted by Hill equation, the IC50 value was yielded to 85 nM (Fig. 3D). The current–voltage (I–V) curve of TTX-S sodium channels is shown in Fig. 4A, in which the initial activated voltage and reversal potential were around 50 and +30 mV, respectively (n = 4). JZTX34 at 100 nM did not change the threshold of activation and the activation voltage of inward peak currents. No shift of the membrane reversal potential was induced either, in accordance with the observation on the conduct–voltage curve of TTX-S that JZTX-34 did not change channel conductance at voltages varying from 80 to 10 mV (Fig. 4B). However, 100 nM JZTX-34 significantly shifted the steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV (Fig. 4C). Using a standard two-pulse protocol as described in Fig. 4D, in which the

second pulse was used to measure the fraction of sodium channels available, we also detected the effect of JZTX-34 on recovery rate of TTX-S neuronal sodium channels from inactivation. DRG neurons were held at 80 mV. Two pulses at 10 mV for 50 ms were separated by an interpulse potential (80 mV) for intervals ranging from 0.5 to 512 ms. As shown in Fig. 4D, 1 mM JZTX-34 did not alter the recovery rate from inactivation, suggesting that JZTX-34 might have no effect on recovery kinetics. On the other hand, recovery of TTX-S sodium channel from JZTX-34 inhibition could be induced by extreme depolarization (+120 mV) (Fig. 5). 3.4. Effects of JZTX-34 on the Kv2.1 channel Since JZTX-34 shares high sequence similarity with several well-characterized spider toxins (v-GsTx SIA, VaTx1, ProTx-I, HmTx1, SGTx1, HaTx1 and JZTX-XI) that show activity toward Kv2.1 channel (Fig. 1B), we asked whether the toxin has the crosschannel activities. Potassium currents were elicited from frog oocytes expressing Kv2.1 channels by a 100-ms depolarizing potential of 0 mV from the holding potential of 80 mV. After application of 10 mM JZTX-34, no significant alteration of Kv2.1 current trace was detected within 1 min (figure not shown). 4. Discussion In this work, we have expressed and characterized a novel neurotoxin JZTX-34 from the Chinese spider C. jingzhao. The full sequence of JZTX-34 was determined by Edman degradation sequencing, MALDI-TOF mass spectrometry and cDNA sequencing. The precursor endoproteolytic sites during the post-translational processes were also detected. Post-translational processings of animal polypeptide toxin appear to be important both for biological activity and for protection from degradation. The toxins

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Fig. 4. Effects of JZTX-34 on the kinetics of TTX-S sodium channels. The I–V curve (A) and conduct (B) of sodium currents shows the relationship between current traces before and after adding 100 nM JZTX-34. (C) Steady-state inactivation of TTX-S: 100 nM JZTX-34 shifts Vh from 59.3  0.1 mV in control to 70.5  0.2 mV (n = 4). The data points obtained from four separated experimental cells are shown as mean  S.E. (D) Effect on the recovery kinetics of TTX-S sodium channel on rat ganglion neurons. A dual-pulse voltage clamp protocol was used two 50-ms pulse from 80 to 10 mV. Data points showing the ratio of Itest/Iprepulse versus the recovery time has been fitted using a dual-exponential equation. 1 mM JZTX-34 (*) compared to control (*) did not change the recovery kinetic of TTX-S sodium channels.

are initially translated as larger precursors that share a similar organization, consisting of an N-terminal signal sequence with a hydrophobic core and a consensus cleavage point for a signal peptidase, a propeptide rich in glutamate residues, and the Cterminal cysteine-rich mature toxin region [8]. Furthermore, there are many precursors like JZTX-34 including extra residues in Cterminus. The rules were also observed in conotoxin [2]. Our previous work has purified and characterized two polypeptides named jingzhaotoxin-IV (JZTX-IV) and jingzhaotoxin-XI

Fig. 5. Reversal of JZTX-34 inhibition by strong depolarization. Cell were held at 80 mV and then with a 50 ms test pulse of 10 mV. A +120 mV strong depolarization implied after cell back held at 80 mV. Finally, a 10 mV pulse to test the currents. After the +120 mV strong depolarization, JZTX-34 could completed reversed.

(JZTX-XI) from the same Chinese tarantula venom (Fig. 1B). JZTX-IV weakly reduces the peak amplitudes and obviously slows the inactivation of TTX-S VGSCs by shifting the voltage dependence of activation curve to more depolarized potentials on DRG neurons. However, JZTX-XI showed no significant effects on the normal activation of both TTX-S and TTX-R VGSCs in DRG neurons. On the other hand, JZTX-XI and JZTX-IV both reduced the peak current amplitude and slowed channel inactivation of Nav1.5 by shifting the steady-state inactivation to hyperpolarized potentials. These results indicated that JZTX-XI and JZTX-IV neither binds to the previously characterized classic site 4, nor site 3 by modifying channel gating [13,25]. Different from the two homologous toxins, JZTX-34 selectively inhibited TTX-S sodium currents from rat DRG neurons and failed to alter the threshold of activation and the activation voltage of inward peak currents. However, JZTX-34 shifted the steady-state inactivation curve of TTX-S sodium channels. These results indicated that JZTX-34 inhibited mammalian neuronal sodium channels through a mechanism similar to HNTX-IV and HWTX-IV [19,27,30], but distinct from other spider toxins such as JZTX-I [28], d-ACTXs [9] and d-atracotoxins [14] which bind to neuronal receptor site 3 to slow the inactivation kinetics of sodium currents [1,9]. In contrast to scorpion b-toxins that are likely to trap the IIS4 voltage sensor in an outward configuration, HWTX-IV interacted with TTX-S sodium channels by docking at DII S3–S4 linker (D816 and E818) to trap the IIS4 voltage sensor in the resting conformation. Recovery of sodium channel from HWTX-IV inhibition could be induced by extreme depolarization (>100 mV) [26]. As seen in Fig. 5, the complex of sodium channel and JZTX-34 could be dissociated by strong depolarization (+120 mV), too. Here we proposed that JZTX-34 might modulate the activities of neuronal sodium channels by docking at DII S3–S4

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linker to trap the IIS4 voltage sensor in the resting conformation through a mechanism similar to HWTX-IV. JZTX-XI and several homologues toxins from spider (v-GsTx SIA, VaTx1, ProTx-I, HmTx1, SGTx1, HaTx1) show activity toward Kv2.1 channel, but JZTX-34 and two homologues, VaTx2 and VaTx3, produce little inhibition on Kv2.1 channel. All of them are typical ICK motif peptides in which three disulfide bridges form a cysteine knot, constraining the molecule into a rigid and compact structure. A short (five to seven residues) hypervariable b-hairpin turn between the fifth and sixth cysteines is believed to be critical for specifying toxin–ion-channel interactions [18,22,24]. Interestingly, VaTx1 and VaTx2 are identical at five of the six residues in this hypervariable turn, whereas their activities toward the Kv2.1 channel are much different [20]. VaTx1 is demonstrated to significantly inhibit Kv2.1 channel. What is the key factor for them exhibiting divergent activities toward Kv2.1 channel? It is notable that the three toxins (JZTX-34, VaTx2 and VaTx3) contain an acidic residue (Glu) situated between the forth and fifth cysteins (Fig. 1B), whereas a basic residue (Lys) emerges in the corresponding region of Kv2.1 channel inhibitors. Here we assumed that the residue E20 might be crucial for JZTX-34 not interacting with Kv2.1 channel. Certainly, further investigation is needed to test this hypothesis. In conclusion, JZTX-34 was successfully expressed and the recombinant form displayed selective inhibition of TTX-S sodium channel currents on rat dorsal root ganglion neurons, which may have the advantage to investigate the mechanism of toxin–channel interaction. Acknowledgments We thank Martin Stocker for providing the Kv2.1 plasmid. This work was supported by the Scientific Research Fund of Hunan Provincial Education Department (07A035), the Key Project of Chinese Ministry of Education (No. 208096), the Huo Ying Dong Education Foundation (No. 111023) and the Program for New Century Excellent Talents in University (No. NCET-07-0279). References [1] Adams ME. Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta. Toxicon 2004;43:509–25. [2] Buczek O, Bulaj G, Olivera BM. Conotoxins and the posttranslational modification of secreted gene products. Cell Mol Life Sci 2005;62:3067–79. [3] Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 2000;26:13–25. [4] Cestele S, Catterall WA. Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 2000;82:883–92. [5] Chen J, Deng M, He Q, Meng E, Jiang L, Liao Z, et al. Molecular diversity and evolution of cystine knot toxins of the tarantula Chilobrachys jingzhao. Cell Mol Life Sci 2008;65:2431–44. [6] Corzo G, Sabo JK, Bosmans F, Billen B, Villegas E, Tytgat J, et al. Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels. J Biol Chem 2007;282:4643–52. [7] Deng M, Luo X, Meng E, Xiao Y, Liang S. Inhibition of insect calcium channels by huwentoxin-V, a neurotoxin from Chinese tarantula Ornithoctonus huwena venom. Eur J Pharmacol 2008;582:12–6.

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