Function and Solution Structure of Huwentoxin-IV, a Potent Neuronal Tetrodotoxin (TTX)-sensitive Sodium Channel Antagonist from Chinese Bird Spider Selenocosmia huwena
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m204063200
ISSN1083-351X
AutoresKuan Peng, Qin Shu, Zhonghua Liu, Songping Liang,
Tópico(s)Venomous Animal Envenomation and Studies
ResumoWe have isolated a highly potent neurotoxin from the venom of the Chinese bird spider, Selenocosmia huwena. This 4.1-kDa toxin, which has been named huwentoxin-IV, contains 35 residues with three disulfide bridges: Cys-2–Cys-17, Cys-9–Cys-24, and Cys-16–Cys-31, assigned by a chemical strategy including partial reduction of the toxin and sequence analysis of the modified intermediates. It specifically inhibits the neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel with the IC50 value of 30 nm in adult rat dorsal root ganglion neurons, while having no significant effect on the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel. This toxin seems to be a site I toxin affecting the sodium channel through a mechanism quite similar to that of TTX: it suppresses the peak sodium current without altering the activation or inactivation kinetics. The three-dimensional structure of huwentoxin-IV has been determined by two-dimensional 1H NMR combined with distant geometry and simulated annealing calculation by using 527 nuclear Overhauser effect constraints and 14 dihedral constraints. The resulting structure is composed of a double-stranded antiparallel β-sheet (Leu-22–Ser-25 and Trp-30–Tyr-33) and four turns (Glu-4–Lys-7, Pro-11–Asp-14, Lys-18–Lys-21 and Arg-26–Arg-29) and belongs to the inhibitor cystine knot structural family. After comparison with other toxins purified from the same species, we are convinced that the positively charged residues of loop IV (residues 25–29), especially residue Arg-26, must be crucial to its binding to the neuronal tetrodotoxin-sensitive voltage-gated sodium channel. We have isolated a highly potent neurotoxin from the venom of the Chinese bird spider, Selenocosmia huwena. This 4.1-kDa toxin, which has been named huwentoxin-IV, contains 35 residues with three disulfide bridges: Cys-2–Cys-17, Cys-9–Cys-24, and Cys-16–Cys-31, assigned by a chemical strategy including partial reduction of the toxin and sequence analysis of the modified intermediates. It specifically inhibits the neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel with the IC50 value of 30 nm in adult rat dorsal root ganglion neurons, while having no significant effect on the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel. This toxin seems to be a site I toxin affecting the sodium channel through a mechanism quite similar to that of TTX: it suppresses the peak sodium current without altering the activation or inactivation kinetics. The three-dimensional structure of huwentoxin-IV has been determined by two-dimensional 1H NMR combined with distant geometry and simulated annealing calculation by using 527 nuclear Overhauser effect constraints and 14 dihedral constraints. The resulting structure is composed of a double-stranded antiparallel β-sheet (Leu-22–Ser-25 and Trp-30–Tyr-33) and four turns (Glu-4–Lys-7, Pro-11–Asp-14, Lys-18–Lys-21 and Arg-26–Arg-29) and belongs to the inhibitor cystine knot structural family. After comparison with other toxins purified from the same species, we are convinced that the positively charged residues of loop IV (residues 25–29), especially residue Arg-26, must be crucial to its binding to the neuronal tetrodotoxin-sensitive voltage-gated sodium channel. Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spiderSelenocosmia huwena.Journal of Biological ChemistryVol. 278Issue 7PreviewPage 47567, Fig. 4 legend: The following sentence is missing from the figure legend. Full-Text PDF Open Access Voltage-gated sodium channels play important roles in electrical signaling in almost all kinds of excitable tissues. They are responsible for the generation of action potentials and nervous influx conduction in sensory nerves. Although the pioneer investigation of tetrodotoxin (TTX) 1The abbreviations used are: TTX, tetrodotoxin; TTX-R, TTX-resistant; TTX-S, TTX-sensitive; DRG, dorsal root ganglion; VGSC, voltage-gated sodium channel; HWTX-IV, huwentoxin IV; ICK, inhibitor cystine knot; Pth, parathyroid hormone; CM, carboxymethyl; R.M.S.D., root mean square difference(s); HPLC, high pressure liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; DQF, double quantum filtered; TOCSY, two-dimensional total correlation spectroscopy on voltage-gated sodium channels (VGSCs) had been performed by Narahashi et al. (1Narahashi T. Deguchi T. Urakama N. Ohkubo Y. Am. J. Physiol. 1960; 198: 934-938Google Scholar, 2Narahashi T. Moore J.W. Scott W.R. J. Gen. Physiol. 1964; 47: 965-974Google Scholar) in 1960s, it was not until early 1990s that people began to classify the voltage-gated sodium channel into two different types (3Roy M.L. Narahashi T. J. Neurosci. 1992; 12: 2104-2111Google Scholar), tetrodotoxin-sensitive (TTX-S) VGSC and TTX-resistant (TTX-R) VGSC. Further investigations on many other neurotoxins from various biological sources such as ciguatoxin (4Strachan L.S. Lewis R.J. Nicholson G.M. J. Pharmacol. Exp. Ther. 1998; 288: 379-388Google Scholar), scorpion toxins (5Possani L.D. Becerril B. Delepierre M. Tytgat J. Eur. J. Biochem. 1999; 264: 287-300Google Scholar), and μ-conotoxins (6Shon K.J. Oliver M.B. 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: 4473-4481Google Scholar, 7Chahine M. Sirois J. Marcotte P. Chen L.Q. Kallen R.G. Biophys. J. 1998; 75: 236-246Google Scholar, 8Li R.A. Ennis I.L. Vélez P. Tomaselli G.F. Marbàn E. J. Biol. Chem. 2000; 275: 27551-27558Google Scholar) have shown that these toxins can affect VGSCs through quite diverse mechanisms. At least six different sites have been proposed to explain the complicated mechanisms of the targeting VGSCs of these neurotoxins. In short, site I toxins such as TTX and μ-conotoxins bind to an external key site of the pore so that sodium ions can no longer pass the channel. Toxins related to sites II–VI differ greatly from each other as they either affect the activation (β-scorpion toxins) or the deactivation (α-scorpion toxins) of sodium channels, and some of them even exert dual effects on both processes (ciguatoxin) (9Meir A. Ginsburg S. Butkevich A. Kachalsky S.G. Kaiserman I. Ahdut R. Demirgoren S. Rahamimoff R. Physiol. Rev. 1999; 79: 1019-1087Google Scholar). At the molecular level, the highly glycosylated α-subunit (260 kDa) is responsible for the pore forming of VGSCs. A number of investigations on drug receptor/channel interaction have enabled us to know many key residues for the function of VGSCs. TTX, saxitoxin, and μ-conotoxins have contributed a great deal in probing the structure-function relationship and modulation of variant VGSCs (10Sampo B. Tricaud N. Leveque C. Seagar M. Couraud F. Dargent B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3666-3671Google Scholar, 11Boccaccio A. Moran O. Irnoto K. Conti F. Biophys. J. 1999; 77: 229-240Google Scholar, 12Penzotti J.L. Fozzard H.A. Lipkind G.M. Dudley S.C. Biophys. J. 1998; 75: 2647-2657Google Scholar, 13Hilber K. Sandtner W. Kudlacek O. Glaaser I.W. Weisz E. Kyle J.W. French R.J. Fozzard H.A. Dudley S.C. Todt H. J. Biol. Chem. 2001; 276: 27831-27839Google Scholar). Another potential striking use of VGSC antagonists comes from their implication with pain. VGSCs expressed in primary sensory neurons (dorsal root ganglion (DRG) neurons) are believed to be important targets in the study of the molecular pathophysiology of pain and in the search for new pain therapies (14Waxman S.G. Dib-Hajj S. Cummins T.R. Black J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7635-7639Google Scholar, 15Porreca F. Lai J. Bian D. Wegert S. Ossipov M.H. Eglen R.M. Kassotakis L. Novakovic S. Rabert D.K. Sangameswaran L. Hunter J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7640-7644Google Scholar, 16Cummins T.R. Waxman S.G. J. Neurosci. 1997; 17: 3503-3514Google Scholar). Peptide toxins with considerable affinity and high selectivity to unique VGSCs are sure to play an important role in this field. In this study, we focus on the structure-function relationship studies of a highly potent neurotoxin purified from Chinese bird spider, Selenocosmia huwena, which is named huwentoxin-IV (HWTX-IV). First, we used patch clamp methods to investigate its effects on VGSCs expressed in adult rat DRG neurons; HWTX-IV selectively inhibits TTX-S VGSCs and shows no significant effect on TTX-R VGSCs. Second, its three-dimensional solution structure was elucidated by using NMR methods; HWTX-IV shares an ancestral global folding pattern with many other neurotoxins called the inhibitor cystine knot (ICK) motif (17Pallaghy P.K. Nielsen K.J. Craik D.J. Norton R.S. Protein Sci. 1994; 3: 1833-1839Google Scholar). Third, comparison with huwentoxin-I and previously known conotoxins bloking at site I provided us some clues on its toxin-receptor interaction. Besides the disulfide-directed backbone, we think positively charged residues (residue Arg-26 especially) in the extrusive loop IV of the compact molecule must play a crucial role in its targeting TTX-S VGSC. The venom from the female Chinese bird spider (S. Huwena) was collected as described in our previous work (18Shu Q. Liang S. J. Pept. Res. 1999; 53: 486-491Google Scholar). HWTX-IV was purified by means of ion-exchange and reverse-phase high performance liquid chromatography. Lyophilized crude venom was loaded onto a Waters Protein-Pak CM 8HR ion-exchange column (5 × 50 mm) initially equilibrated with 0.02m sodium phosphate buffer, pH 6.25 (buffer A). The column was eluted with a linear gradient of 0–50% of buffer B (1m sodium chloride, 0.02 m sodium phosphate, pH 6.25) over 40 min at a flow rate of 0.7 ml min−1. The fraction of interest was then applied to a Vydac C18 analytical reverse-phase HPLC column (218TP54, 4.6 × 250 mm) and eluted at a flow rate of 0.8 ml min−1 using a gradient of 0–20% buffer B (0.1% v/v trifluoroacetic acid in acetonitrile) over 8 min after an equilibrium period of 2 min followed by a gradient of 20–35% buffer B over 40 min. (Buffer A was 0.1% v/v trifluoroacetic acid in water.) Once purified to >95% homogeneity (assessed by reverse-phase HPLC and mass spectrometry), peptide was lyophilized and stored at −20 °C until further use. The molecular mass was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Bruker ProFlex-III mass spectrometer, and the entire amino acid sequence was obtained from a single sequencing run on an Applied Biosystems/PerkinElmer Life Sciences Procise 491-A protein sequencer. HWTX-IV (0.1 mg) was solubilized in 10 μl of 0.1 mol/liter citrate buffer (pH 3) containing 6 mol/liter guanidine-HCl. Partial reduction of HWTX-IV disulfide bonds was carried out by adding 10 μl of 0.1 mol/liter Tris (2-carboxyethyl) phosphine at 40 °C for 10 min at pH 3, and the intermediates were isolated by reverse-phase HPLC (column: 218TP54, 4.6 × 250 mm) with linear gradient elution (20∼35% acetonitrile in 50 min). The intermediates of partial reduction were collected, and their masses were determined by MALDI-TOF. Appropriate intermediates containing free thiols were dried and then alkalized by adding 100 μl of 0.5 mol/liter 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. Insect toxicity was determined by intraperitoneal injection of HWTX-IV into the abdomen of adult American cockroaches Periplaneta americana at doses of 10–200 μg g−1. Insects were monitored for 48 h after injection. Rat DRG neurons were acutely dissociated and maintained in short term primary culture using the method described by Hu and Li (22Hu H.Z. Li Z.W. J. Physiol. 1997; 501: 67-75Google Scholar) Briefly, 30-day-old adult Sprague-Dawley rats of either sex were decapitated, and the dorsal root ganglia were isolated quickly from the spinal cord. Then they were transferred into Dulbecco's modified Eagle's medium containing trypsin (0.5 mg ml−1, type III, Sigma), collagenase (1.0 mg ml−1, type IA, Sigma), and DNase (0.1 mg ml−1, type III, Sigma) for incubation at 34 °C for 30 min. Trypsin inhibitor (1.5 mg ml−1, type II-S, Sigma) was used to terminate enzyme treatment. The DRG cells were transferred into 35-mm culture dishes (Corning, Sigma) with the cultured medium (95% Dulbecco's modified Eagle's medium, 5% newborn calf serum, hypoxanthine aminopterin thymidine supplement, and penicillin-streptomycin) and incubated in CO2 incubator (5% CO2, 95% air, 37 °C) for 1–4 h before the patch clamp experiment. DRG cells with large diameters (around 50 picosiemens in slow capacitance) and those with relatively small diameters (around 20 picosiemens for slow capacitance) were chosen for study of TTX-S and TTX-R sodium currents, respectively. Meanwhile, TTX (final concentration at 0.2 μm) was used to separate TTX-R sodium current from TTX-S sodium current. Drug-containing solutions of 10-μl volume were applied by pressure injection with a microinjector (IM-5B, Narishige, Tokyo, Japan) through a micropipette (20-μm tip diameter) placed about 100 μm away from the cells under study. Patch clamp experiments were performed at room temperature (20–25 °C) under the whole cell patch clamp configuration. Suction pipettes (2.0–3 microohms) were made of borosilicate glass capillary tubes with a two-step pulling from a vertical micropipette puller (PC-10, Narishige). The pipette solution contained: 145 mm CsCl, 4 mmMgCl2·6H2O, 10 mm HEPES, 10 mm EGTA, 10 mm glucose, 2 mm ATP (the pH was adjusted to 7.2 with KOH). The external solution contained: 145 mm NaCl, 2.5 mm KCl, 1.5 mmCaCl2, 1.2 mmMgCl2·6H2O, 10 mm HEPES, 10 mm glucose (the pH was adjusted to 7.4 with NaOH). Experimental data were collected and analyzed by using the program Pulse/Pulsefit 8.0(HEKA Electronics, Lambrecht/Pfalz, Germany), and macroscopic TTX-S or TTX-R sodium currents were filtered at 10 kHz and digitized at 3 kHz with an EPC-9 patch clamp amplifier (HEKA Electronics, Germany). Series resistance was kept near 5 microohms and compensated 65–70%, and linear capacitative and leakage currents were digitally subtracted by using the P/4 protocol. An NMR sample was prepared by dissolving ∼10 mg of HWTX-IV in 500 μl of 20 mm phosphate buffer (H2O/D2O, 9/1, v/v) containing 0.002% NaN3 and 0.1 mm EDTA. The pH was adjusted to 4.5 with HCl, and sodium 3-(trimethyl-silyl) propionate-2, 2, 3, 3-D4 was added at a final concentration of 200 μm as an internal chemical shift reference. The final concentration of HWTX-IV was ∼5 mm. For experiments in D2O, the sample used in H2O experiments was lyophilized and then redissolved in 500 μl of 99.996% D2O (Cambridge Isotope Laboratories). All NMR experiments were recorded on a 500 MHz Bruker DMX-500 spectrometer. All two-dimensional spectra were recorded in phase-sensitive mode by the time-proportional phase incrementation method following standard pulse sequences and phase cycling. Solvent suppression was achieved by the presaturation method. Two-dimensional NMR spectra were recorded at a temperature of 300 K, including COSY, DQF-COSY, and TOCSY with a mixing time of 37 and 73 ms, as well as NOESY with a mixing time of 100, 200, and 300 ms. The recording data points of t1 × t2 were 512 × 2048 for COSY and TOCSY, 700 × 4096 for DQF-COSY, and 512 × 2048 for all NOESY spectra except 768 × 4096 for the 100 ms spectrum. The hydrogen-deuterium exchange experiments were carried out by recording a series of one-dimensional spectra after redissolving the lyophilized H2O experiment sample in D2O at time scalar of 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. A TOCSY spectrum was recorded after 8 h of exchanging. Spectra were processed and viewed using the software XWINNMR (Bruker) or Felix 98.0 (Biosym Technologies) on the O2 work station (Silicon Graphics). All data were zero-filled to produce a 2K × 4K real matrix to COSY, DQF-COSY and NOESY or 1K × 2K to TOCSY. Before Fourier transformation, sine bell or sine bell square window functions were used with a phase shift of π/2. Distance constraints were derived from the intensities of cross-peaks in NOESY spectra with mixing times of 100 or 200 ms. Observed NOE data were classified into strong, medium, and weak, corresponding to upper bound interproton distance restraints of 2.7, 3.5, and 5.0 Å, respectively. Lower distance bounds were taken, and the sum of the van der Waals radii of proton was 1.8 Å. Pseudo-atom corrections were applied to non-stereo measurements specifically assigned to methyl and methylene protons according to the method of Wüthrich (23Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Google Scholar). Backbone dihedral constraints were derived from3JHNHα coupling constants measured from either one-dimensional NMR spectra or the anti-phase cross-peak splitting in a high digital resolution DQF-COSY spectrum. 14 φ dihedral angles were restrained to −120 ± 40 (degree) for a3JHNHα ≥ 8 Hz and −65 ± 25 (degree) for 3JHNHα ≤ 5.5 Hz. HWTX-IV contains 6 cysteine residues paired as Cys-2–Cys-17, Cys-9–Cys-24, and Cys-16–Cys-31, assigned by partial reduction and sequence analysis. Nine additional fake distance constraints were added to define the three disulfide bonds involved in HWTX-IV. For each disulfide bond, three distance constraints, S(i) − S(j), S(i) − Cβ(j), and S(j) − Cβ(i), were set to 2.02 ± 0.02, 2.99 ± 0.5, and 2.99 ± 0.5 Å, respectively. Irredundant distance constraints derived from NOEs and dihedral constraints derived from 3JHNαcoupling constants have been used to calculate the structure of HWTX-IV by distance geometry and simulated annealing calculation with the program XPLOR (24Brünger A.T. X-PLOR Manual, Version 3.1. Yale University, New Haven, CT1992Google Scholar). HWTX-IV was purified by a combined use of ion-exchange HPLC and reverse-phase HPLC as described before (Fig. 1). Its molecular mass is 4107.5 Da, read from its mass spectrometry. The amino acid sequence of HWTX-IV, as shown in Fig. 4, is composed of 35 amino acids residues including six cysteine residues.Figure 4Comparison of amino acid sequence of HWTX-IV with HWTX-I and previously known conotoxins (CTX) blocking at site I (O = 4-trans-l-hydroxyproline. The proposed key residues important for their functions are displayed in the frame boxes. Conotoxin GS (38Hill J.M. Alewood P.F. Craik D.J. Structure. 1997; 5: 571-583Google Scholar, 39Yanagawa Y. Abe T. Satake M. Odani S. Suzuki J. Ishikawa K. Biochemistry. 1988; 27: 6256-6262Google Scholar), μ-conotoxin PIIIA (6Shon K.J. Oliver M.B. 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: 4473-4481Google Scholar), μ-conotoxin GIIIA (7Chahine M. Sirois J. Marcotte P. Chen L.Q. Kallen R.G. Biophys. J. 1998; 75: 236-246Google Scholar, 35Li R.A. Ennis I.L. French R.J. Dudley Jr., S.C. Tomaselli G.F. Marbàn E. J. Biol. Chem. 2001; 276: 11072-11077Google Scholar, 36Sato K. Ishida Y. Wakamatsu K. Kato R. Honda H. Ohizumi Y. Nakamura H. Ohya M. Lancelin J.M. Kohda D. Inagaki F. J. Biol. Chem. 1991; 266: 16989-16991Google Scholar), and μ-conotoxin GIIIB (8Li R.A. Ennis I.L. Vélez P. Tomaselli G.F. Marbàn E. J. Biol. Chem. 2000; 275: 27551-27558Google Scholar, 37Hill J.M. Alewood P.F. Craik D.J. Biochemistry. 1996; 35: 8824-8835Google Scholar) block at site I. HWTX-I was suggested to be an N-type calcium channel inhibitor (33Peng K. Chen X. Liang S. Toxicon. 2001; 39: 491-498Google Scholar). Like HWTX-IV and conotoxin GS, it adopts a 1–4, 2–5, 3–6 disulfide pattern and cystine knot motif (32Zhang D. Liang S. J. Protein Chem. 1993; 12: 735-740Google Scholar).View Large Image Figure ViewerDownload (PPT) Fig.2 shows the HPLC separation of the mixture obtained from the partial reduction under controlled reducing conditions. Five chromatographic peaks contain intact peptide and partially reduced intermediates, as determined by MALDI-TOF analysis. The main peak (marked 1) represents intact HWTX-IV, whose observed MALDI mass is the same as the calculated average mass of native HWTX-IV. The mass of peak 2 has 2 Da more in comparison with that of native HWTX-IV, indicating that peak 2 has one of the three disulfide bonds reduced. Peak 3 and peak 4 represent species that are each 4 Da heavier than intact peptide, suggesting two reduced disulfide bonds. Peak 5 represents completely reduced peptide, whose mass is 6 Da heavier than intact HWTX-IV. Peaks 2–4 were collected and alkalized by adding iodoacetamide before further purification by analytical reverse-phase HPLC. There is 58-Da shift from the original molecular mass after the alkyl group was added to single free thiol upon alkylation. The masses of the three alkalized intermediates determined by MALDI-TOF correspond to the above molecular mass results very well (data not shown). In Fig. 3 A, Pth-CM-Cys signal was observed in the chromatograms at the 2nd and 17th cycles after Edman degradation of alkalized peak 1, whereas no Pth-CM-Cys signals were shown at other cysteine cycles. The above result indicates that the only reduced disulfide bond is Cys-2–Cys-17. When sequencing alkalized peak 3, Pth-CM-Cys signals were just observed in the chromatograms at the 2nd, 16th, 17th, and 31st cycles in the HPLC profiles of cysteine cycles (Fig. 3 B), indicating that Cys-9 is still linked to Cys-24 by a disulfide bond. For alkalized peak 4, Pth-CM-Cys signals were just observed at the 2nd, 9th, 17th, and 24th sequencing cycles, as showed in Fig.3 C. The data indicate that the remaining disulfide bond is Cys-16–Cys-31. All these results indicate that the disulfide linkage of HWTX-IV is Cys-2–Cys-17, Cys-9–Cys-24, and Cys-16–Cys-31, adopting a 1–4, 2–5, 3–6 disulfide pattern. The amino acid sequence of HWTX-IV is shown in Fig. 4. It is widely accepted that two different types of VGSCs exist in rat DRG neurons. TTX-S sodium currents activate and inactivate quickly, whereas TTX-R sodium currents activate and inactivate slowly. TTX at a dose of around 200 nm can suppress TTX-S sodium currents, but TTX-R sodium currents remain almost unchanged at that dose. When the membrane was held near its resting potential at −80 mV, TTX-S sodium currents were much more sensitive to the blocking action of HWTX-IV than TTX-R currents. HWTX-IV at a dose of 10 nm blocked 15–55% of the TTX-S sodium currents (Figs.5 a and 7), whereas TTX-R sodium currents were left intact after the application of HWTX-IV at a concentration up to 100 nm (Fig. 5 b). The effects of HWTX-IV on the current-voltage relationship are illustrated in Fig. 6. As can be seen from the current-voltage curve, HWTX-IV blocked TTX-S sodium currents to the same degree in the entire membrane potential range (Fig. 6,a–c). No significant shift in current-voltage relationship was observed. It seemed that HWTX-IV had no significant effect on TTX-R sodium currents (Fig. 6, d–f). The inhibition of HWTX-IV on TTX-S sodium currents was dose-dependent, and the IC50 estimated from data collected from six to eight DRG neurons was about 30 nm (Fig.7), which indicates a rather striking potency among all known VGSC antagonists.Figure 7Dose-dependent inhibition of HWTX-IV on TTX-S sodium current. Currents were elicited by 20-ms voltage steps to −20 mV. Cells with a rundown of sodium current above 5% in 3 min were excluded from further statistics. Data points (mean ± S.E., six to eight cells per point) show currents relative to control current amplitudes. The block was determined after toxin had been applied for >1 min. The inset shows an illustration of the gradual inhibition of HWTX-IV on peak amplitude of TTX-S current in which peaks 1–6 represent the effect of HWTX-IV at doses from 0.1 nm to 10 μm with a 10-fold increment.View Large Image Figure ViewerDownload (PPT)Figure 6Effects of HWTX-IV on I-V relationships of TTX-S and TTX-R sodium currents. TTX-S (a andb) and TTX-R (d and e) sodium currents were recorded before and after the application of 100 nmHWTX-IV. Currents were elicited by 20-ms test pulses from a holding potential of −80 mV to variant potentials from −70 mV to 70 mV with an increment of 10 mV. c and f, alterations in I-V relationships resulted from the application of HWTX-IV for TTX-S and TTX-R sodium currents, respectively.View Large Image Figure ViewerDownload (PPT) Some peptide neurotoxins such as α-scorpion toxins (5Possani L.D. Becerril B. Delepierre M. Tytgat J. Eur. J. Biochem. 1999; 264: 287-300Google Scholar) and funnel-web spider toxins (26Nicholson G.M. Willow M. Howden M.E.H. Narahashi T. Eur. J. Physiol. 1994; 428: 400-409Google Scholar, 27Nicholson G.M. Little M.J. Tyler M. Narahashi T. Toxicon. 1996; 34: 1443-1453Google Scholar, 28Nicholson G.M. Walsh R. Little M.J. Tyler M. Eur. J. Physiol. 1998; 436: 117-126Google Scholar) that interact with a variety of receptor sites on the VGSC can produce repetitive firing of nerves. This symptom is due to toxin-induced alteration in activation and/or inactivation kinetics. Sodium channels are maintained in the open state, and thus the prolonged depolarizing post-potential results in repetitive activity. Unlike these toxins, HWTX-IV shows no effect on the activation and inactivation kinetics of both TTX-S and TTX-R VGSCs. Its action on TTX-S current is very similar to that of TTX. Thus it is reasonable to take HWTX-IV as a site I toxin, although we have not conducted an isotope-labeled toxin binding assay to test whether it shares the same binding site with TTX. The sequence-specific assignment of proton resonances was performed according to standard procedures developed by Wüthrich (23Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Google Scholar). Spin systems for methyl-containing residues such as Leu-3, Ile-5, Ala-8, Leu-22, Val-23, Thr-28, and Ile-35 were identified through the combined analysis of DQF-COSY, TOCSY, and NOESY spectra. They were used as the start points for the sequential assignment process. The spin system of residue Pro-11 was realized by the observation of strong NOE cross peaks between the α proton of Asn-10 and the δ proton of Pro-11, which also suggests that residue Pro-11 in HWTX-IV takes the trans configuration. All backbone and more than 95% of side chain proton resonances belonging to spin systems for each of the 35 amino acid residues were assigned. Fig. 8 shows the sequential dαN(i,i+1) connectivities on the CαH-NH fingerprint region of the NOESY spectrum with a mixing time of 200 ms. Table I shows the summary of the chemical shifts of proton resonances of HWTX-IV.Table IChemical shifts of the assigned 1H-NMR resonances of HWTX-IVResidueNHCαHCβHOthersE14.1752.13, 2.15C3H2 2.22, 2.45C28.745.063.25, 3.35L38.724.431.42, 1.78CγH 1.60; CδH3 0.89, 1.00E47.8254.141.82, 2.26CγH2 2.32, 2.41I57.893.271.40CγH2 0.06, 0.59; CγH3 0.74; CδH3 0.31F68.603.802.94, 3.20C2, 6 H 6.60; C3, 5H 7.27K77.834.421.82, 2.15CγH2 1.33, 1.61; CδH2 1.71; CɛH2 3.04A88.344.871.57C98.394.883.02, 3.12N109.135.182.80, 3.04NδH2 7.13, 7.78P113.951.83, 2.10CγH2 2.02; CδH2 3.88S127.824.443.84, 3.95N137.634.672.62, 2.69NδH2 6.85, 7.55D148.674.132.53, 3.04Q158.084.641.80, 2.75CγH2 2.36, 2.44; NɛH2 6.86, 7.11C169.314.952.66, 2.76C179.414.562.78, 3.19K188.984.261.94CγH2 1.63; CδH2 1.73; CɛH2 3.10S198.994.283.96, 4.04S206.784.713.68, 4.16K218.033.872.00, 2.20CγH21.38; CδH2 1.72; CɛH2 3.03L226.975.180.85, 1.77CγH 1.44; CδH3 0.35V239.164.351.89CγH3 0.80, 0.88C249.674.572.56, 3.15S257.724.403.81, 4.03R269.213.881.85CγH2 1.66, 1.77; CδH2 3.23; NɛH2 7.45K277.944.171.75CγH2 1.30, 1.42; CδH2 1.64; CɛH2 2.96T287.493.952.56CγH3 0.62R298.003.812.37, 2.06CγH2 1.30, 1.45; CδH2 3.24; NɛH2 7.33W307.135.623.20, 2.80C2H 7.04; C4H 7.63; C5H 7.22 C6H 7.32; C7H 7.60; N1H 10.55C318.574.933.19, 2.57K329.534.801.95CγH2 1.45, 1.59; CδH2 1.74; CɛH2 3.03Y338.264.723.07, 2.81C2, 6H 6.82; C3, 5H 7.17Q348.774.251.82, 2.20CγH2 1.97, 2.24; NɛH2 6.88, 7.43I358.074.081.83CγH2 1.21, 1.50; CγH3 0.95; CδH3 0.94 Open table in a new tab 527 irredundant distance constraints derived from NOEs and 14 dihedral constraints derived from3JHNα coupling constants had been used to calculate the structure of HWTX-IV by distance geometry and simulated annealing calculation with the program XPLOR. The initial structures were refined by two rounds of simulated annealing with force constants 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2 for NOE distance and dihedral angel constraints, respectively. An ensemble of 20 structures with lower energy and better Ramachandran plots were chosen to represent the three-dimensional solution fold of HWTX-IV. NOE violations of all of these 20 structures are less than 0.3 Å, and dihedral violations are less than 2 degrees. The 20 structures exhibit no significant deviation from ideal covalent geometry, satisfy the experiment constraints with minimal violations, and have good non-bonded contacts as evidenced by the low values of the mean Lennard-Jones potential. Structural statistics are shown in Table II. Analysis of the family of 20 structures using the program PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Google Scholar) reveals that 81.6% of all the non-Pro/Gly residues lie in
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