Jingzhaotoxin-I, a Novel Spider Neurotoxin Preferentially Inhibiting Cardiac Sodium Channel Inactivation
2004; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m411651200
ISSN1083-351X
AutoresYucheng Xiao, Jianzhou Tang, Weijun Hu, Jinyun Xie, Chantal Maertens, Jan Tytgat, Songping Liang,
Tópico(s)Synthetic Organic Chemistry Methods
ResumoJingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3′- and 5′-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is ∼30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an α-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms. Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3′- and 5′-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is ∼30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an α-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms. Voltage-gated sodium channels (VGSCs) 1The abbreviations used are: VGSC, voltage-gated sodium channel; Kv, voltage-gated potassium channel; TTX, tetrodotoxin; TTX-R, TTX-resistant; TTX-S, TTX-sensitive; DRG, dorsal root ganglion; RACE, rapid amplification of cDNA ends; ICK, inhibitor cystine knot; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; TCEP, Tris-(2-carboxyethyl)phosphine; HNTX, Hainantoxin.1The abbreviations used are: VGSC, voltage-gated sodium channel; Kv, voltage-gated potassium channel; TTX, tetrodotoxin; TTX-R, TTX-resistant; TTX-S, TTX-sensitive; DRG, dorsal root ganglion; RACE, rapid amplification of cDNA ends; ICK, inhibitor cystine knot; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; TCEP, Tris-(2-carboxyethyl)phosphine; HNTX, Hainantoxin. are integral plasma membrane proteins composed of a pore-forming α-subunit (260 kDa) associated with up to four auxiliary β subunits (21–23 kDa) (1.Ogata N. Tatebayashi H. J. Physiol. (Lond.). 1993; 466: 9-37Google Scholar, 2.Catteral W.A. Neuron. 2002; 26: 13-25Abstract Full Text Full Text PDF Scopus (1640) Google Scholar). VGSCs play a vital role in the initiation and propagation of exciting signals on most excitable tissues. Similar to the "shaker" potassium channel, the three-dimensional structure of sodium channel is a bell-shaped molecule (3.Sokolova O. Kolmakova-Partensky L. Grigorieff N. Structure. 2001; 9: 215-220Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 4.Sato C. Ueno Y. Asai K. Takahashi K. Sato M. Engel A. Fujiyoshi Y. Nature. 2001; 409: 1047-1051Crossref PubMed Scopus (224) Google Scholar). From mammals, over ten mammalian subtypes (Nav1.1–1.9 and Navx) exhibiting relatively similar pharmacological properties in different expression systems have been identified and characterized. Sequence analysis demonstrates that they have been highly conserved during evolution. Furthermore, to be beneficial for catching their prey, the properties of sodium channels in many animals even have evolved geographically in a coevolutionary arms race with their toxic prey (5.Geffeney S. Brodie Jr., E.D. Ruben P.C. Brodie III, E.D. Science. 2002; 297: 1336-1339Crossref PubMed Scopus (134) Google Scholar, 6.Huey R.B. Moody W.J. Science. 2002; 297: 1289-1290Crossref PubMed Scopus (9) Google Scholar). Many polypeptides targeting VGSCs have been identified in many kinds of animal venoms, such as those from scorpions, spiders, snakes, and marine animals. With different critical residues and distinct functional characterization, these naturally occurring toxins are proved to be important tools in distinguishing the different subtypes and disclose the function-structure relationships of VGSCs. Over six neuronal receptor sites are suggested to elucidate such relationships (7.Cestele 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). Based on the analysis of precursor organization and gene structure combined with a three-dimensional fold, Zhu et al. (8.Zhu S. Darbon H. Dyason K. Verdonck F. Tytgat J. FASEB J. 2003; 17: 1765-1767Crossref PubMed Scopus (143) Google Scholar) suggest that inhibitor cystine knot (ICK) peptides from animals shared a common evolutionary origin. Compared with scorpion and snake toxins, spider peptides are shorter ones containing around 35 residues with three/four disulfide bonds. Despite distinct amino acid compositions, most spider peptides have also been found to share a clear homological ICK fold determined structurally by NMR and homology modeling techniques (9.Shu Q. Lu S.Y. Gu X.C. Liang S.P. Protein Sci. 2002; 11: 245-252Crossref PubMed Scopus (36) Google Scholar). The majority of them such as δ-atracotoxins (δ-ACTXs) and μ-agatoxins share a common mode of inhibiting inactivation kinetics of sodium currents on vertebrate or/and insect VGSCs by binding to receptor site 3 (10.Grolleau F. Stankiewicz M. Birinyi-strachan L. Wang X.H. Nicholson G.M. Pelhate M. Lapied B. J. Exp. Biol. 2001; 204: 711-721Crossref PubMed Google Scholar, 11.Nicholson G.M. Walsh R. Little M.J. Tyler M.I. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 117-126Crossref PubMed Scopus (72) Google Scholar, 12.Omecinsky D.O. Holub K.E. Adams M.E. Reily M.D. Biochemistry. 1996; 35: 2836-2844Crossref PubMed Scopus (69) Google Scholar). Functional convergence widely occurs among animal toxins with different origins during evolution (8.Zhu S. Darbon H. Dyason K. Verdonck F. Tytgat J. FASEB J. 2003; 17: 1765-1767Crossref PubMed Scopus (143) Google Scholar, 14.Zhu S. Bosmans F. Tytgat J. J. Mol. Evol. 2004; 58: 145-153Crossref PubMed Scopus (69) Google Scholar). Many significant evidences appear in scorpions, snakes, and marine animals, but similar evidence still remains to be found in spiders. The remarkable functional difference between spider site-3 toxins and other animal toxins is that spider site 3 toxins evidently depress current amplitudes, whereas other animal toxins enhance current amplitudes evoked under whole-cell patch clamp recording (10.Grolleau F. Stankiewicz M. Birinyi-strachan L. Wang X.H. Nicholson G.M. Pelhate M. Lapied B. J. Exp. Biol. 2001; 204: 711-721Crossref PubMed Google Scholar, 11.Nicholson G.M. Walsh R. Little M.J. Tyler M.I. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 117-126Crossref PubMed Scopus (72) Google Scholar, 12.Omecinsky D.O. Holub K.E. Adams M.E. Reily M.D. Biochemistry. 1996; 35: 2836-2844Crossref PubMed Scopus (69) Google Scholar). Spider site 3 toxins show no effects on tetrodotoxin-resistant (TTX-R) sodium channels such as Nav1.5 and Nav1.8–1.9. New emerging ligands will play an important role in elucidating their subtle difference. Moreover, they have been proven to be potential valuable pharmaceutics or insecticides (13.Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (124) Google Scholar). Here we report the isolation, cDNA sequence determination, and functional characterization of a novel sodium channel toxin, Jingzhaotoxin-I (JZTX-I), from the venom of Chinese tarantula Chilobrachys jingzhao (Araneae:Theraphosidae:Chi-lobrachys) (15.Zhu M.S. Song D.X. Li T.H. J. Baoding Teachers College. 2001; 14: 1-6Google Scholar). The toxin is composed of 33 residues stabilized by three disulfide bridges (I-IV, II-V, and III-VI) assigned by partial reduction, sequencing, and multi-enzymatic digestion. JZTX-I shows no effect on neuronal TTX-R VGSCs and Kv1 channels, but it inhibits channel inactivation of neuronal TTX-S subtypes and cardiac TTX-R subtypes. To the best of our knowledge, JZTX-I is an α-like toxin first reported to date from spider venoms. It provides an important witness for studying the convergent and divergent functional evolution of animal venoms. Toxin Purification and Sequencing—JZTX-I was fractionated from Chinese tarantula C. jingzhao venom using a combination of ion-exchange chromatography and reverse-phase high pressure liquid chromatography (HPLC) as previous described (13.Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (124) Google Scholar). Lyophilized venom (10 mg in 2 ml in distilled water) was applied to a Waters protein-Pak CM 8 H column (5 × 50 mm) initially equilibrated with 0.2 m sodium phosphate buffer, pH 6.25 (buffer A). The column then was eluted with a linear gradient (see Fig. 1A) at a flow rate of 3.0 ml/min. The fraction of interest collected was applied to a Vydac C18 (C4) analytical reverse-phase HPLC column (218TP54, 4.6 × 250 mm) and eluted at a flow rate of 0.7 ml/min by a linear gradient of acetonitrile containing 0.1% v/v trifluoroacetic acid (see Fig. 1B). The molecular mass and purity of toxin were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The full amino acid sequence (Swiss-Prot accession numbers P83974 and AJ854060) was obtained from a single sequencing run on an Applied Procise™ 491-A protein sequencer by automated Edman degradation. Assignment of the Disulfide Bridges of JZTX-I—Native peptide (0.1 mg) was partially reduced in 20 μl of citrate buffer (1 m, pH 3.0) containing 6 m guanidine-HCl and 0.05 m Tris (2-carboxyethyl)phosphine (TCEP) at 40 °C for 10 min. The fractions monitored at 280 nm were separated on a C18 reverse-phase HPLC column with a linear gradient elution (25–50% acetonitrile in 40 min). The masses of all of the fractions collected were determined by MALDI-TOF mass spectrometry. The intermediate with free thiols were lyophilized and then alkalized by adding 100 μl of 0.5 m iodoacetamide, pH 8.3. The alkalized peptide was desalted by reverse-phase HPLC and then submitted to an Applied Biosystems Model 491 gas-phase sequencer. The Edman degradation was performed with a normal automatic cycle program. Concerning the protease digestion strategy, native JZTX-I (0.1 mg) was dissolved in 0.2 ml of Tris-HCl (0.2 m, pH 7.5) buffer containing trypsin (4 μg), chymotrypsin (4 μg), and V8 protease (4 μg). The mixture was incubated at 37 °C for 16 h. The masses of enzymolytic products then were analyzed using MALDI-TOF mass spectrometry. Identification of JZTX-I cDNA—The full-length of JZTX-I cDNA was obtained using rapid amplification of cDNA ends (RACE) methods as described previously (27.Diao 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 instruction, the total RNA was extracted from 0.1 g of fresh venom glands of female spiders using TRIzol reagent kit. 5 μg of RNA was taken to convert mRNA into cDNA using the Superscript II reverse transcriptase with a universal oligo(dT)-containing adapter primer (5′-GGCCACGCGTCGACTAGTAC(dT)17-3′). The cDNA then was used as template for PCR amplification in 3′-RACE. Degenerate primer 1 (5′-GC(A/T/C/G)AA(C/T)TT(T/C)GC(A/C/G/T)TG(T/C)AA(G/A)AT(A/C/T)-3′) was designed corresponding to the N-terminal residues (18ANFACKI24) of mature JZTX-I. The partial cDNA of mature toxin was amplified by PCR technique using primer 1. Second, based on the partial cDNA sequence of JZTX-I determined by 3′-RACE, the antisense primers were designed and synthesized for 5′-RACE as gene-specific primer 2 (5′-GGCCTAAGGGCTCCAGATACA-3′). With the strategy described by the RACE kit supplier, the 5′-end cDNA of JZTX-I was amplified using its gene-specific primer 2. Amplified products in both 3′- and 5′-RACE were precipitated and cloned into the pGEM-Teasy vector for sequencing. DNA sequencing was performed by Bioasia Inc. Nucleic acid sequences were analyzed using the software of DNAclub (by Xiongfong Chen) and DNAman (by Nynnon Biosoft). Cell Preparation—Rat DRG neurons were acutely dissociated and maintained in a short-term primary culture according to the procedures adapted from Xiao et al. (16.Xiao Y. Tang J. Yang Y. Wang M. Hu W. Xie J. Zeng X. Liang S. J. Biol. Chem. 2004; 279: 26220-26226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 30-day-old adult Sprague-Dawley rats of either sex, in adherence with protocols approved by the Hunan Normal University Animal Care and Use Committee, were killed by decapitation without anesthetization, the dorsal root ganglia were removed quickly from the spinal cord, and then they were transferred into Dulbecco's modified Eagle's medium containing trypsin (0.5 g/liter, type III), collagenase (1.0 g/liter, type IA), and DNase (0.1 g/liter, type III) to incubate at 34 °C for 30 min. Trypsin inhibitor (1.5 g/liter, 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 °C) for 1–4 h before the patch clamp experiment. Single ventricular cardiomyocytes were enzymatically dissociated from adult rats according to the procedures adapted from Xiao and Liang (17.Xiao Y.C. Liang S.P. Eur. J. Pharmacol. 2003; 477: 1-7Crossref PubMed Scopus (60) Google Scholar). Sprague-Dawley rats (∼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; and 1.8 CaCl2 at pH 7.2. The heart then 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 ((in mm) 70.0 l-glutanic; 25.0 KCl; 20.0 taurine; 10.0 KH2PO4; 3 MgCl2; 0.5 EGTA; 10.0 glucose; and 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 a 200-μm nylon mesh. All of the cells were used within 8 h of isolation. Cotton bollworm central nerve ganglion neurons were acutely dissociated and maintained in a short-term primary culture as described previously (18.He B.J. Liu A.X. Chen J.T. Sun J.S. Rui C.H. Meng X.Q. Acta Entomol. Sin. 2001; 44: 422-427Google Scholar). 10-day-old cotton bollworms were killed in 75% alcohol and washed in saline containing the following (in mm): 90 NaCl; 6 KCl; 2 MgCl2; 10 HEPES; and 140 d-glucose at pH 6.6. After being wiped off the enteron, the central nerve ganglia were removed quickly. The nerve ganglia torn then were incubated at room temperature (20–25 °C) in enzyme solution for 20 min containing the following: 90 mm NaCl; 6 mm KCl; 10 mm HEPES; 25 mm d-glucose; 115 mm d-mannitol; 0.3% trypsin; and 0.15% collagenase IV at pH 6.6. Their enzymatic digestion was stopped in 35-mm-culture dishes containing 45% TC-100 (Invitrogen), 45% Dulbecco's modified Eagle's medium (Sigma), 10% fetal bovine serum, 100 mm d-glucose, 0.6 mm glutamine, glutathione, and penicillin-streptomycin at pH 6.6. After the cells in the ganglia were gently dispersed using a suction tube, they were incubated in CO2 incubator (5% CO2, 95% air, 28 °C) for 2–3 h before the patch clamp experiment. Whole-cell Recording—Sodium currents were recorded from experimental cells using whole-cell patch clamp technique at room temperature (22–25 °C). Recording pipettes (2–3 μm in diameter) were made from borosilicate glass capillary tubing, and its resistances were 1.0–2.0 megohms when filled with internal solution contained the following (in mm): 135 CsF; 10 NaCl; and 5 HEPES at pH 7.0. The external bathing solution contained the following (in mm): 30 NaCl; 5 CsCl; 25 d-glucose; 1 MgCl2; 1.8 CaCl2; 5 HEPES; 20 triethanolamine chloride; and 70 tetramethylammonium chloride at pH 7.4. After establishing the whole-cell recording configuration, the resting potential was held at –80 mV for at least 4 min to allow adequate equilibration between the micropipette solution and the cell interior. Ionic currents were filtered at 10 kHz and sampled at 3 kHz on EPC-9/10 patch clamp amplifier (HEKA, Lambrecht, Germany). The P/4 protocol was used to subtract linear capacitive and leakage currents. Experimental data were acquired and analyzed by the program Pulse+Pulsefit8.0 (HEKA). 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). Concerning DRG neurons containing TTX-S and TTX-R sodium channels, the cells with diameters of 30–40 μm were chosen for the experiments. Larger DRG cells (>30 μm) tended to express TTX-S VGSCs, whereas the smaller ones (<10 μm) tended to express TTX-R VGSCs (19.Su X. Wachtel R.E. Gebhart G.F. Am. J. Physiol. 1997; 277: 1180-1188Google Scholar). TTX-R currents were separated from total currents using 0.2 μm TTX blocking TTX-S channels completely. Evolutionary Tree of Spider Sodium Channel Toxins—The phylogeny of spider sodium channel toxins was constructed as described previously (14.Zhu S. Bosmans F. Tytgat J. J. Mol. Evol. 2004; 58: 145-153Crossref PubMed Scopus (69) Google Scholar). In our study, we focused on the spider toxins from the species in the family Mygalomorphae. Two groups of well known spider toxins (μ-agatoxin I-VI and δ-paluIT 1–4) were chosen as typical representations of the family Araneomorphae to outline the phylogeny clearly (12.Omecinsky D.O. Holub K.E. Adams M.E. Reily M.D. Biochemistry. 1996; 35: 2836-2844Crossref PubMed Scopus (69) Google Scholar, 20.Corzo G. Escoubas P. Stankiewicz M. Pelhate M. Kristensen C.P. Nakajima T. Eur. J. Biochem. 2000; 267: 5783-5795Crossref PubMed Scopus (62) Google Scholar). Multiple sequences of spider toxins were edited using the Bioedit Sequence Alignment Editor software and then aligned and refined manually using ClustalW1.8 program (21.Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (34817) Google Scholar). A pairwise distance matrix was calculated on the basis of the proportions of different amino acids. The matrix was then used to construct trees by the neighbor-joining method (MEGA2.1) (22.Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar). The reliability of branching patterns was assessed using 1000 bootstrap replications. Purification and Sequence Analysis of JZTX-I—The fraction of interest (JZTX-I) was purified using a combination method of ion-exchange HPLC and reverse-phase HPLC (Fig. 1) based on its ability to evidently increase the strength and the rate of vertebrate heartbeats (figure not shown). The molecular mass of naturally occurring toxin was determined to be 3675.64 Da by MALDI-TOF mass spectrometry. Its full amino acid sequence was performed by N-terminal Edman degradation and found to be composed of 33 residues including six cysteines (Fig. 3A). Six cysteines were assayed to form three disulfide bridges by the molecular mass of alkalized sample, which increased 58 × 6 Da. The calculated molecular mass (3675.4 Da) corresponding to the primary sequence was consistent with the measured mass, suggesting that the C-terminal residue (Pro33) was not amidated. JZTX-I was a novel tarantula toxin exhibiting limited sequence similarity with any reported peptide but 50.0% with JZTX-III. Despite the significant sequence divergence, sequence alignment indicated that all six cysteines in JZTX-I were strictly conserved at similar positions in most spider peptides adopting a typical ICK fold such as HWTX-I, ProTx-I, and HNTX-I. Assignment of the Disulfide Bridges of JZTX-I—Fig. 2A showed the typical reverse-phase HPLC separation of the partially reduced mixture of JZTX-I by TECP. As MALDI-TOF mass spectrometry analysis pointed out, only one intermediate was obtained and further resolved to contain one disulfide bridge in peak II, whereas peaks I and III represented intact peptide and completely reduced peptide, respectively, because their molecular masses increased by 0 (peak I), 4 (peak II), and 6 Da (peak III) compared with that of native peptide, respectively. Peak II then was collected and alkalized immediately with iodoacetamide followed by further purification using reverse-phase HPLC. Molecular mass determination and sequencing indicated that the free thiols of the fraction of interest had been alkylated. The sequencing results showed that the signals of Pth-CM-Cys signals were observed except at the 16th and 29th cycles, positively supporting that the remaining disulfide bridge was cross-linked by Cys16-Cys29. In Fig. 2B, when JZTX-I was exposed to multi-enzymes (trypsin, chromotrypsin, and V8 protease) in the buffering solution, a series of smaller enzymolytic fragments was produced and their molecular masses were measured by MALDI-TOF mass spectrometry. The reverse-phase HPLC separation of the enzymolytic mixture could produce a 2176-Da fragment, which was sequenced to be Ala1-Trp6 (containing Cys2), Gly12-Phe20 (Cys16 and Cys17), and Leu28-Trp31 (Cys29), as described in Fig. 2B, inset. Thus two disulfide bridges should be paired among Cys2, Cys16, Cys17, and Cys29. Because a disulfide bridge between Cys16 and Cys29 had been determined above, we concluded that the second was cross-linked between Cys2 and Cys17. Accordingly, by process of elimination, the third disulfide bond was between Cys9 and Cys22. Therefore, the disulfide linkage of JZTX-I was homologous to that for ICK peptides from spider venoms, such as ProTxs, HWTX-IV, and HNTX-I, as well as conotoxins (δ-, μ-, κ-, and ω-), although the positions of six cysteines were not conserved between them (13.Peng K. Shu Q. Liu Z. Liang S. J. Biol. Chem. 2002; 49: 47564-47571Abstract Full Text Full Text PDF Scopus (124) Google Scholar, 23.Middleton 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. et al.Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (192) Google Scholar, 24.Li D. Xiao Y. Hu W. Xie J. Bosmans F. Tytgat J. Liang S. FEBS Lett. 2004; 555: 616-622Crossref Scopus (71) Google Scholar, 25.Shon K.J. Olivera B.M. Watkins M. Jacobsen R.B. Gray W.R. Floresca C.Z. Cruz L.J. Hillyard D.R. Brink A. Terlau H. Yoshikami D. J. Neurosci. 1998; 18: 4473-4481Crossref PubMed Google Scholar). Cloning and Sequencing JZTX-I cDNA—The full-length cDNA sequence of JZTX-I was completed by overlaying two fragments resulting from 3′- and 5′-RACE. As shown in Fig. 3B, the oligonucleotide sequence of the cDNA was a 383-bp bond found to comprise a 5′-untranslated region, open reading frame, and 3′-untranslated region. The open reading frame encoded a 63-residue peptide (Swiss-Prot accession number AJ854060) corresponding to the JZTX-I precursor that contained a signal peptide of 21 residues, a propeptide of 8 residues, and a mature peptide of 33 residues. The pre-proregion composed of a signal peptide and a propeptide is a hydrophobic peptide common to all spider toxins. Furthermore, its homology is important proof that spider toxins can be grouped into different superfamilies to analyze their evolutionary relationship. The pre-propeptide of JZTX-I precursor showed limited sequence identity with that of other reported precursors but 66.7 and 57.1% with JZTX-III and GsMTx-4 (16.Xiao Y. Tang J. Yang Y. Wang M. Hu W. Xie J. Zeng X. Liang S. J. Biol. Chem. 2004; 279: 26220-26226Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26.Ostrow K.L. Mammoser A. Suchyna T. Sachs F. Oswald R. Kubo S. Chino N. Gottlieb P.A. Toxicon. 2003; 42: 263-274Crossref PubMed Scopus (65) Google Scholar), respectively. Different from most spider sodium channel toxins, JZTX-I had no extra amino acid residues Gly or (Gly + Arg/Lys) at its C terminus known to allow "post-modification" and α-amidation at the C-terminal residue (27.Diao J.B. Lin Y. Tang J.Z. Liang S.P. Toxicon. 2003; 42: 715-723Crossref PubMed Scopus (35) Google Scholar), also implying that the C-terminal residue of mature toxin is not amidated. A polyadenylation signal (AATAAA) emerged in the 3′-untranslated region at position 17 upstream of the poly(A). Effects of JZTX-I on Sodium Currents—TTX-S and TTX-R VGSCs co-express in adult rat DRG neurons (1.Ogata N. Tatebayashi H. J. Physiol. (Lond.). 1993; 466: 9-37Google Scholar), whereas only TTX-S type is distributed in cotton bollworm central nerve ganglion neurons (18.He B.J. Liu A.X. Chen J.T. Sun J.S. Rui C.H. Meng X.Q. Acta Entomol. Sin. 2001; 44: 422-427Google Scholar). TTX-R VGSCs are the primary type in adult rat ventricular myocytes, although Maier et al. (28.Maier S.K. Westenbroek R.E. Schenkman K.A. Feigl E.O. Scheuer T. Catterall A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4073-4078Crossref PubMed Scopus (240) Google Scholar) suggest that some brain TTX-S subtypes are situated in its transverse tubules (28.Maier S.K. Westenbroek R.E. Schenkman K.A. Feigl E.O. Scheuer T. Catterall A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4073-4078Crossref PubMed Scopus (240) Google Scholar). The sodium channels expressing in these tissues have been defined as different isoforms based on their divergent amino acid sequences. Generally, TTX-R sodium currents activated and inactivated more slowly than TTX-S types under whole-cell configuration. JZTX-I at 1 μm in the bath solution was resistant to control TTX-R sodium currents in rat DRG neurons (Fig. 4A, n = 5) but sensitive to TTX-R currents in ventricular myocytes and TTX-S currents in sensory neurons (Fig. 4, B–D). JZTX-I evidently inhibited channel inactivation with distinct affinity on three tested tissues without changing peak current amplitudes or the time of current peak. A similar result was detected after the application of β-pompilidotoxin, which was considered to selectively interfere with sodium channel inactivation process without affecting the activation (29.Kinoshita E. Maejima H. Yamaoka K. Konno K. Kawai N. Shimizu E. Yokote S. Nakayama H. Seyama I. Mol. Pharmacol. 2001; 59: 1457-1463Crossref PubMed Scopus (41) Google Scholar). The efficiency of the toxin then was assayed by measuring the I5ms/Ipeak ratio, which gave an estimate of the probability for the channel not to be inactivated after 5 ms. JZTX-I at 1 μm inhibited channel inactivation of TTX-S currents in rat DRG neurons by 51.2 ± 8.0% (Fig. 4B, n = 20), whereas an equal inhibition of TTX-R currents was achieved in rat cardiac myocytes exposed to the toxin only at 50 nm (Fig. 4C, n = 5). The toxin at 1 μm could slow the decay of TTX-S sodium currents in cotton bollworm neurons by 52.4 ± 6.8% (Fig. 4D, n = 11). All of the inhibitions above were in a time-dependent and concentration-dependent manner, and their IC50 values were assessed to be 0.13 μm, 31.6 nm, and 0.76 μm, respectively (Fig. 4E). Furthermore, we checked the ion component of the inward currents maintained at the ending of depolarizing pulse. Because they could be eliminated in both rat DRG and insect neurons by TTX (0.2 μm) completely in Fig. 4B (n = 8), ion currents were still conducted through sodium chan
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