BmP09, a “Long Chain” Scorpion Peptide Blocker of BK Channels
2005; Elsevier BV; Volume: 280; Issue: 15 Linguagem: Inglês
10.1074/jbc.m412735200
ISSN1083-351X
AutoresJing Yao, Xiang Chen, Hui Li, Yang Zhou, Lijun Yao, Gong Wu, Xiaoke Chen, Naixia Zhang, Zhuan Zhou, Tao Xu, Houming Wu, Jiuping Ding,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoA novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na+ channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na+ channel blocker toaK+ channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca2+- and voltage-dependent K+ channels (BK) but not Na+ channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 α-subunits in Xenopus oocytes. The half-maximum concentration EC50 of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced ∼70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic π -π interactions around the center. A novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na+ channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na+ channel blocker toaK+ channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca2+- and voltage-dependent K+ channels (BK) but not Na+ channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 α-subunits in Xenopus oocytes. The half-maximum concentration EC50 of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced ∼70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic π -π interactions around the center. Large conductance Ca2+- and voltage-dependent potassium (MaxiK, BK) channels are thought to play a primary role in a link between the membrane potential and the cellular calcium homeostasis. BK channels are very abundant in many tissues from pancreas to smooth muscle to brain (1Schreiber M. Salkoff L. Biophys. J. 1997; 73: 1355-1363Abstract Full Text PDF PubMed Scopus (341) Google Scholar). Natural toxins are among the most potent and important tools for studying the functions and structure of ion channels. Various species such as the sea anemone, snakes, cone snails, spiders, and scorpions possess ion channel toxins in their venom (2Goudet C. Chi C.W. Tytgat J. Toxicon. 2002; 40: 1239-1258Crossref PubMed Scopus (236) Google Scholar). κ conotoxin BtX (κ-BtX) coming from the venom of a worm-hunting cone snail enhances the currents of BK (3Fan C.X. Chen X.K. Zhang C. Wang L.X. Duan K.L. He L.L. Cao Y. Liu S.Y. Zhong M.N. Ulens C. Tytgat J. Chen J.S. Chi C.W. Zhou Z. J. Biol. Chem. 2003; 278: 12624-12633Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Another toxin from the medicine herb dehydrosoyasaponin-1 (DHS-1) also increases the BK currents when the α-subunit coexists with its β-subunit (4McManus O.B. Harris G.H. Giangiacomo K.M. Feigenbaum P. Reuben J.P. Addy M.E. Burka J.F. Kaczorowski G.J. Garcia M.L. Biochemistry. 1993; 32: 6128-6133Crossref PubMed Scopus (181) Google Scholar). Scorpion venoms are rich sources of fascinating neurotoxins, which bond with high affinity and specificity to various ion channels and thus widely serve as useful tools in probing the protein mapping of ion channels and clarifying the molecular mechanism involved in the signal transmission and channel gating. Some of the peptidyl scorpion toxins such as Charybdotoxin (ChTX), 1The abbreviations used are: ChTX, Charybdotoxin; MACCs, mouse adrenal medulla chromaffin cells; MS, mass spectrometry; ESI-MS, electrospray ionization-mass spectrometry; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TEA, tetraethylammonium; HPLC, high pressure liquid chromatography; BmK, B. martensi Karsch. Iberiotoxin, and Slotoxin also block the BK currents encoded by both the Slo1 α-subunits and the β-subunits but with a higher EC50 (5Kaczorowski G.J. Knaus H.G. Leonard R.J. McManus O.B. Garcia M.L. J. Bioenerg. Biomembr. 1996; 28: 255-267Crossref PubMed Scopus (266) Google Scholar, 6Garcia-Valdes J. Zamudio F.Z. Toro L. Possani L.D. Possan L.D. FEBS Lett. 2001; 505: 369-373Crossref PubMed Scopus (51) Google Scholar). Those toxins have in common very poor reversibility, which makes it difficult to study the functions of BK currents, especially in current clamp experiments, even though this property is often used to identify the existence of β-subunits (7Xia X.M. Ding J.P. Lingle C.J. J. Neurosci. 1999; 19: 5255-5264Crossref PubMed Google Scholar, 8Wallner M. Meera P. Toro L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4137-4142Crossref PubMed Scopus (325) Google Scholar, 9Meera P. Wallner M. Toro L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5562-5567Crossref PubMed Scopus (336) Google Scholar). Recently, Xu et al. (10Xu C.Q. Brône B. Wicher D. Bozkurt Ö. Lu W.Y. Huys I. Han Y.H. Tytgat J. Van Kerkhove E. Chi C.W. J. Biol. Chem. 2004; 279: 34562-34569Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) found another scorpion toxin BmBKTx1 that blocks pSlo (82 nm) and dSlo (194 nm), but not hSlo. According to its specific characteristics, BmBKTx1 can be used to identify different subunits involved in BK channels. So far more than 120 peptide modulators of ion channels have been isolated from scorpion venoms. Most of the scorpion toxins blocking K+ channels (KTx) are short peptides (22–43 amino acids residues) with a well conserved three-dimensional structure stabilized by three or four disulfide bridges (11Rodriguez de la Vega R.C. Possani L.D. Toxicon. 2004; 43: 865-875Crossref PubMed Scopus (307) Google Scholar). The Chinese scorpion Buthus martensi Karsch (BmK) has been used as traditional medicine in China for more than 1000 years, especially for treatments in neural diseases such as apoplexy, hemiplegia, and facial paralysis (12Li H.M. Wang D.C. Zeng Z.H. Jin L. Hu R.Q. J. Mol. Biol. 1996; 261: 415-431Crossref PubMed Scopus (91) Google Scholar). Indeed, over the past decade, more than 70 different peptides, toxins, or homologues have been isolated. Among them, 14 short chain peptides are associated with the K+ channel toxin family; 51 long chain peptides are related to the Na+ channel toxin family, and only one long chain peptide is identified as a blocker of voltage-dependent K+ channels (KV) (2Goudet C. Chi C.W. Tytgat J. Toxicon. 2002; 40: 1239-1258Crossref PubMed Scopus (236) Google Scholar). In our previous paper (13Wu H.M. Wu G. Huang X.L. Jiang S.K. Pure Appl. Chem. 1999; 71: 1157-1162Crossref Scopus (21) Google Scholar), a systematic isolation has been achieved, and 11 short chain peptides have been characterized from the venom of the Chinese scorpion BmK. Solution structures of some short chain scorpion toxins (less than 40 amino acid residues long) have been described previously (14Zhang N. Li M. Chen X. Wang Y. Wu G. Hu G. Wu H. Proteins. 2004; 55: 835-845Crossref PubMed Scopus (11) Google Scholar, 15Zhang N. Chen X. Li M. Cao C. Wang Y. Wu G. Hu G. Wu H. Biochemistry. 2004; 43: 12469-12476Crossref PubMed Scopus (12) Google Scholar, 16Ji Y.H. Wang W.X. Ye J.G. He L.L. Li Y.J. Yan Y.P. Zhou Z. J. Neurochem. 2003; 84: 325-335Crossref PubMed Scopus (27) Google Scholar). Here we report on the purification, characterization, and sequence determination of a novel BK potassium channel blocker BmP09, which is composed of a long chain (66 amino acid residues long) with four disulfide bridges. By assaying the effects of BmP09 on voltage-gated channels in MACCs and on BK channels (mSlo) expressed in Xenopus oocytes, we found that BmP09 was a better specific blocker of BK channels encoded by mSlo α-subunits with perfect reversibility; in other words, it only took less than 5 s for a full recovery. Compared with the Charybdotoxin (ChTX), the superior selectivity and reversibility makes BmP09 a better tool for the structural and functional studies on BK channels. Crude venom was collected by electrical stimulation of the telson of scorpion BmK bred in captivity in Henan Province, China. The peptide was purified as described previously (13Wu H.M. Wu G. Huang X.L. Jiang S.K. Pure Appl. Chem. 1999; 71: 1157-1162Crossref Scopus (21) Google Scholar). Lyophilized crude venom was dissolved in NH4HCO3 buffer (50 mm, pH 8.5) and centrifuged at 4000 × g for 15 min. The supernatant was loaded onto a Sephadex G-50 column (2.5 × 150 cm, Amersham Biosciences), which was equilibrated and eluted with the same buffer (Fig. 1A). Fraction III from the Sephadex G-50 column was loaded onto a Mono S cation exchange column (HR 5/5, Amersham Biosciences), eluted with a step gradient of solution A to solution B at pH 5.0 (Fig. 1B). Solution A contained NaAc (20 mm), and solution B contained NaAc (20 mm) and NaCl (1 m). It was followed by similar separation on another Sephadex G-50 column (Fig. 1C). The final purification of BmP09 (G3512) was performed by using a reversephase HPLC column (C18 column, 4.6 × 250 mm, 5 μm bead size, Alltech) eluted with a linear gradient from solution C to 50% solution D in solution C at a flow of 1 ml/min. Solution C contained CH3CN (10%) and trifluoroacetic acid (0.1%) in H2O, and solution D contained H2O (20%) and trifluoroacetic acid (0.1%) in CH3CN (Fig. 1D). The molecular weight of BmP09 was measured using a LCMS-2010A ESI-MS instrument (Shimadazu, Japan). Amino acid analysis was performed on a Beckman 6300 apparatus (Beckman) after hydrolysis of the sample in HCl (6 m) under vacuum at 110 °C for 18 h. The N-terminal sequence of BmP09 was achieved by Edman degradation using a Beckman LF3200 protein-peptide sequencer. Because of the existence of disulfide bonds in the sequence, the sample of BmP09 was subjected to dithiothreitol reduction and iodoacetamide derivatization before MS analysis. Peptide BmP09 was reduced with a 250-fold molar excess of dithiothreitol in 0.25 m Tris-HCl buffer (pH 8.5) containing 6 m guanidine HCl and 4 mm EDTA. Reduction was carried out in the dark under nitrogen at 37 °C for 1 h. Free thiols were alkylated by addition of a 500-fold molar excess of iodoacetamide held at room temperature for 30 min in the dark. The sample was load onto a 10% Tris-Tricine gel for SDS-PAGE to remove reagents. Peptide mapping study was performed on S-alkylated BmP09 as the l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin took place in situ in the gel after electrophoresis. AutoMS-Fit automation software was used to analyze the masses of peptide fragments obtained by digestion. A sample of BmP09 (100 μl, 50 ng/μl) was digested with carboxypeptidase P and Y (Sigma, 0.2 ng/μl, enzyme/substrate ratio was 1:500, w/w) in aqueous solution at 25 °C. Every 6.0-μl digested sample was taken out at 20, 40, and 60 s, 2, 5, 10, 15, 20, 30, and 60 min, and 2 and 4 h after the onset of incubation, and each aliquot was acidified with 1% trifluoroacetic acid and lyophilized immediately. Each lyophilized sample was mixed at the ratio of 1:1 with a 4 mg/ml solution of α-cyano-4-hydroxycinnamic acid (CH3OH:H2O, 0.1% trifluoroacetic acid) and performed by a matrix-assisted laser desorption ionization time-of-flight MS spectrum using a Voyager-DE STR (Applied Biosystems) operated in the reflection mode with time lag focusing. Chromaffin Cell Preparation—Based on several early studies (17Neely A. Lingle C.J. J. Physiol. (Lond.). 1992; 453: 97-131Crossref Scopus (90) Google Scholar, 18Ding J.P. Lingle C.J. Biophys. J. 2002; 82: 2448-2465Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), MACCs were isolated and maintained as described previously. To compare the effects of BmP09 and BmK AS-1 in the patch clamp experiments, we used rat adrenal chromaffin cells (3Fan C.X. Chen X.K. Zhang C. Wang L.X. Duan K.L. He L.L. Cao Y. Liu S.Y. Zhong M.N. Ulens C. Tytgat J. Chen J.S. Chi C.W. Zhou Z. J. Biol. Chem. 2003; 278: 12624-12633Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Ji Y.H. Wang W.X. Ye J.G. He L.L. Li Y.J. Yan Y.P. Zhou Z. J. Neurochem. 2003; 84: 325-335Crossref PubMed Scopus (27) Google Scholar, 19Wu J.J. He L.L. Zhou Z. Chi C.W. Biochemistry. 2002; 41: 2844-2849Crossref PubMed Scopus (29) Google Scholar). Dispersion of chromaffin cells was typically done on adrenal medullas from two or three Wistar mice (20–30 g) ∼1 month of age. Cells were cultured with Dulbecco's modified Eagle's medium in a standard CO2 incubator at 37 °C, and currents were recorded 1–5 days after plating. Mutagenesis—Point mutations of mSlo, F266L and F266A, were produced by using QuikChange protocol (Stratagene). In brief, PCRs were performed by using the wild-type mSlo as a template and a pair of complementary mutagenesis primers (F266L, 5′-CAGGGGACCCATGGGAAAATCTTCAAAACAACCAGGCACTTAC-3′ and 5′-GTAAGTGCCTGGTTGTTTTGAAGATTTTCCCATGGGTCCCCTG-3′; F266A, 5′-CAGGGGACCCATGGGAAAATGCTCAAAACAACCAGGCACTTACG-3′ and 5′-CGTAAGTGCCTGGTTGTTTTGAGCATTTTCCCATGGGTCCCCTG-3′). The PCR mixture was then cut with the enzyme DpnI to digest the template wild-type mSlo. After DpnI digestion, the PCR product was transformed into competent bacterial cells to amplify the mutant plasmid of mSlo. Both mutant constructs were verified by sequencing. Expression in Xenopus Oocytes—Methods of expression in stage V–VI Xenopus oocytes were as described previously (7Xia X.M. Ding J.P. Lingle C.J. J. Neurosci. 1999; 19: 5255-5264Crossref PubMed Google Scholar). Oocytes were defolliculated by treatment with 2 mg/ml collagenase I (Sigma) in zero calcium ND-96 solution. Between 2 and 24 h after defolliculation, 1–2 ng (mSlo) cRNA (a gift of Dr. Christopher Lingle, Washington University, St. Louis, MO) were injected into Xenopus oocytes using a Drummond Nanoject II (Drummond Scientific Co.). After injection, oocytes were then incubated in ND-96 solution at 18 °C. Currents were recorded 2–7 days after RNA injection. ND-96 solution (pH 7.5) containing the following concentrations (in mm), 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 2.5 sodium pyruvate, and 10 H+-HEPES, were supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (only for incubation). Solutions—For MACCs, the normal extracellular solution contained the following (in mm): 150 NaCl, 5.4 KCl, 1.8 CaCl2, 2 MgCl2, and 10 H+-HEPES (pH 7.4) titrated with NaOH. For whole-cell recording, the standard pipette solution contained the following (in mm): 130 potassium glutamine, 30 KCl, 0.1 EGTA, 10 H+-HEPES, 0.05 GTP, and 2 MgATP (pH 7.4). The "high tetraethylammonium chloride (TEA)" solution was the same as the standard bath solution except that the 20 mm NaCl was replaced by 20 mm TEA. The "high Cs+" solution contained the following (in mm): 130 cesium glutamine, 30 CsCl, 0.1 EGTA, 10 H+-HEPES, 0.05 GTP, and 2 MgATP (pH 7.4). To record Na+ currents, the high TEA solution was used as a bath solution, and pipettes were backfilled with high Cs+ solution. For Ca2+ current recording solutions, the bath solution contained the following (in mm): 160 TEA, 5 BaCl2, 10 H+-HEPES, and 0.1 EGTA, with pH adjusted to 7.4 with tetraethylammonium hydroxide, and the pipette solution was the high Cs+ solution. For oocytes, during recordings, oocytes were bathed in the solution containing the following (in mm): 160 MeSO3K, 10 H+-HEPES, and 2 MgCl2, adjusted to pH 7.0 with MeSO3H. Pipettes were filled with a solution containing the following (in mm): 160 MeSO3K, 10 H+-HEPES, and 5 N-hydroxyethylenediaminetriacetic acid (HEDTA) with added Ca2+ to make 10 μm free Ca2+, as defined by the EGTAETC program (E. McCleskey, Vollum Institute, Portland, OR), with the pH adjusted to 7.0. All of the chemicals were obtained from Sigma. Patch Clamp Recording from Single Cells—Patch pipettes pulled from borosilicate glass capillaries had resistances of 2–6 megohms when filled with internal solution. An outside-out patch was obtained by excising the patch from a cell in the whole-cell configuration. Experiments were performed and recorded using an EPC-9 patch clamp amplifier and PULSE software (HEKA Electronics, Germany). Currents were typically digitized at 20 kHz. Macroscopic records were filtered at 2.9 kHz during digitization. Single-channel records were filtered at 10 kHz. During recording, drugs and control/wash solutions were puffed locally onto the cell via a puffer pipette containing seven solution channels. The tip (300 μm diameter) of the puffer pipette was located about 120 μm from the cell. As determined by the conductance tests, the solution around a cell under study was fully controlled by the application solution with a flow rate of 100 μl/min or greater. All pharmacological experiments met this criterion. All these experiments were done at room temperature (22–25 °C). Data were analyzed with IGOR (Wavemetrics, Lake Oswego, OR), Clampfit (Axon Instruments, Inc.), SigmaPlot (SPSS Inc.), and QUB (State University of New York, Buffalo) software. Unless stated otherwise, the data are presented as means ± S.E.; significance was tested by Student's t test, and differences in the mean values were considered significant at a probability of ≤0.05. Dose-response curve for the percent block of BK currents was drawn according to the Hill equation I = Im/(1 + ([toxin]/EC50)n), where Im is maximum blocking percentage of BK currents, and [toxin] is the concentration of BmP09. EC50 and n denote the toxin concentration of half-maximal effect and the Hill coefficient, respectively. The model of BK channel (20Morita T. Hanaoka K. Morales M.M. Montrose-Rafizadeh C. Guggino W.B. Am. J. Physiol. 1997; 273: F615-F624PubMed Google Scholar) was generated by homology modeling on the basis of the crystal structure of the bacterial KcsA channel (Protein Data Bank code 1BL8) (21Doyle D.A. Morais C.J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5770) Google Scholar), using the software SYBYL6.3 (Tripos Associates). The sequence alignment between KcsA and BK channel was obtained using the same criteria as those described by Gao and Garcia (22Gao Y.D. Garcia M.L. Proteins. 2003; 52: 146-154Crossref PubMed Scopus (63) Google Scholar). The homology model of BK channel was further subjected to Powell minimization (2000 steps) using Kollman force field. The models of toxin BmP09 and BmK AS-1 (23Lan Z.D. Dai L. Zhuo X.L. Feng J.C. Xu K. Chi C.W. Toxicon. 1999; 37: 815-823Crossref PubMed Scopus (21) Google Scholar) were generated by homology modeling on the basis of the crystal structure of the toxin neurotoxin 2 (Protein Data Bank code 1JZB) (24Cook W.J. Zell A. Watt D.D. Ealick S.E. Protein Sci. 2002; 11: 479-486Crossref PubMed Scopus (20) Google Scholar). The sequence alignment between them is described in Fig. 3. The homology models of toxins BmP09 and BmK AS-1 were further subjected to energy minimization and dynamic simulation. The surface electrostatic distribution analysis indicated that BmP09 preferred association with the entryway of the K+ channel by using the positively charged patch with the side chain of Lys-41 in the center. The program "O" (version 8.0.6) (25Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) was used for the docking experiment. BmP09 was docked manually into the outer entryway of the BK channel model along with the dipole direction. As expected, the mouth of the K+ channel bears a large negative charge, whereas the surface of the toxin BmP09 has a positive charge. The electrostatic potential between the toxin BmP09 and the K+ channel attracted the positively charged toxin to the entryway of the channel. In order to obtain favorable toxinchannel clusters, the toxin molecule was allowed to rotate during the docking process. The most stable cluster with the best fit between the toxin and the K+ channel was used to analyze the contacts between the BmP09 and the BK channel. The BmP09-BK channel cluster docked most favorably was further subjected to energy minimization for 2000 steps to achieve the gradient tolerance 0.05 kcal/(mol Å) using the Powell algorithm and the Kollman force field in the software SYBYL6.3. Molecular dynamics simulation using the Powell algorithm was then carried out for the complex for 100 fs at 300 K. Kollman force field constraints were applied on the backbones of the channel in the region comprising residues His-254 to Val-0278, whereas the remaining part of the channel was kept fixed during the simulation. The structure of the peptide was completely unconstrained. A cut-off distance of 8 Å was used for nonbounded interactions. An integration time step of 0.1 fs was used, and coordinate sets of the trajectory were saved every 2 fs. Every structure obtained from the coordinate sets over the 100 fs of simulation was performed with 500 steps of minimization. Finally, the average structure was energy-minimized with 1000 steps of Powell minimization. Purification of BmP09 —The crude venom was initially separated into four fractions (I–IV) by gel filtration chromatography on a Sephadex G-50 column (Fig. 1A). Separation of the fraction III on a Mono S cation exchange column gave five fractions (Fig. 1B). Among the five fractions, fraction 5 was further separated on another Sephadex G-50 column, and two sub-fractions 351 and 353 were obtained (Fig. 1C). A pure peptide was obtained after the separation of fraction 351 on a reverse-phase HPLC column (Fig. 1D). Primary Sequence of BmP09 —The molecular weight of BmP09 was 7721 Da, as determined by ESI-MS (Fig. 1E). The results of the amino acid analysis (Table I), the N-terminal 14-residue sequence analysis (DNGYLLNKYTGCKI), and peptide mapping studies (Fig. 2) are consistent with those calculated from the mature peptide BmK AS-1 derived from cDNA (GenBank™ accession number AF079061). The difference in molecular weight between the ESI-MS data (7721 Da) and calculated value (7704.8 Da) according to the sequence of BmK AS-1 could be attributed to the oxidation of the C-terminal Met residue (26Vogt W. Free Radic. Biol. Med. 1995; 18: 93-105Crossref PubMed Scopus (793) Google Scholar). The oxidative modification could be validated by the MS analysis of the carboxypeptidase-digested products. Actually, a principal ion with the MS value of 7575 Da was observed under the molecular ion in the MS spectrum of the carboxypeptidase-digested products. The mass difference (147 Da) is well accounted for a Met residue with a sulfoxide group. Therefore, the sequence of BmP09 is the same to that of BmK AS-1, and only differs at the C terminus by an oxidative modification (see Fig. 3).Table IThe amino acid compositions of BmP09Amino acidNo. residuesAsx8.77 (9)Thr1.87 (2)Ser2.06 (3)Glx5.57 (5)Gly5.93 (6)Ala3.06 (3)Val1.56 (1)Met0.85 (1)Ile1.98 (2)Leu5.02 (5)Tyr5.37 (7)Phe1.35 (1)Arg2.04 (2)Lys6.57 (7)Pro1Trp3Cys-Cys3.17 (4)Mh (calculated)7704.81Mh (experimental)7721 Open table in a new tab Effects of BmP09 on Voltage-gated Channels in Chromaffin Cells—MACCs are excitable cells and express variety of voltage-gated channels such as voltage-gated Na+, K+, and Ca2+ channels. They are widely used as a neuronal model for studying the features of channel behaviors and searching the targets of toxins (27Zhou Z. Neher E. J. Physiol. (Lond.). 1993; 469: 245-273Crossref Scopus (356) Google Scholar, 28Zhou Z. Misler S. J. Biol. Chem. 1995; 270: 3498-3505Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 29Fenwick E.M. Marty A. Neher E. J. Physiol. (Lond.). 1982; 331: 599-635Crossref Scopus (632) Google Scholar, 30Elhamdani A. Zhou Z. Artalejo C.R. J. Neurosci. 1998; 18: 6230-6240Crossref PubMed Google Scholar, 31Neely A. Lingle C.J. J. Physiol. (Lond.). 1992; 453: 133-166Crossref Scopus (38) Google Scholar). To study whether the BmP09 affects voltagegated ion channels, we started to screen for its effects on the MACCs. As shown in Fig. 4A, whole-cell currents were elicited by 60-ms voltage ramps from –90 to +100 mV within the normal extracellular solution in the presence and absence of 100 nm BmP09. With the augmentation of the membrane potential by the voltage ramp, inward currents of Na+ and Ca2+ channels first emerged at ∼0 mV, and the outward currents of voltage-gated K+ channels, including both the KV and the KCa channels, started to increase gradually (28Zhou Z. Misler S. J. Biol. Chem. 1995; 270: 3498-3505Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 31Neely A. Lingle C.J. J. Physiol. (Lond.). 1992; 453: 133-166Crossref Scopus (38) Google Scholar). In Fig. 4A, the dark trace shows a 67% reduction of outward maximum currents, by the application of 100 nm BmP09, with a slight increase in inward currents. The reduction of outward currents may result in the slight increase of inward currents. In addition, after removal of BmP09, the current trace indicated in Fig. 4A is almost back to the control level, which hints that the blocking behavior of BmP09 is reversible. Voltage-gated sodium channels play a critical role in the repeated firing of action potentials and propagation in excitable cells (2Goudet C. Chi C.W. Tytgat J. Toxicon. 2002; 40: 1239-1258Crossref PubMed Scopus (236) Google Scholar). Most of the long chain peptides have been proved to be the blockers of Na+ channels such as BmK AS-1 (32Tan Z.Y. Mao X. Xiao H. Zhao Z.Q. Ji Y.H. Neurosci. Lett. 2001; 297: 65-68Crossref PubMed Scopus (33) Google Scholar). However, the traces in Fig. 4B, activated by voltage steps to 0 mV after a prepulse to –90 mV to remove inactivation, are overlaid to emphasize that there is no inhibition on Na+ channels before, during, and after the application of the toxin 100 nm BmP09. In contrast, BmK AS-1 has an inhibitive effect on the Na+ channel in chromaffin cells (32Tan Z.Y. Mao X. Xiao H. Zhao Z.Q. Ji Y.H. Neurosci. Lett. 2001; 297: 65-68Crossref PubMed Scopus (33) Google Scholar) but no effect on the K+ currents (n = 5, data not shown). In chromaffin cells, most types of calcium channels exist, and the L-type calcium channel is clustered with the BK channels (33Prakriya M. Lingle C.J. J. Neurophysiol. 1999; 81: 2267-2278Crossref PubMed Scopus (91) Google Scholar). There were three possibilities for the inhibition of BmP09 on outward K+ currents. The first possibility was directly blocking on Ca2+-activated K+ currents; the second possibility was directly blocking K +V channels; and the third possibility was indirectly blocking on the Ca2+ channels. Therefore, we intended to verify whether BmP09 blocked voltage-gated calcium channels first. In Fig. 4C, Ca2+ currents were elicited by 200 ms of depolarization from a holding potential –70 mV to 0 mV. Three traces of Ca2+ currents are nearly at the same level for 0, 100, and 0 nm BmP09, which suggests that BmP09 has negligible effects on the inward Ca2+ currents. This result also hinted that the BmP09 blocked K +V/BK channels. Selectivity of BmP09 among K+Channels—K+-selective channels with a tremendous diversity are found in probably all the cells. K+ channels, when they are open, set the resting potential, keep fast action potential short, etc. (34Yellen G. Nature. 2002; 419: 35-42Crossref PubMed Scopus (540) Google Scholar). However, we often do not know which types are present in cells, e.g. in MACCs. As we know, there are at least two types of KV channels, i.e. a delayed-rectified channel and an A-type transient channel, and two types of Ca2+-dependent K+ channels (KCa) in MACCs, i.e. a small conductance Ca2+-dependent K+ channel (SK) and a BK channel. It is hard to distinguish their individual types in MACCs, but it is easy to separate KV and KCa channels by applications with alternating calcium concentration in the bath solution. In the Fig. 5, A and B, all traces were activated by 100 ms of depolarization to +80 mV from a holding potential of –70 mV, which was designed to avoid the calcium influx induced by the opening of calcium channels at ∼0 mV. In Fig. 5A, 100 nm BmP09 obviously blocked the "KV" currents within the normal bath solution, i.e. 1.8 mm Ca2+ in the normal saline. But as applied with Ca2+-free
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