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• W2126923168 abstract "We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3′-rapid amplification of cDNA ends and 5′-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC50 value of 0.38 μm in rat cardiac myocytes. Different from scorpion β-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 μm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion α-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype. We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3′-rapid amplification of cDNA ends and 5′-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC50 value of 0.38 μm in rat cardiac myocytes. Different from scorpion β-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 μm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion α-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype. Voltage-gated sodium channels (VGSCs) 1The abbreviations used are: VGSC, voltage-gated sodium channel; VGPC, voltage-gated potassium channel; VGCC, voltage-gated calcium channel; TTX, tetrodotoxin; TTX-R, TTX-resistant; TTX-S, TTX-sensitive; JZTX-III, jingzhaotoxin-III; HNTX-IV, hainantoxin-IV; HWTX-IV, Huwentoxin; DRG, dorsal root ganglion; RACE, rapid amplification of cDNA ends; ICK, inhibitor cystine knot; BMK-I, B. martensi Karsch I; RP-HPLC, reverse phase-high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; TCEP, Tris (2-carboxyethyl) phosphine. are trans-membrane proteins distributed widely in the most excitable tissue. Similar to the shaker potassium channel (1Sokolova O. Kolmakova-Partensky L. Grigorieff N. Structure. 2001; 9: 215-220Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), the three-dimensional structure of sodium channel is a bell-shaped molecule determined by helium-cooled cryo-electron microscopy and single particle image analysis (2Sato C. Ueno Y. Asai K. Takahashi K. Sato M. Engel A. Fujiyoshi Y. Nature. 2001; 409: 1047-1051Crossref PubMed Scopus (228) Google Scholar). In terms of tetrodotoxin (TTX), they can be classified into TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) types. More recently, from mammals including human, more than 10 mammalian (Nav1.1–Nav1.9 and Navx) subtypes have been identified, cloned, functionally expressed, and characterized (3Lopreato G.F. Lu Y. Southwell A. Athinson N.S. Hillis D.M. Wilcox T.P. Zakon H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7588-7592Crossref PubMed Scopus (78) Google Scholar). Most of them can express in dorsal root ganglion (DRG) neurons, except for Nav1.4 in skeletal muscles and Nav1.5 in cardiac myocytes (4Ogata N. Ohishi Y. Jpn. J. Pharmacol. 2002; 88: 365-377Crossref PubMed Scopus (155) Google Scholar). These subtypes have been highly conserved during evolution (5Goldin A.L. Barchi R.L. Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Berwald N. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 6Goldin A.L. J. Exp. Biol. 2002; 205: 575-584PubMed Google Scholar). With more than 75% sequence identity among one another, they exhibit relatively similar pharmacological properties in different expression systems. However, it is the divergent residues among the sequences of these VGSC isoforms that determine their response to distinct ligands. For instance, after tyrosine 371 is substituted by serine in rNav1.6 and rNav1.3 (wild types), which are TTX-S phenotypes, the mutants become resistant to TTX (7Cummins T.R. Aglieco F. Renganathan M. Herzog R.I. Dib-Hajj S.D. Waxman S.G. J. Neurosci. 2001; 21: 5952-5961Crossref PubMed Google Scholar, 8Herzog R.I. Liu C.J. Waxman S.G. Cummins T.R. J. Neurosci. 2003; 23: 8261-8270Crossref PubMed Google Scholar). As the major contributors to the initiation and propagation of action potentials, VGSCs become the main targets attacked by many spider toxins. With specific pharmacological properties and higher affinity with VGSCs, spider peptides have attracted the interests of many scientists. Until now, more than 30 sodium channel toxins have been purified and well characterized from venoms of various species. NMR and homology modeling techniques indicate that, irrespective of the different composition of amino acids, most of them adopt a typical inhibitor cystine knot (ICK) fold distinct from the α/β scaffold emerging in scorpion toxins (9Escoubas P. Diochot S. Corzo G. Biochimie (Paris). 2000; 82: 893-907Crossref PubMed Scopus (213) Google Scholar, 10Goudeta C. Chi C.W. Tytgata J. Toxicon. 2002; 40: 1239-1258Crossref PubMed Scopus (236) Google Scholar). Most residues in their primary structure are believed to support the peptide framework, whereas only a few charged residues situated at the loop domains of ICK motifs are critical to interact with sodium channels. Recently, scorpion toxin determinants demonstrate that some conserved aromatic residues (Phe, Tyr, and Trp) also play an important role in modifying the sodium channel activities (11Sun Y.M. Bosmans F. Zhu R.H. Goudet C. Xiong Y.M. Tytgat J. Wang D.C. J. Biol. Chem. 2003; 278: 24125-24131Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Spider toxins exhibit limited sequence identity in the sodium channel toxins from other origins, such as marine animals, scorpions, and snakes, revealing that there is a perspective for searching for new valuable ligands to dissect variant VGSCs. Furthermore, spider toxins have been shown to lead to new insecticides and pharmaceuticals. On the functional α subunit of VGSCs, more than six sites (sites 1–6) have been disclosed to bind certain ligands (12Cestele S. Ben Khalifa R.B. Pelhate M. Rochat H. Gordon D. J. Biol. Chem. 1995; 270: 15153-15161Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Spider toxins mainly interact with three of them, corresponding to blocking channel pore (site 1, hainantoxin-IV (HNTX-IV) and Huwentoxin-IV (HWTX-IV)), slowing channel inactivation (site 3, μ-agatoxins and δ-atracotoxins), and inhibiting channel activation (site 4, Magi 5 and ProTx I–II), respectively (13Omecinsky D.O. Holub K.E. Adams M.E. Reily M.D. Biochemistry. 1996; 35: 2836-2844Crossref PubMed Scopus (71) Google Scholar, 14Nicholson G.M. Walsh R. Little M.J. Tyler M.I. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 117-126Crossref PubMed Scopus (76) Google Scholar, 15Corzo G. Gilles N. Satake H. Villegas E. Dai L. Nakajima T. Haupt J. FEBS Lett. 2003; 547: 43-50Crossref PubMed Scopus (85) Google Scholar, 16Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 17Middleton R.E. Warren V.A. Kraus R.L. Hwang J.C. Liu C.J. Dai G. Brochu R.M. Kohler M.G. Gao Y.D. Garsky V.M. Bogusky M.J. Mehl J.T. Cohen C.J. Smith M.M. Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (200) Google Scholar, 18Xiao Y.C. Liang S.P. Eur. J. Pharmacol. 2003; 477: 1-7Crossref PubMed Scopus (62) Google Scholar). Most of them are found to have high affinity with the subtypes of VGSCs localizing on sensory neurons, but a few affect the cardiac isoform. In this study, we report the isolation, cDNA sequence clone, and functional characterization of a novel cardiotoxin from the spider Chilobrachys jingzhao, which was identified as a new species recently (19Zhu M.S. Song D.X. Li T.H. J. Baoding Teachers Coll. 2001; 14: 1-6Google Scholar). The crude venom is lethal to mice with an intrapertoneal LD50 of 4.4 mg/kg. The spider toxin, denoted jingzhaotoxin-III (JZTX-III), is composed of 36 amino acid residues including 6 cysteines cross-linked in a pattern of I-IV, II-V, and III-VI. The toxin shows no effect on voltage-gated potassium channels (Kv1.1–1.3) expressed in Xenopus laevis oocytes or VGSCs and voltage-gated calcium channels (VGCCs) distributed in DRG neurons. However, it can selectively inhibit activation of TTX-R VGSCs in cardiac myocytes followed by shifting activated voltage in a depolarizing direction. We further assume that JZTX-III binds to site 4 on sodium channel proteins, which is formed by amino acid residues in the extracellular linker between domain II-S3 and domain II-S4. Toxin Purification and Sequencing—The venom from female C. jingzhao spiders was collected as described earlier (16Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (129) Google Scholar). Lyophilized venom (1 mg in 0.2 ml in distilled water) was applied to a Vydac C18 analytical reverse-phase (RP) HPLC column (218TP54, 4.6 × 250 mm) and eluted at a flow rate of 1 ml/min by a linear gradient of 0–40% of buffer B (acetonitrile containing 0.1% v/v trifluoroacetic acid) over 50 min after an equilibrium period of 3 min with buffer A (distilled water containing 0.1% v/v trifluoroacetic acid). The fraction containing JZTX-III was purified further on the same RP-HPLC column by a slower linear gradient of 30–35% buffer B over 20 min. Once purified to >99% homogeneity assessed by RP-HPLC and mass spectrometry, the peptide sample was lyophilized and stored at -20 °C until use. The molecular mass was determined by MALDI-TOF mass spectrometry on a Voyager-DE™ STR Biospectrometry™ work station. The entire amino acid sequence was obtained from a single sequencing run on an Applied Biosystems/PerkinElmer Life Sciences Procise 491-A protein sequencer. Identification of JZTX-III cDNA—The characterization of JZTX-III cDNA was performed using 3′- and 5′-RACE methods described previously (20Diao J.B. Lin Y. Tang J.Z. Liang S.P. Toxicon. 2003; 42: 715-723Crossref PubMed Scopus (35) Google Scholar). First, according to the manufacturer's instructions, the total RNA was extracted from 0.1 g of fresh venom glands of female spiders using the TRIzol reagent kit. 5 μg of RNA was taken to convert mRNA into cDNA using the Superscript II reverse transcriptase with an universal oligo(dT)-containing adapter primer (5′-GGCCACGCGTCGACTAGTAC (dT)17-3′). The cDNA was then used as a template for PCR amplification in 3′-RACE. A degenerate primer 1 (5′-GG(A/T/C/G)CA(G/A)TT(T/C)TGGTGGAA(A/G)TG(T/C)-3′) was designed corresponding to the N-terminal residues (5GQFWWKC11) of mature JZTX-III. The cDNA of mature toxin was amplified using primer 1 and an abridged universal adapter primer containing an additional HindIII restriction site (5′-CGAAGCTTGGCCACGCGTCGACTAGTAC-3′). Second, based on the partial cDNA sequence of JZTX-III determined by 3′-RACE, the antisense primers were designed and synthesized for 5′-RACE as follows: the genespecific primer 2 (5′-GCAGGCATACCCTTTGCAGCA-3′) corresponding to the C-terminal residues (18CCKGYAC24). With the strategy described by the RACE kit supplier, the 5′-end cDNA of JZTX-III was amplified using its gene-specific primer 2. Amplified products in both 3′- and 5′-RACE were precipitated and cloned into the pGEM-T easy vector for sequencing. DNA sequencing was performed by Bioasia Inc. Nucleic acid sequences were analyzed using the software DNAclub (by Xiongfong Chen) (www.imtech.res.in/pub/nsa/dnaclub/dos/) and DNAman (by Nynnon Biosoft) (www.Lynnon.com). Assignment of the Disulfide Bonds of JZTX-III—To determine the disulfide connections of JZTX-III, partial reduction by Tris (2-carboxyethyl) phosphine (TCEP) at low pH was employed (16Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 21Li D.L. Xiao Y.C. Hu W.J. Xie J.Y. Bosmans F. Tytgat J. Liang S.P. FEBS Lett. 2003; 555: 616-622Crossref PubMed Scopus (71) Google Scholar). 0.1 mg of JZTX-III, dissolved in 10 μl of 0.1 m citrate buffer (pH 3) containing 6 m guanidine-HCl, was partially reduced by adding 10 μl of 0.1 m TCEP at 40 °C for 8 min at pH 3. The intermediates were isolated by RP-HPLC, and their masses were measured by MALDI-TOF mass spectrometry. Appropriate intermediates containing free thiols were dried and then alkylated by adding 100 μl of 0.5 m iodoacetamide (pH 8.3). The alkylated peptide was desalted by RP-HPLC and then submitted to an Applied Biosystems 491 protein sequencer. Preparation of Cardiac Myocytes—Single ventricular cardiomyocytes were enzymatically dissociated from adult rats by a previously described method (22Cao C.M. Xia Q. Chen Y.Y. Zhang X. Shen Y.L. Pfluegers Arch. Eur. J. Physiol. 2002; 443: 635-642Crossref PubMed Scopus (18) Google Scholar) with minor modifications. Briefly, Sprague-Dawley rats (about 250 g) of either sex were killed by decapitation without anesthetization, and the heart was rapidly removed and rinsed in ice-cold Tyrode's solution containing (in mm): 143.0 NaCl, 5.4 KCl, 0.3 NaH2PO4, 0.5 MgCl2, 10.0 glucose, 5.0 HEPES, 1.8 CaCl2 at pH 7.2. Then the heart was mounted on a Langendorff apparatus for retrograde perfusion via the aorta with non-recirculating Ca2+-free Tyrode's solution bubbled at 37 °C by 95% O2 and 5% CO2. After 10 min, perfusate was switched to a Ca2+-free Tyrode's solution supplemented with 0.3% collagenase IA and 0.7% bovine serum albumin, and the hearts were perfused in a recirculated mode for 5 min. After the enzymatic solution was replaced by KB buffer containing (in mm): 70.0 l-glutanic, 25.0 KCl, 20.0 taurine, 10.0 KH2PO4, 3 MgCl2, 0.5 EGTA, 10.0 glucose, 10.0 HEPES at pH 7.4, the partially digested hearts were cut, minced, and gently triturated with a pipette in the KB buffer at 37 °C for 10 min. The single cells were obtained after undigested tissues filtered through 200-μm nylon mesh. All cells were used within 8 h of isolation. Electrophysiological Studies—Whole cell cardiac sodium currents were recorded from rod-shaped cells with clear cross-striations at room temperature (20–25 °C). Recording pipettes (2–3-μm diameter) were made from borosilicate glass capillary tubing, and their resistances were 1–2 megaohms when filled with internal solution containing (in mm): 135.0 CsF, 10.0 NaCl, 5.0 HEPES at pH 7.0. External bath composition was (in mm): 30 NaCl, 5 CsCl, 25 d-glucose, 1 MgCl2, 1.8 CaCl2, 5 HEPES, 20 triethanolamine-chlorine, 70 tetramethylammonium chloride at pH 7.4. Ionic currents were filtered at 10 kHz and sampled at 3 kHz on EPC-9 patch clamp amplifier (HEKA Electronics). Linear capacitive and leakage currents were subtracted by using a P/4 protocol. Experimental data were acquired and analyzed by the program pulse+pulsefit8.0 (HEKA Electronics). The needed concentrations of toxin dissolved in external solution were applied onto the surface of experimental cells by low pressure injection with a microinjector (IM-5B, Narishige). Data Analysis—Data analysis was performed using Pulsefit (HEKA Electronics) and Sigmaplot (Sigma). All data are presented as means ± S.E., and n is the number of independent experiments. The fitted curves of concentration-dependent inhibition were obtained by using the following form of the Boltzmann equation: Inhibition% = 100/[1+ exp(C - IC50)/k], in which IC50 is the concentration of toxin at half-maximal inhibition, k is the slope factor, and C is the toxin concentration. Purification and Sequence Analysis of JZTX-III—A typical RP-HPLC chromatogram of the female spider venom was shown in Fig. 1A, in which more than 20 fractions eluted were monitored at 280 nm. The fraction with the retention time of 38 min, containing JZTX-III, was further purified by a repeated RP-HPLC (Fig. 1B). Two purifications yielded about 0.05 mg of JZTX-III/mg of crude venom with a purity over 99%. Its molecular mass was determined to be 3919.4 Da by MALDI-TOF mass spectrometry. The complete amino acid sequence of the toxin was obtained by Edman degradation and found to contain 36 residues including 6 cysteines (see Fig. 4A). After being reduced by dithiothreitol and then alkylated with iodoacetamide, the molecular mass of JZTX-III increased 348 Da (58 Da × 6), implying that all 6 cysteines were involved in forming three disulfide bridges. Since the primary structure had a mass of 3919.52 Da, consistent with the measured mass, the C-terminal residue could not be amidated. JZTX-III is a basic peptide sharing less than 50% sequence identity with any know peptides, although its 6 cysteines were highly conserved at corresponding positions in many toxins, such as HWTX-IV, ProTx-I, and ProTx-II (16Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (129) Google Scholar, 17Middleton R.E. Warren V.A. Kraus R.L. Hwang J.C. Liu C.J. Dai G. Brochu R.M. Kohler M.G. Gao Y.D. Garsky V.M. Bogusky M.J. Mehl J.T. Cohen C.J. Smith M.M. Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (200) Google Scholar).Fig. 4Amino acid sequence (A) and cDNA sequence (B) of JZTX-III.A, comparison of the primary structures of six sodium channel toxins: JZTX-III, ProTx-I, ProTx-II, HNTX-I, HNTX-IV, and HWTX-IV. Except for JZTX-III and ProTxs, others had an amidated C terminus. ProTx-I and ProTx-II were isolated from the venom of the tarantula Thrixopelma pruriens. HNTX-I, HNTX-IV, and HWTX-IV were isolated from the venoms of the Chinese bird spiders Selenocosmia hainana and S. huwena, respectively. The identical residues are shaded in black. The disulfide bridge pattern of the toxins is indicated under their sequences. JZTX-III inhibited activation of TTX-R VGSCs in rat cardiac myocytes. ProTx I–II inhibited the activation of Nav1.2, 1.5, 1.7, and 1.8 expressed in human embryonic kidney (HEK) cells. HNTX-I blocked rNav1.2 and para/tipE expressed in Xenopus laevis oocytes. Both HWTX-IV and HNTX-IV blocked TTX-S VGSCs in DRG neurons. B, the cDNA sequence of JZTX-III. The precursor deduced from the cDNA is indicated below the nucleotide acid sequence. The amino acid sequence of mature toxin is underlined. The possible endoprotelytic sites are pointed out with down arrows. An asterisk indicates that the C-terminal carboxyl group is amidated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Determination of the Disulfide Bridges in JZTX-III—Because vicinal cysteines (18CC19) emerge in the primary structure of JZTX-III, a chemical strategy composed of partial reduction and sequence analysis was introduced instead of a traditional enzymatic method. As shown in Fig. 2, four main peaks were obtained from the RP-HPLC separation of the partial reduced mixture of JZTX-III by TCEP. MALDI-TOF mass spectrometry analysis points out that the two intermediates yielded were resolved to contain one or two disulfide bridges in peak II and I, respectively, whereas peaks R and N represent completely reduced peptide and intact peptide, respectively. Peaks I–II were collected and alkylated rapidly with iodoacetamide followed by further purification using analytical RP-HPLC. Molecular mass determination and sequencing indicated that the free thiols of these two peptides had been alkylated. In Fig. 3A, Pth-CM-Cys signals were observed at the 4th and 19th cycles in the sequencing chromatograms of alkylated peak I, whereas no signals emerged at other cysteine cycles. The result indicates that the only reduced disulfide bond is Cys4–Cys19. When sequencing alkylated peak II, Pth-CM-Cys signals were observed at the 4th, 11th, 19th, and 24th cycles in the profiles of cysteine cycles (Fig. 3B), indicating that Cys18 was still linked to Cys31 by a disulfide bond. The above results indicate that two of three disulfide bridges in JZTX-III were determined to be Cys4–Cys19 and Cys18–Cys31. Accordingly, the third one is cross-linked between Cys11 and Cys24. Thus, JZTX-III has a conserved disulfide connectivity emerging among ICK motifs where 6 cysteines were linked in a pattern of I-IV, II-V, and III-VI (9Escoubas P. Diochot S. Corzo G. Biochimie (Paris). 2000; 82: 893-907Crossref PubMed Scopus (213) Google Scholar). Cloning and Sequencing of JZTX-III cDNA—The full-length cDNA sequence of JZTX-III was completed by overlapping two fragments resulting from 3′- and 5′-RACE. As shown in Fig. 4B, the oligonucleotide sequence of the cDNA was a 373-bp bond in which the first ATG was assumed to serve as the translation start codon. The open reading frame, ending before the first stop codon TGA at 3′-terminal position, encoded 63 residues corresponding to the JZTX-III precursor. It comprised a signal peptide of 21 residues, a pro-peptide of 5 residues, and a mature peptide of 36 residues. The deduced mature peptide sequence was consistent with that of native JZTX-III determined by Edman degradation. Unlike huwentoxin-IV, JZTX-III had no extra Gly or Gly + Arg/Lys residues at the C terminus, which are known to allow “post-modification” α-amidation at the C-terminal residue (20Diao J.B. Lin Y. Tang J.Z. Liang S.P. Toxicon. 2003; 42: 715-723Crossref PubMed Scopus (35) Google Scholar). The prepro-regions common to all spider toxins are a hydrophobic peptide and can be processed at a common signal site-X-Arg-before mature peptide sequences, which is recognized by special endoprotelytic enzymes. In general, this region is composed of over 40 residues. Interestingly, further analysis indicated that JZTX-III had a very small pre-pro-region that exhibits no similarity to those of other spider toxins from diverse species including the Chinese bird spider Selenocosmia huwena Wang (also known as Ornithoctonas huwena Wang) (20Diao J.B. Lin Y. Tang J.Z. Liang S.P. Toxicon. 2003; 42: 715-723Crossref PubMed Scopus (35) Google Scholar, 21Li D.L. Xiao Y.C. Hu W.J. Xie J.Y. Bosmans F. Tytgat J. Liang S.P. FEBS Lett. 2003; 555: 616-622Crossref PubMed Scopus (71) Google Scholar). Furthermore, it is worth noting that the signal site anterior to mature JZTX-III was an uncommon one (-X-Ser-) (20Diao J.B. Lin Y. Tang J.Z. Liang S.P. Toxicon. 2003; 42: 715-723Crossref PubMed Scopus (35) Google Scholar, 23Cardoso F.C. Pacifico L.G. Carvalho D.C. Victoria J.M. Neves A.L. Chavez-Olortegui C. Gomez M.V. Kalapothakis E. Toxicon. 2003; 41: 755-763Crossref PubMed Scopus (28) Google Scholar, 24Wang C.Z. Jiang H. Ou Z.L. Chen J.S. Chi C.W. Toxicon. 2003; 42: 613-619Crossref PubMed Scopus (21) Google Scholar, 25Alami M. Vacher H. Bosmans F. Devaux C. Rosso J.P. Bougis P.E. Tytgat J. Darbon H. Martin-Eauclaire M.F. Biochem. J. 2003; 375: 551-560Crossref PubMed Scopus (41) Google Scholar). A polyadenylation signal, AATAAA, was found in the 3′-untranslated region at position 16 upstream of the poly(A). Effects of JZTX-III on VGSCs—Using whole cell patch clamp technique, the actions of JZTX-III were characterized on VGSCs in rat DRG neurons and ventricular myocytes, in which both TTX-S and TTX-R types are co-expressed. TTX (200 nm) was added to the external bath solution to separate TTX-R type from mixture currents. Although Maier et al. (26Maier S.K. Westenbroek R.E. Schenkman K.A. Feigl E.O. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4073-4078Crossref PubMed Scopus (244) Google Scholar) suggested that some brain TTX-S subtypes were situated in transverse tubules of ventricular myocytes, in our experiments, the induced sodium currents were not changed in the absence or presence of TTX at 0.2 μm (data not shown, n = 4). Therefore, the effects of JZTX-III on cardiac myocytes were assayed in bath solution without TTX. After establishing whole cell configuration, 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. As shown in Fig. 5, A and B, 1 μm JZTX-III showed no evident effects on the normal activities of both TTX-S and TTX-R VGSCs in DRG neurons (n = 3). However, cardiac TTX-R currents were sensitive to the novel toxin. 1 μm JZTX-III reduced the control peak amplitude to a maximum effect by 64.7 ± 4.7% (Fig. 5C, n = 8). JZTX-III up to 10 μm completely eliminated the remaining currents within less than 1 min (Fig. 5, D and E, n = 4). The rapid inhibition was dose-dependent with an IC50 value of 0.38 ± 0.04 μm (Fig. 5F). It was observed that similar to ProTx I–II (15Corzo G. Gilles N. Satake H. Villegas E. Dai L. Nakajima T. Haupt J. FEBS Lett. 2003; 547: 43-50Crossref PubMed Scopus (85) Google Scholar), JZTX-III failed to alternate channel inactivation, although most spider toxins (e.g. δ- and μ-toxins) identified to date share a common mode of slowing channel inactivation similar to scorpion α-toxins (7Cummins T.R. Aglieco F. Renganathan M. Herzog R.I. Dib-Hajj S.D. Waxman S.G. J. Neurosci. 2001; 21: 5952-5961Crossref PubMed Google Scholar). ProTx I–II have been suggested to bind to VGSC site 3 or site 4. To further determine the detailed site for the toxin of interest, a simple competitive assay was introduced between JZTX-III and site 3 toxins. Buthus martensi Karsch I (BMK-I), acting on site 3, is a typical α-like scorpion toxin isolated from the Asian scorpion, B. martensi Karsch. It can slow the inactivation of VGSCs expressing in both mammalian sensory neurons and ventricular myocytes without significantly affecting the peak amplitudes (10Goudeta C. Chi C.W. Tytgata J. Toxicon. 2002; 40: 1239-1258Crossref PubMed Scopus (236) Google Scholar, 27Ji Y.H. Mansuelle P. Terakawa S. Kopeyan C. Yanaihara N. Hsu K. Rochat H. Toxicon. 1996; 34: 987-1001Crossref PubMed Scopus (111) Google Scholar). Exposed to 10 μm JZTX-III, the slowing currents induced by 10 μm BMK-I were eliminated completely, suggesting that the spider toxin modulated cardiac VGSCs through a mechanism distinct from site 3 toxins. Fig. 6 shows the current-voltage (I-V) curve of cardiac TTX-R VGSCs, yielding that initial activated voltage and reversal potential are -50 mV and +25 mV, respectively (n = 4). After 1 μm JZTX-III treatment for 1 min, the inhibition of currents could be observed at tested potential from -40 mV to +20 mV. JZTX-III shifted the threshold of initial activation more than +10 mV in a depolarizing direction, but no change was observed significantly in the membrane reversal potential, implying that it did not change the ion selectivity of channels. Effects of JZTX-III on VGCCs and Voltage-gated Potassium Channels (VGPCs)—There are two main categories of VGCCs distributed in rat DRG neurons: high voltage-activated channels and low voltage-activated channels, which can be discriminated by their voltage dependence and kinetics. JZTX-III (1 μm) was not found to affect VGCCs (Fig. 7, n = 3). Three different VGPC isoforms (Kv1.1, Kv1.2, and Kv1.3) were expressed in Xenopus Laevis oocytes and checked for toxins using the two-electrode voltage clamp technique as described previously (28Huys I. Tytgat J. Eur. J. Neurosci. 2003; 17: 1786-1792Crossref PubMed Scopus (9) Google Scholar). No effects were detected with JZTX-III at 1 μm (Fig. 8, n = 4).Fig. 8Effects of JZTX-III on VGPCs expressed in X. laevis oocytes. Kv1.1 (A), Kv1.2 (B), and Kv1.3 (C) current traces were evoked by depolarizations to +10 mV from a holding potential of -90 mV. After exposure to 1 μm JZTX-III, no changes of the currents were detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this work, we have isolated and characterized a 3.9-kDa toxin named JZTX-III from the Chinese spider C. jingzhao (19Zhu M.S. Song D.X. Li T.H. J. Baoding Teachers Coll. 2001; 14: 1-6Google Scholar). The full sequence of the toxin was performed by Edman degradation and found to contain 36 residues including 6 cysteines. No amidation at it" @default.
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• W2126923168 title "Jingzhaotoxin-III, a Novel Spider Toxin Inhibiting Activation of Voltage-gated Sodium Channel in Rat Cardiac Myocytes" @default.
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