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• W2022675045 abstract "The isolation of the peptide inhibitor of M-type K+ current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277–280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K+ channels was also studied. BeKm-1 inhibited hERG1 channels with an IC50 of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.AF276623 The isolation of the peptide inhibitor of M-type K+ current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277–280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K+ channels was also studied. BeKm-1 inhibited hERG1 channels with an IC50 of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels. AF276623 ether-a-go-go gene EAG-related EAG-like KQT-like K+ channel IK, SK, high-, intermediate-, small-conductance Ca2+-activated K+ channels rapid amplification of cDNA ends reverse transcriptase open reading frame base pair(s) high performance liquid chromatography polymerase chain reaction K+ channels comprise a large, diverse group of integral membrane proteins, which are found in all cells. K+ channels are involved in neuroendocrine secretion, cell volume regulation, electrolyte balance, and regulation of levels of excitability. K+ channels have been classified according to their biophysical and pharmacological characteristic (2Christie M.J. Clin. Exp. Pharmacol. Physiol. 1995; 22: 944-951Crossref PubMed Scopus (52) Google Scholar). Recent molecular cloning of a large number of K+ channels has resulted in a classification into structural classes including the 2-transmembrane K+ channels (inward rectifiers), the 4-transmembrane K+ channels (2P domain channels), and the 6-transmembrane K+ channels (voltage-dependent, Ca2+-activated, or cyclic nucleotide gated) (seee.g. Ref. 3Coetzee W.A. Amarillo Y. Chiu J. Chow A. Lau D. McCormack T. Moreno H. Nadal M.S. Ozaita Pountney A. Saganich M. Vega-Saenz de Miera E. Rudy B. Ann. N. Y. Acad. Sci. 1999; 868: 233-285Crossref PubMed Scopus (982) Google Scholar). Natural toxins are useful probes for evaluating the involvement of K+ channels in cell activity, and for investigating K+ channel structure and localization. In recent years, peptide toxins that block various K+ channels with high affinity have been purified from diverse animal venoms (see Refs. 4Rowan E.G. Harvey A.L. Braz. J. Med. Biol. Res. 1996; 29: 1765-1780PubMed Google Scholar, 5Miller C. Neuron. 1995; 15: 5-10Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 6Harvey A.L. Vatanpour H. Rowan E.G. Pinkasfeld S. Vita C. Menez A. Martineauclaire M.F. Toxicon. 1995; 33: 425-436Crossref PubMed Scopus (29) Google Scholarfor review). The largest group of K+ channel peptide inhibitors is the family of neurotoxic peptides found in scorpion venoms. These peptides block, in nanomolar concentrations, both voltage-gated and Ca2+-activated K+ channels in a wide variety of cell types, and generally contain 31–40 amino acid residues cross-linked by three or four disulfide bridges (5Miller C. Neuron. 1995; 15: 5-10Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 7Olamendi-Portugal T. Gomez-Lagunas F. Gurrola G. Possani L.D. Biochem. J. 1996; 315: 977-981Crossref PubMed Scopus (80) Google Scholar, 8Kharrat R. Marbouk K. Crest M. Darbon H. Oughideni R. Martineauclaire M.F. Jacquet G. Elayeb M. Vanrietschoten J. Rochat H. Sabatier J.M. Eur. J. Biochem. 1996; 242: 491-498Crossref PubMed Scopus (96) Google Scholar). These toxins have been intensively studied using biochemical, structural, and electrophysiological methods. Both natural and mutated recombinant scorpion short chain toxins have also been used to identify the pore region (9MacKinnon R. Miller C. Science. 1989; 245: 1382-1385Crossref PubMed Scopus (298) Google Scholar, 10MacKinnon R. Heginbotham L. Abramson T. Neuron. 1990; 5: 767-771Abstract Full Text PDF PubMed Scopus (189) Google Scholar), determine the subunit stoichiometry of K+channels (11MacKinnon R. Nature. 1991; 350: 232-235Crossref PubMed Scopus (768) Google Scholar), and elucidate the topology of the extracellular face of the channel pore (12Hidalgo P. MacKinnon R. Science. 1995; 268: 307-310Crossref PubMed Scopus (425) Google Scholar, 13Naranjo D. Miller C. Neuron. 1996; 16: 123-130Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Aiyar J. Rizzi J.P. Guttman G.A. Chandy K.G. J. Biol. Chem. 1996; 271: 31013-31016Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 16Lu Z. MacKinnon R. Biochemistry. 1997; 36: 6936-6940Crossref PubMed Scopus (39) Google Scholar). Despite rapid advances in the molecular biology of K+channels, the subunit composition and the physiological role of several K+ channel subtypes are still unclear. That is why the identification and characterization of ligands that interact specifically with ion channels is critical not only for defining their structural and functional organization, but also to elucidate the contribution of specific ion channels to certain physiological phenomena. A score of years ago Brown and co-workers (17Brown D.A. Adams P.R. Nature. 1980; 283: 673-676Crossref PubMed Scopus (1003) Google Scholar) discovered the M-current, a voltage-dependent potassium current in sympathetic neurons that is suppressed by muscarinic acetylcholinic receptor activity. M-channels open at resting potentials and are slowly activated by membrane depolarization. They play a key role in controlling repetitive firing in many neurons (and hence may be of significance to normal cognitive function, dementia, and epilepsy), and are regulated by a variety of G protein-coupled receptors. The 4-kDa polypeptide BeKm-1 was previously isolated from the venom of the Central Asian scorpion Buthus eupeus, and characterized as the first peptide inhibitor of the M-like current in NG108-15 mouse neuroblastoma × rat glioma cells (1Filippov A.K. Kozlov S.A. Pluzhnikov K.A. Grishin E.V. Brown D.A. FEBS Lett. 1996; 384: 277-280Crossref PubMed Scopus (12) Google Scholar). It was shown that BeKm-1 affected neither transient and delayed rectifier K+ current nor Na+ current. The molecular constituents underlying the native M-current and M-like currents have remained an intriguing puzzle. Recently, the ether-a-go-go gene(EAG)1 K+ channel was suggested to contribute to the mammalian M-channel underlying the native M-current in neurons (18Stansfeld C. Ludwig J. Roeper J. Weseloh R. Brown D. Pongs O. Trends Neurosci. 1997; 20: 13-14Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Later, heteromeric KCNQ2/KCNQ3 channels were demonstrated to contribute to the native M-current (19Wang H.S. Pan Z. Shi W. Brown B.S. Wymore R.S. Cohen I.S. Dixon J.E. McKinnon D. Science. 1998; 282: 1890-1893Crossref PubMed Scopus (1030) Google Scholar). During the last year, the M-like current described in NG108-15 cells has been reported to contain two components: a “fast” and a “slow,” which have been suggested to be carried by KCNQ2/KCNQ3 and channels encoded by the mouse ether-a-go-go-related gene(mERG1), respectively (20Selyanko A.A. Hadley J.K. Wood I.C. Abogadie F.C. Delmas P. Buckley N.J. London B. Brown D.A. J. Neurosci. 1999; 19: 7742-7756Crossref PubMed Google Scholar, 21Meves H. Schwarz J.R. Wulfsen I. Br. J. Pharmacol. 1999; 127: 1213-1223Crossref PubMed Scopus (51) Google Scholar). The study of the action of BeKm-1 on these different K+-channel types might throw light upon the true toxin target in the cell. In heart, both KCNQ1 channels and ERG channels play significant roles in the repolarization of the action potential. Mutations in either channel gene result in a prolongation of the QT-interval on the electrocardiogram and give rise to long QT syndrome. Long QT syndrome is a disorder that may cause syncope and sudden death resulting from episodic ventricular arrhytmias and ventricular fibrillation. Inherited long QT type 2 results from mutaions in the human ERG, originally referred to as hERG. As novel members of the ERG family emerge, the terminology hERG1 seems more appropriate. This paper presents the total amino acid sequence of BeKm-1 deduced from cDNA sequencing and the biological properties of the recombinant BeKm-1 and of the three mutants containing point changes in the COOH-terminal region of the toxin. In the present study, we have investigated the action of recombinant BeKm-1 on native M-like current in NG108-15 cells, and directly on cloned human KCNQ- and hERG1 potassium channels. Native BeKm-1 toxin was purified from the scorpion venom as described previously (1Filippov A.K. Kozlov S.A. Pluzhnikov K.A. Grishin E.V. Brown D.A. FEBS Lett. 1996; 384: 277-280Crossref PubMed Scopus (12) Google Scholar). Disulfide bonds of the purified toxin were reduced with dithiothreitol, and SH-groups were modified with 4-vinylpyridine (22Kawasaki I. Itano H.A. Anal. Biochem. 1972; 48: 546-556Crossref PubMed Scopus (33) Google Scholar). The modified toxin was digested with Staphylococus aureusprotease V8 (Roche Molecular Biochemicals) at 37 °C for 12–14 h in 50 mm ammonium acetate buffer, pH 4.0, at a ratio of 1 μg of enzyme per 25 μg of toxin or with trypsin at 37 °C for 16 h in 50 mm Tris buffer, pH 8.5 (ratio enzyme:toxin 1:20). The digests were fractionated by reverse-phase HPLC on an Ultrasphere ODS column (2 × 250 mm) using an acetonitrile gradient in 0.1% trifluoroacetic acid. The NH2-terminal amino acid sequence of BeKm-1 and its internal peptides were determined by automated Edman degradation using an Applied Biosystems Sequencer (470A protein sequencer) on-line with the phenylthiohydantoin analyzer (120A analyzer). Total RNA was isolated by a guanidinium thiocyanate/phenol chloroform method (23Kiyatkin N. Dulubova I. Grishin E. Eur. J. Biochem. 1993; 213: 121-127Crossref PubMed Scopus (58) Google Scholar) from scorpion venom glands frozen in liquid nitrogen immediately after sacrificing. Poly(A)-rich RNA was prepared from total RNA by two-cycle chromatography on oligo(dT)-cellulose as described (24Aviv H. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 1408-1413Crossref PubMed Scopus (5183) Google Scholar). Reverse transcriptase (RT) reaction was performed with 2 μg of mRNA (pre-heated at 70 °C for 5 min) in a total reaction volume of 20 μl of 1 × RT buffer (U.S. Biochemical). The downstream primer used was the primer RLdT with the sequence 5′-GAGAATTCGGATCCCTGCAGAAGCTTTTTTTTTTTTTTTTT-3′. Other components of the RT reaction were 0.5 mm of each dNTP, 1 unit/μml RNAsin, 100 units of Moloney murine leukemia virus-reverse transcriptase. The mixture was incubated for 1 h at 37 °C. 1 μl of this reaction mixture was used in 50 μl of polymerase chain reaction. PCR was carried out with denaturation for 30 s at 94 °C, annealing for 1 min at 50 °C, and extension for 1 min at 72 °C. Oligonucleotide primers used were sense T1 (codes for the predicted amino acid sequence from Arg1 to Lys6of BeKm-1 with codon degeneracy), 5′-GGAATTCG(G/A/T/C)CC(G/A/T/C)AC(G/A/T/C)GA(C/T)ATAAA(A/G)TG-3′, and antisense RL, 5′-GAGAATTCGGATCCCTGCAGAAGCTT-3′. The 220-bp fragment was gel purified, digested with EcoRI/PstI, and ligated to similarly prepared vector pBluescript SK+ (Stratagene).Escherichia coli MH1 was used for plasmid propagation. The recombinant clones were analyzed with the standard technique (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and sequenced on both strands using the Sanger (26Sanger F. Nisclen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52655) Google Scholar) dideoxynucleotide chain termination method. 5′-RACE was performed according to the anchored PCR technique described (27Loh E.Y. Elliott J.F. Cwirla S. Lanier L.L. Davis M.M. Science. 1989; 243: 217-220Crossref PubMed Scopus (525) Google Scholar). A poly(dG) tail sequence was introduced to the first strand cDNA with terminal deoxynucleotidyl transferase (U. S. Biochemical) in 1 × terminal deoxynucleotidyl transferase buffer with 1 mm dGTP for 15 min at 37 °C; the reaction was stopped by heating at 70 °C for 10 min. Amplification was performed using one specific primer T2 corresponding to the 3′-flanking region of the cDNA of BeKm-1 (5′-GCATTACATACTTTCATTATAAATCTG-3′) and poly(dC) primer C13 (5′-GTGAATTCCTTAACCCCCCCCCCCCC-3′). The denaturation step was at 94 °C for 30 s, the annealing step was at 57 °C for 1 min, and the extension was at 72 °C for 1 min. Amplification was performed for 30 cycles and the product was analyzed. An appropriate size region was cut from the gel and directly reamplified with a mixture of universal primer M13 and M13-C13 (5′-GTAAAACGACGGCCAGTGAATTCCTTAACCCCCCCCCCCCC-3′) as the primers for the poly(dG) end at the ratio of 10:1 and the T2 primer. Amplification was performed in the same conditions for 12 cycles. Two PCR bands ∼250 and 300 bp were reamplified separately with M13 and the T3 primer, corresponding to C terminus of BeKm-1 (5′-CCATTCACGCACCTTCCATTAGTC-3′). The PCR products were cloned using AdvanTAgeTM PCR Cloning Kit (CLONTECH) according to the manufacturer's procedure and sequenced. The following nucleotide probes were used for mutagenesis: R27K, 5′-CAAAAACCATT-CACGCACTTTCCATTAGTCTTCCC-3′; F32K, 5′-CGAATTCTAAAAACAGTCGCACTTACCATTCACGCAC-3′. Mutated codons are underlined. Mutagenesis of Arg27 was achieved using two sequential polymerase chain reactions as described (28Park C.-S. Miller C. Neuron. 1992; 9: 307-313Abstract Full Text PDF PubMed Scopus (171) Google Scholar). The cDNA encoding BeKm-1 was amplified by PCR. The forward primer was nondegenerate oligonucleotide E1, 5′-GGAATTCGGACGACGACGACAAGCGACCTACAGATATAAAATGCAG-3′, containing an EcoRI restriction enzyme site (italicized) and corresponding to five codons encoding an enterokinase cleavage site and NH2-terminal residues 1–6 of BeKm-1. Primer E2, 5′-CGAATTCTAAAAACAGTCGCAAAAACCATTCACGC-3′, or F32K primer in the case of F32K-contained mutants, were used as the reverse primers. Both of them carried an EcoRI restriction enzyme site (italicized) and corresponded to the stop codon and COOH-terminal residues 28–36 of BeKm-1. The PCR fragments encoding mature and mutated BeKm-1 were gel purified, digested with EcoRI, and cloned into the expression vector pEZZ18 (Protein A gene Fusion Vector, Amersham Pharmacia Biotech). Clones were screened for the presence and orientation of the inserts by PCR. The resulting constructs were checked by sequencing and used to transform E. coli HB101 stain for protein production (29Boyer H.W. Roulland-Dussoix D. J. Mol. Biol. 1969; 41: 459-472Crossref PubMed Scopus (2571) Google Scholar). The wild type and mutated genes of BeKm-1 were expressed in the periplasm of E. coli as a fusion protein with two IgG-binding domains (ZZ) of staphylococcal Protein A. E. coli HB101 cells harboring the expression vectors were cultured at 37 °C in LB medium containing 100 μg/ml ampicillin. After 30 h the cells were harvested, resuspended in TS solution (50 mm Tris buffer, pH 7.6, 150 mm NaCl), and lysed by ultrasonication. After ultrasonication, the mixture was centrifuged for 15 min at 15,000 rpm to remove any remaining insoluble particles. The supernatant was applied to an IgG-Sepharose 6FF column (Amersham Pharmacia Biotech). The column was washed first with TS solution, containing 1 m NaCl and 0.05% Tween 20 and then with TS solution. The bound proteins were eluted with 0.5 m acetic acid, pH 3.4, and immediately lyophilized. Purity of the hybrid proteins was checked by SDS-polyacrylamide gel electrophoresis (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207180) Google Scholar). The toxins were cleaved from fusion proteins by enterokinase (31Mihailova A.G. Rumsh L.D. FEBS Lett. 1999; 442: 226-230Crossref PubMed Scopus (23) Google Scholar) (1 μg/50 μg of fusion protein) at 37 °C in 50 mmTris buffer, pH 8.0, for up to 36 h. The recombinant toxins were purified from the cleavage mixture by chromatography on a reverse phase HPLC column (Delta Pak C18 300-Å pore, 3.9 × 300 mm, Waters) using an acetonitrile gradient in 0.1% trifluoroacetic acid. The fractions containing recombinant toxins were rechromatographed on an ODS Ultrasphere column (4.6 × 150 mm, Beckman). Mass spectrometry and NH2-terminal amino acid determination verified the composition of the purified material. The peptide content was determined using the bicincholinic acid method (32Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18638) Google Scholar) with bovine serum albumin as the standard. Mass analysis of the recombinant toxins was performed in a VISION 2000-time of flight mass spectrometer with matrix-assisted laser desorption ionization, Thermo Bioanalysis Corp. (United Kingdom). NG108-15 mouse neuroblastoma × rat glioma cells were cultured and differentiated as described previously (32Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18638) Google Scholar). Recordings were made in the whole cell configuration of the patch clamp, at room temperature (20–25 °C). The perfusing solution comprised (mm): NaCl 144, KCl 2.5, CaCl2 2, MgCl2 0.5, HEPES 5, and glucose 10, adjusted to pH 7.4 with Tris base Electrodes (2–4 MΩ) were filled with a solution containing (mm): K acetate 90, KCl 20, Hepes 40, MgCl2 3, EGTA 3, Na2ATP 3, and Na-GTP 0.3, adjusted to pH 7.2 with NaOH. Recombinant and mutated BeKm-1 were dissolved in water at 100 μm, and added to the circulating bath solution to give a final concentration of 100 nm. To record the M-like current, cells were voltage-clamped with discontinuous (“switching”) amplifier (Axoclamp-2A, Axon Instruments, Inc.) with sampling a voltage at 6–8 kHz (50% duty cycle). Commands were generated via Digidata 1200 interface using pClamp 6 software (Axon Instruments). A standard voltage step protocol for M-current recording was used (33Robbins J. Trouslard J. Marsh S.J. Brown D.A. J. Physiol. 1992; 451: 159-185Crossref PubMed Scopus (72) Google Scholar, 34Adams P.R. Brown D.A. Constanti A. J. Physiol. 1982; 332: 223-262Crossref PubMed Scopus (278) Google Scholar, 35Brown D.A. Higashida H. J. Physiol. 1988; 397: 167-184Crossref PubMed Scopus (63) Google Scholar), in which cells were held at −28 mV and stepped for 1 s to potentials between −18 and −128 mV, by 10-mV negative increments. Current amplitudes at the end of each step were measured for subsequent construction of current-voltage relations using pClamp 6 software. The leak component of current was estimated by extrapolating a line fitted by eye from the region of the current-voltage relationship (negative to −60 mV) where only linear (ohmic) currents were observed. The resulting numerical data for control and BeKm-1-inhibited current records were processed in Quattro Pro version 5.0, and percentage reduction of the current at −28 mV was calculated. Graphs were created in Microcal Origin version 4.1. In addition, the current was monitored during toxin application by repeated steps from −28 mV to −68 and −108 mV, to ascertain when a steady-state block was reached. HEK-293 cells were incubated for 3–5 h in Opti-MEM medium with a transfection mixture containing: 2.0 μg of the appropriate K+ channel cDNA (α-subunits: KCNQ2-KCNQ4, hEAG, rELK1, and hERG1; β-subunits: KCNE1 and KCNE2), LipofectAMINE (Life Technologies), and Plus reagent (Life Technologies). In experiments with transiently transfected cells, recordings were performed 24–72 h post-transfection using enhanced green fluorescent protein as a marker of successful transfection. Some experiments were performed on HEK-293 cells stably expressing K+ channels (hSK1, rSK2, hIK, hBK, or hERG1+KCNE1). For details see Refs. 36Jensen B.S. Strøbæk D. Christophersen P. Jørgensen T.D. Hansen C. Silahtaroglu A. Olesen S.-P. Ahring P.K. Am. J. Physiol. 1998; 275: 848-856Crossref PubMed Google Scholar, 37Strøbæk D. Jørgensen T.D. Christophersen P. Ahring P.K. Olesen S.-P. Br. J. Pharmacol. 2000; 129: 991-999Crossref PubMed Scopus (162) Google Scholar, 38Strøbæk D. Christophersen P. Holm N.R. Moldt P. Ahring P.K. Johansen T.E. Olesen S.-P. Neuropharmacology. 1996; 35: 903-914Crossref PubMed Scopus (79) Google Scholar. The patch-clamp set-up and whole cell recordings were as previously described (36Jensen B.S. Strøbæk D. Christophersen P. Jørgensen T.D. Hansen C. Silahtaroglu A. Olesen S.-P. Ahring P.K. Am. J. Physiol. 1998; 275: 848-856Crossref PubMed Google Scholar, 37Strøbæk D. Jørgensen T.D. Christophersen P. Ahring P.K. Olesen S.-P. Br. J. Pharmacol. 2000; 129: 991-999Crossref PubMed Scopus (162) Google Scholar). SK, IK, and BK currents were recorded after application of voltage ramps ranging from −80 mV to +80 mV (duration 200 ms, holding potential 0 mV). The bath solution was an extracellular K+solution. Cells expressing KCNQ2/KCNQ3 channels were bathed in an extracellular Na+ solution and the currents were activated by a 1-s step from a holding potential of −90 mV to −30 mV. In the initial experiments with the hERG1 expressing cells, the currents were activated by a voltage protocol reassembling a cardiac action potential. In the protocol the cells were held at −90 mV and depolarized to +30 mV followed by 8 hyperpolarizing ramps altogether shaping an action potential with a duration of 315 ms. The composition of solutions used in experiments performed on HEK-293 cells consisted of extracellular Na+ solution (mm): NaCl 140, KCl 4, CaCl2 2, MgCl2 1, and Hepes 10 (pH 7.4, titrated with NaOH), the extracellular K+ solution (mm): KCl 144, CaCl2 2, MgCl2 1, and Hepes 10 (pH 7.4, titrated with KOH); and the intracellular solutions (mm): KCl 110, CaCl2 5.1–7.6, MgCl2 1.2–1.4, Na2ATP 4, EGTA 10/KOH 30, and Hepes 10 (pH 7.2, titrated with KOH). The partial amino acid sequence of BeKm-1 was determined from the NH2-terminal sequence and the sequences of the peptides derived from proteolytic digestion. The sequence of the NH2-terminal amino acid fragment was obtained after reducing and modification of cysteine residues. The hydrolysis of the modified toxin chain using V8 proteinase fromS. aureus resulted in two peptides, which were sequenced. The sequences of these peptides had an overlapping region, and the primary structure of the BeKm-1 fragment Arg1-Cys28 was determined. Amino acid sequences of some peptides obtained from tryptic hydrolysis of native BeKm-1 corresponded to the established regions of the toxin molecule. To determine the full amino acid sequence and to predict the structure of the toxin precursor, the cDNA encoding BeKm-1 was isolated. Briefly, the first strand cDNA was synthesized using poly(A)-rich RNA by Moloney murine leukemia virus reverse transcriptase and first-strand primer (RLdT) containing restriction sites for cloning. Degenerate oligonucleotide primer T1 was designed using amino acid sequence information and codes for the toxin region from Arg1 to Lys6. PCR using RL and T1 primers yielded a single band of the expected size (220 bp). This PCR product was subcloned in pBluescript between thePstI and EcoRI sites and sequenced. All cDNA sequences had one major open reading frame (ORF), encoding an amino acid sequence corresponding to the partial amino acid sequence of BeKm-1 obtained from primary structural analysis of the native toxin. 5′-RACE was used to identify the unknown sequence of the 5′-end of the mRNA. 5′-Flanking sequence information was obtained using the anchored PCR technique (27Loh E.Y. Elliott J.F. Cwirla S. Lanier L.L. Davis M.M. Science. 1989; 243: 217-220Crossref PubMed Scopus (525) Google Scholar). In this procedure, mRNA was first transcribed with reverse transcriptase and a poly(dG) tail was added to the 3′-end of the strand with terminal deoxynucleotidyl transferase. The product was then amplified with a specific 3′-primer (in our case a T2 oligonucleotide) and another oligonucleotide consisting of a poly(dC) tail (C13 and M13-C13 primers). Series of consistent PCR generated two bands of about 300 and 250 bp with cloned cDNA sequences that had long and short 5′-untranslated region, respectively, that may be a result of partial degradation of the mRNA. The full-length cDNA of BeKm-1, together with its 5′- and 3′-untranslated region, is shown in Fig.1. The full-length of the cDNA, excluding the poly(A) tail, was 365 bp and contained a 171-bp ORF encoding a 57-amino acid peptide. The first ATG was located at position 121. The 3′-untranslated region of the cDNA contained a putative polyadenylation signal (AATAAA) ∼16 nucleotides upstream from the poly(A) tail. The ORF encoded a polypeptide precursor for BeKm-1 in which the first 21 amino acid residues are predicted to be a signal peptide followed by the mature 36-amino acid peptide. The signal peptide has structural features characteristic of secreted proteins, and shows homology to the published leader peptides of KTx, KTx2 (39Legros C. Bougis P.E. Martin-Eauclaire M.F. FEBS Lett. 1997; 402: 45-49Crossref PubMed Scopus (28) Google Scholar), and Ts κ (40Legros C. Oughuideni R. Darbon H. Rochat H. Bougis P.E. Martin-Eauclaire M.F. FEBS Lett. 1996; 390: 81-84Crossref PubMed Scopus (50) Google Scholar), K+ channel blockers from scorpion venoms. To obtain significant quantities of the peptide and its mutated forms for structure-function investigation, the E. coli expression system was adopted. We expressed the wild-type BeKm-1 and three mutated toxins (R27K, F32K, and R27K/F32K) as fusion proteins with two IgG-binding domains of Protein A from S. aureus. Sense and antisense primers, both with a specific restriction enzyme site, were used to amplify the BeKm-1 toxin cDNA for cloning into the pEZZ18 expression vector. The pentapeptide sequence DDDDK, which is recognized by the restriction protease enterokinase, was inserted immediately upstream of the BeKm-1 sequence. A translation termination codon was inserted at the end of the BeKm-1 cDNA. Site-directed mutants were constructed using PCR, and each mutated plasmid was verified by sequencing. The final constructs were transformed into the E. coli strain HB101. Fusion proteins were directly secreted into the periplasm of HB101, making them easy to purify by affinity chromatography on an IgG-Sepharose column. The size of the affinity purified proteins observed from SDS-polyacrylamide gel electrophoresis was in accordance with that expected from a fusion protein of ZZ and BeKm-1. The yield of fused toxins varied from 2 to 6 mg/liter of culture. The fusion proteins were treated with enterokinase and purified by reverse-phase HPLC to obtain the pure recombinant toxins. The recombinant, enterokinase-digested BeKm-1 had the same retention time as the native toxin when fractionated by reverse-phase HPLC (Fig.2). Homogeneity of recombinant BeKm-1 and of each mutant was further confirmed by analytical reverse-phase HPLC employing an Ultrasphere ODS column. The total yield of purified recombinant peptides was" @default.
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• W2022675045 date "2001-03-01" @default.
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• W2022675045 title "An ERG Channel Inhibitor from the Scorpion Buthus eupeus" @default.
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