An ERG Channel Inhibitor from the Scorpion Buthus eupeus*

Yuliya V. KorolkovaDagger §, Sergey A. KozlovDagger , Aleksey V. LipkinDagger , Kirill A. PluzhnikovDagger , Jennifer K. Hadley, Alexander K. Filippov, David A. Brown, Kamilla Angelo||, Dorte Strøbæk**, Thomas Jespersen||, Søren-Peter Olesen||**DaggerDagger, Bo S. Jensen||**, and Eugene V. GrishinDagger

From the Dagger  Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, 117997, GSP-7, Moscow, Russia, the  Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom, the ||  Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark, and the ** NeuroSearch A/S, 93 Pederstrupvej, DK-2750 Ballerup, Denmark

Received for publication, July 7, 2000, and in revised form, November 30, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (2). 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) (see e.g. Ref. 3).

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. 4-6 for 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 (5, 7, 8). 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 (9, 10), determine the subunit stoichiometry of K+ channels (11), and elucidate the topology of the extracellular face of the channel pore (12-16).

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 (17) 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 (1). 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 (18). Later, heteromeric KCNQ2/KCNQ3 channels were demonstrated to contribute to the native M-current (19). 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 (20, 21). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of BeKm-1 from B. eupeus Venom

Native BeKm-1 toxin was purified from the scorpion venom as described previously (1).

Determination of Amino Acid Sequence of BeKm-1

Disulfide bonds of the purified toxin were reduced with dithiothreitol, and SH-groups were modified with 4-vinylpyridine (22). The modified toxin was digested with Staphylococus aureus protease 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).

Preparation of mRNA from Venom Glands

Total RNA was isolated by a guanidinium thiocyanate/phenol chloroform method (23) 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 (24).

PCR Amplification of BeKm-1 DNA, Cloning, and DNA Sequencing

RT-PCR-- 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 Lys6 of 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 (25) and sequenced on both strands using the Sanger (26) dideoxynucleotide chain termination method.

5'-RACE-PCR-- 5'-RACE was performed according to the anchored PCR technique described (27). 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.

Site-directed Mutagenesis and Expression Vectors Construction

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 (28). 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 (29).

Expression and Purification of Recombinant Toxins

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 (30).

The toxins were cleaved from fusion proteins by enterokinase (31) (1 µg/50 µg of fusion protein) at 37 °C in 50 mM Tris 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 (32) with bovine serum albumin as the standard.

Mass Spectrometry

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).

Electrophysiology

Measurement of M-type Currents in NG108-15 Cells-- NG108-15 mouse neuroblastoma × rat glioma cells were cultured and differentiated as described previously (32). 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 MOmega ) 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 (33-35), 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.

Stably and Transiently Transfected HEK-293 Cell Lines-- 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 (alpha -subunits: KCNQ2-KCNQ4, hEAG, rELK1, and hERG1; beta -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. 36-38.

Measurements in HEK-293 Cells-- The patch-clamp set-up and whole cell recordings were as previously described (36, 37). 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.

Solutions Used in HEK-293 Cells-- 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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amino Acid Sequence of BeKm-1-- 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 from S. 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.

Cloning and Sequencing of BeKm-1 cDNA-- 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 the PstI 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 (27). 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.


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Fig. 1.   Nucleotide sequence of the cDNA encoding the BeKm-1 precursor. ORF sequence is boxed; open boxes represent the leader sequence, followed by filled-in box carrying the nucleotide sequence of the mature BeKm-1 toxin. The deduced amino acid sequence of the ORF is indicated by the three-letter code below the appropriate nucleotide codon. The putative polyadenylation signal AATAAA is underlined.

Analysis of the cDNA Sequence-- 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 (39), and Ts kappa  (40), K+ channel blockers from scorpion venoms.

Expression of Recombinant BeKm-1 and Its Mutants-- 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 ~200 µg of toxin/liter of culture. The molecular mass of the recombinant products obtained by matrix-assisted laser desorption ionization mass spectrometry were in accordance with the expected theoretical mass calculated from sequence data (determined mass 4092, 4064, 4074, and 4046; calculated mass 4091.69, 4063.67, 4072.69, and 4044.68 Da for the recombinant wild-type and native toxins, mutants R27K, F32K, and R27K/F32K, respectively).


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Fig. 2.   Reverse phase HPLC of recombinant and native BeKm-1. A, sample of fusion protein digested with enterokinase (1 µg of enterokinase/50 µg of fusion protein in 50 mM Tris buffer, pH 8.0, for 30 h at 37 °C); B, native BeKm-1. Column C18 Delta Pak 3.9 × 300 mm, 300-Å pore (Waters). Elution was performed with a gradient of solvent B (0.1% trifluoroacetic acid in acetonitrile) in solvent A (0.1% trifluoroacetic acid in water) at flow rate 2 ml/min.

Effect of the Recombinant BeKm-1 and of the Mutants on the M-like Current in NG108-15 Cells-- The effect of the recombinant BeKm-1 and all mutated toxins were tested for inhibition of the M-like current in mouse neuroblastoma × rat glioma NG108-15 cells. The M-like current in NG108-15 cells is a sustained, voltage-activated, outward potassium current observed at potentials positive to -70 mV (33). This current is similar to the M-current of sympathetic ganglia in many respects, but differs from it in certain pharmacological properties (33), so we refer to it here as an "M-like" current. We used the standard protocol for M-like current recording (for example, see Refs. 33 and 35). The cell was voltage-clamped at holding potential of -28 mV, to pre-activate the M-like current, and the current was then deactivated by 1-s voltage steps to more hyperpolarized levels (see also "Experimental Procedures").

The recombinant BeKm-1 toxin clearly inhibited the M-like current by reducing the standing (holding) outward current at -28 mV holding potential, the amplitude of its relaxation on deactivating and the absolute current amplitude at the end of the steps (Fig. 3A). Fig. 3B shows the inhibition of the current at -28 mV by 100 nM recombinant BeKm-1 (n = 3) and mutants R27K, F32K, and R27K/F32K (n = 4 for each). A one-tailed t test (Quattro Pro) gave a marginal significance (p = 0.049) in favor of the hypothesis that mutant R27K had marginally lower activity that the wild-type toxin. Nevertheless, its blocking activity was still appreciable at >40%. Inhibition by the other two mutants did not differ significantly from that by the nonmutated toxin; all were >50%. The results demonstrate that the blocking effect of the recombinant peptides on the M-like current in NG108-15 cells is not less than that of the native toxin (about 45% (see Ref. 1)). Since the M-like current in NG108-15 cells has been suggested to be carried by both KCNQ2/KCNQ3 and mErg1 channels (20, 21), the partial blocking effect of the toxin could mean that the toxin blocked selectively only one of the component channels.


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Fig. 3.   Effect of the recombinant BeKm-1 and mutants on the M-like current in neuroblastoma × glioma NG108-15 cells. A, inset records show an example of current responses of a cell to a series of voltage steps from a holding potential of -28 mV to command potentials between -18 and -128 mV in 10 mV increments, recorded before and 3 min after starting superfusion with 100 nM recombinant BeKm-1. Dotted line indicates zero current. An arrow indicates were current was measured for a graph. The graph shows the absolute current levels (current measured from zero current) attained at the end of each voltage step, plotted against the command potential, before and during superfusion with toxin. B, the columns show the mean percentage inhibition of M-like currents by 100 nM recombinant BeKm-1 and the three mutated variants. The bars show the standard error of the mean in each case, n is number of experiments. Of the differences between mean values, only that between recombinant BeKm-1 and mutant R27K attained marginal statistical significance. M-like current amplitudes were measured at -28 mV after leak subtraction from the current-voltage curves obtained as in A (see also "Experimental Procedures").

The Effect of BeKm-1 on Cloned Human KCNQ and hERG1 Channels-- To elucidate the molecular composition of the current blocked by BeKm-1, the toxin was applied to HEK-293 cells transiently expressing human KCNQ2/KCNQ3 channels, which have been suggested to underlie the M-current (19). In whole cell experiments, the KCNQ2/KCNQ3 current was visualized as an outward current activating slowly upon a voltage step from -90 to -30 mV. The step was repeated every 5 s and after establishment of a stable baseline, the Na+ solution was changed to a Na+ solution containing 100 nM BeKm-1. The presence of BeKm-1 in the bath solution for 2-3 min did not change the amplitude of the current (n = 3, data not shown). The toxin was tested in a similar way on other members of the KCNQ family, KCNQ4 (n = 3, not shown) and KCNQ1 coexpressed with KCNE1 (n = 3, not shown). BeKm-1 did not modulate any of these currents.

Since the M-like current in NG108-15 cells has been reported to include both a KCNQ and an ERG component (20, 21), BeKm-1 was also tested on human ERG1 channels. In preliminary experiments, BeKm-1 was added at a concentration of 100 nM to a HEK-293 cell line stably transfected with hERG1 and KCNE1. BeKm-1 inhibited the hERG1 + KCNE1 current activated by a cardiac action potential protocol (see "Experimental Procedures"). The block was fast and reversible with an IC50 value estimated at 10-30 nM (n = 7, data not shown). The three mutants of BeKm-1 inhibited the hERG1 + KCNE1 current with similar potencies (n = 3 for each toxin-mutant, data not shown).

The inhibition of hERG1 by BeKm-1 was characterized further in HEK-293 cells transiently transfected with the hERG1-cDNA alone. Fig. 4A shows a whole cell experiment with 144 mM [K+]0, where the hERG1 current was fully activated by stepping to +40 mV for 400 ms every 5 s from a holding potential of -80 mV. After each step the voltage was returned to -120 mV and large inward tail currents could be recorded. Characteristic of hERG1, relief from channel inactivation is apparent at the peak of the tail currents shown. In Fig. 4B the size of the tail currents is plotted as a function of time (same experiment as in Fig. 4A). The hERG1 current was blocked as 4 nM and subsequently 100 nM BeKm-1 was added to the bath solution for the periods indicated by bars. After wash out of the toxin for 2 min, the tail current recovered totally and reached the control level. Fig. 4C depicts the mean of normalized tail currents as a function of the BeKm-1 concentration from four experiments. The IC50 values for the effect of BeKm-1 on the hERG1 channel was calculated by fitting the normalized tail currents to the function Itail = 1 - ([BeKm-1]n/[BeKm-1]n + IC50), where Itail is the fraction of unblocked current and n is the Hill coefficient. From the fitted dose-response curve an IC50 value of 3.3 nM and a Hill coefficient of 0.9 was obtained (n = 4, error bars represents S.E.).


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Fig. 4.   The effect of BeKm-1 on the cloned hERG1 channel. A, whole cell current traces recorded from HEK cells transfected with the cDNA encoding hERG1. The experiment was performed with high K+ solutions on both sides of the membrane at a holding potential of -80 mV. Tail currents were elicited by applying a +40 mV pre-pulse followed by a step to -120 mV for 500 ms applied every 5 s. The traces represent recordings at superfusion with high K+ solution alone, in the presence of 4 nM, and 100 nM BeKm-1, respectively. B, time course of the same experiment as in A. Current was measured at the arrow shown in A. C, mean dose-response curve from four experiments (error bars represent S.E.). The points were fitted with a modified Hill equation.

Two beta -subunits, KCNE1 and KCNE2, are known to interact with the hERG1 channel (41). To test if association of the hERG1 alpha -subunit with its beta -subunits would influence the effect of BeKm-1, co-transfection studies were performed. 10 nM BeKm-1 was applied to HEK cells transiently transfected with hERG1 + KCNE1 and hERG1 + KCNE2, respectively. All experimental conditions was as in Fig. 4A. Kd values were estimated by exponential fitting to the time course of blockade (37). The Kd values 5 and 7 nM obtained from recordings with hERG1 + KCNE1 and hERG1 + KCNE2 were not found to be significantly different from the block by BeKm-1 on hERG1 channel alone.

Specificity of the BeKm-1 Toxin-- The specificity of BeKm-1 was tested at a variety of cloned K+ channels and, as stated in Table I, addition of 100 nM of the toxin in whole cell experiments was without effect at four members of the family of calcium-activated potassium channels. These channels were activated by 100-300 nM free calcium in the pipette solution and a ramp-protocol ranging from -80 to +80 mV was used. The hSK1, rSK2, and hIK channels are not voltage-dependent, and weakly inward rectifying currents are measured upon application of voltage ramps with a 140 mM K+ solution in the bath (see Refs. 37 and 38). The voltage-dependent hBK channels were activated at potentials more positive than +30 mV. After the addition of 100 nM BeKm-1 and a washing period, the specificity of the current measured in each of the experiments was verified by the inhibition induced by apamin (hSK1 and rSK2), charybdotoxin (hIK), or iberiotoxin (hBK) at 100 nM.

                              
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Table I
Specificity of BeKm-1
The effect of BeKm-1 on cloned K+ channels was tested using a single dose of toxin, 100 nM, for initial screening. Calcium-activated channels were tested for the effect of BeKm-1 on the voltage-independent, calcium-activated K+ channels, hSK1, rSK2, and hIK was tested, using symmetrically high K+ solutions with the intracellular solution containing 100-300 nM free Ca2+ and applying a voltage-ramp (-80 to +80 mV, 200 ms duration). The KCNQ1/KCNE1, KCNQ2/KCNQ3, or the KCNQ4 current was visualized as an outward current activating slowly upon a voltage step from -90 to -30 mV recorded in asymmetrical Ringer's solution (high extracellular Na+, high intracellular K+). The step was repeated every 5 s and after establishment of a stable baseline, 100 nM was applied, and the current amplitude recorded. The ether-a-go-go like channel experiments were performed with high K+ solutions on both sides of the membrane at a holding potential of -80 mV. Tail currents were elicited by applying a +40 mV pre-pulse followed by a step to -120 mV for 500 ms applied.

To determine whether the BeKm-1 toxin is a general blocker of the ether-a-go-go family, or if it is specific to hERG1, the toxin was tested on the cloned hEAG and rELK1 channels. Whole cell experiments on transiently transfected HEK-293 cells were performed in 140 mM [K+]o solution applying the same protocol as in the experiments of the hERG1 channel in Fig. 4A. To prevent blockade of the hEAG channel by intracellular Ca2+ (42), no CaCl2 was added to the pipette solution in these experiments. When performing recordings on rELK1 expressing HEK cells, the holding potential was set to -100 mV, due to the low threshold activation of these channels (43). The inward tail currents were followed on-line while the cells were superfused with 100 nM BeKm-1 for 3-6 min. No effect was observed on the hEAG tail current (n = 3), however, the rELK1 current was slightly inhibited by the toxin. The tail current recorded in the presence of 100 nM toxin was reduced by 9.4 ± 2.6% (n = 3, S.D) as compared with the control current level.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The discovery of high-affinity K+ channel toxins has increased the understanding of the K+ channel structure, distribution, and subcellular localization in different cell types significantly. In this study we investigated the structure and function of BeKm-1, a toxin from venom of the scorpion B. eupeus (1). It is demonstrated here that BeKm-1 is a novel specific blocker of the hERG1 potassium channel (IC50 = 3.3 nM) with potency 5 times higher than that of the previously reported peptide ERG-Tx (44). Recombinant BeKm-1, like the native toxin (1), also reduced the M-like current in NG108-15 mouse neuroblastoma × rat glioma cells, part of which is carried by mERG1 channels (20, 21). It did not, however, inhibit KCNQ channels, in agreement with the lack of effect of the native toxin on the KCNQ2/KCNQ3 M-currents in rat sympathetic neurons (1).

Amino Acid Alignment Analysis-- The combination of protein chemistry methods and the RT-PCR technique enabled us to obtain the primary structure of BeKm-1. BeKm-1 is a 36-amino acid peptide containing 6 cysteines and 6 positively charged residues.

On the basis of analysis of its amino acid sequence, BeKm-1 belongs to a family of closely related scorpion toxins. The amino acid sequence of BeKm-1 was compared with those of other known scorpion peptide blockers of K+ channels (Fig. 5). The largest degree of amino acid identity is observed between BeKm-1, IbTx, and TsTx Kalpha (40.5%). The conserved position of the 6 cysteine residues among all toxins suggests a common disulfide bridging pattern as shown for ChTx, IbTx, MgTx, KTx, and other scorpion toxins, whose spatial structures were resolved by NMR (45-48). It is possible that the folding and general conformation of BeKm-1 is homologous to the previously determined folding of scorpion venom toxins. Despite the homology between BeKm-1 and other scorpion toxins, it cannot be assigned to one of the 12 distinct subfamilies of scorpion K+ channel blocking peptides proposed by Tytgat (49). Thus, BeKm-1 belongs to a new subfamily of scorpion toxins. Comparison of the deduced amino acid sequence of BeKm-1 with those of other K+ channel-specific scorpion toxins revealed that BeKm-1 displays several important sequence differences, which could explain its specific effect on the hERG1 K+ channel. The principal differences are in the COOH-terminal part that is highly conserved among all scorpion toxins. BeKm-1 contains Arg27, Val29, and Phe32 instead of Lys, Met, and Lys, respectively.


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Fig. 5.   Sequence homology between BeKm-1 and other scorpion toxins acting on K+ channel. Representatives of some alpha -KTx subgroups (49): ChTx, charybdotoxin from Leiurus quinquestriatus hebraeus; IbTx, iberiotoxin from Buthus tamulus; TsTx-Kalpha , tityustoxin Kalpha from Tityus serrulatus, NxTx, noxiustoxin from C. noxius; KTx, kaliotoxin from Androctonus mauretanicus mauretanicus; ScTx, scyllotoxin from Leiurus quinquestriatus hebraeus, Pi1 pandinotoxin 1 from P. imperator. Sequences are aligned at Cys residues (boxes). Identical residues are filled. The last line is the ERG channels inhibitor ERG-Tx, ergtoxin from C. noxius (44).

The functional role of the highly conserved Lys27 was previously studied by individual chemical modification and by site-directed mutagenesis of ChTx. It has been identified that the positively charged Lys27 is an important toxin element implicated in the recognition of K+ channels, and the critical residue for voltage-dependent block of the BK channel (50) and Shaker K+ channel (51). Furthermore, even a minor change of a functionally important Lys27, such as mutation to Arg, was unfavorable. For example, in the Shaker channel, a conservative mutation of this lysine to arginine destabilized ChTx binding over 1000-fold (39). Arginine instead of lysine in a position equivalent to 27 was found only in Pi7 toxin from scorpion Pandinus imperator. This toxin, however, was absolutely inactive on the Shaker B channel, whereas the highly homologous toxin Pi4 from the same venom containing a lysine residue completely blocked this type of K+ channel (53).

On the other hand, BeKm-1 displays no homology with ergtoxin (ERG-Tx) isolated from the scorpion Centruroides noxius (44). ERG-Tx has an another structure (Fig. 5) that differs from those previously known for K+ channel toxin blockers. ERG-Tx specifically inhibits only ERG channels with IC50 of 16 nM. It is interesting that ERG-Tx and BeKm-1 have only two homologous residues, namely Arg and Asp (positions 1 and 4 in BeKm-1 and 2, 3 in ERG-Tx) in the NH2-terminal part of the toxins. As Arg1 in BeKm-1 is unique among all K+ channel blockers from scorpion venom, it may have an important role in hERG1 channel inhibition.

Choice of the Mutations-- Previously, it was suggested that the highly conserved cluster of about 10 amino acid residues in the COOH-terminal region of scorpion toxins may play a prominent role in toxin-channel interactions. We hypothesized that the COOH-terminal 26-32 sequence of BeKm-1 may contribute the core of the toxin-binding site that recognizes K+ channels. To elucidate this point, site-directed mutagenesis of BeKm-1 was undertaken. The DNA segment encoding BeKm-1 was mutated to obtain a set of variants of the toxin to study structure-function relationships. Mutations were chosen so as to produce a change in the original biological function. Two amino acids, Arg27 and Phe32, were substituted by the Lys found in most K+ channel toxins. Since Lys27 is highly conserved among all the toxins and has an important role in K+ channel inhibition, it was interesting to assess its contribution to the block of hERG1 channels by BeKm-1. Phe32 has not been found in other potassium channel blockers from scorpion venom, suggesting that its role may be important for hERG1 inhibition. The double mutant R27K/F32K had the same sequence of active region as many others scorpion toxins.

Expression of Recombinant Toxins-- We successfully produced BeKm-1 and its altered analogues in the periplasm of E. coli, using the plasmid pEZZ18 as a fusion protein with two IgG-binding domains of Protein A from S. aureus. Previous studies showed that this type of expression system is particularly well adapted to disulfide-rich proteins. It has been used to express erabutoxin A (54) and BotXIV, insect-specific alpha  toxin of the scorpion Buthus occitanus tunetanus (55). The combination of secretion and single step affinity purification has obvious advantages. The yield of the desired fusion product was 2-6 mg/liter of culture, giving about 200 µg of recombinant toxin after cleavage from the fusion protein by enterokinase and purification by HPLC.

Heterologous expression of cDNAs encoding other small scorpion toxins, like charybdotoxin (ChTx) from Leiurus quinquestriatus (56) and margatoxin (MgTx) from Centruroides margaritatus (57) has been described in E. coli. The fusion toxins with the gene 9 protein of T7 bacteriophage produced in the cytoplasm were cleaved by factor Xa after purification and the resulting toxins refolded in vitro. The major drawback of the production procedure is that the recovery yield is rather low as compared with those reached using the procedure mentioned above. Recently KTx2 was successfully produced as a fusion protein with maltose-binding protein in the periplasm of E. coli (58) with a yield comparable to that obtained in the present study.

Effect on K+ Channels-- Experiments presented here demonstrate that recombinant BeKm-1 has structural and functional characteristics identical to those of the native toxin. Recombinant BeKm-1 inhibits the M-like K+ current in NG108-15 cells in a manner very similar to that of the native toxin (see Ref. 1). These results indicate that the recombinant and native toxins are functionally identical in their binding properties. Furthermore, all mutants inhibited the M-like potassium current in NG108-15 cells similar to that of the recombinant wild-type BeKm-1, although the mutant R27K was found to be marginally less potent than BeKm-1.

The examination of the action of BeKm-1 on the cloned K+ channels revealed that BeKm-1 potently blocked the human ERG1 channel. Coexpression of the hERG1 channel with its putative beta -subunits, KCNE1 and KCNE2, did not change the toxin affinity. The results show that recombinant BeKm-1 does not interact with hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, or KCNQ4 channels. As BeKm-1 did not affect KCNQ2/KCNQ3 channels it is clear that the blocking effect of BeKm-1 on M-like current can be fully accounted for by inhibition of mERG1 channels in NG108-15 cells. Thus, BeKm-1 is a highly specific inhibitor of ERG channels.

Point substitutions in the COOH-terminal region of BeKm-1 did not change the efficacy of the toxin to block M-like currents or cloned hERG1 channels indicating that the BeKm-1 mutants have the same affinity for hERG1 channels as the wild-type toxin. Therefore, the highly conserved sequence found in K+ channel-selective scorpion toxins, which constitutes a common high affinity binding site recognizing both voltage-gated and Ca2+-activated K+ channels, is not essential for ERG channels. The high affinity of the BeKm-1 toxin and the specificity toward hERG1 channels might result from interactions different from other pore-blocking toxins and probably involve the NH2-terminal toxin region. Further efforts will include this subject of analysis.

By using expression systems in combination with site-directed alterations in the peptide sequence, the molecular basis of BeKm-1 recognition and inhibition of ERG channels may be addressed. With the feasibility of producing large amounts of BeKm-1 in vitro, it will be possible to study the three-dimensional structure of the peptide. The study of the interaction of BeKm-1 with its channel receptor promises to reveal new information regarding ERG channels structure and function, and to clarify how scorpion toxins act on diverse members of the K+ channel superfamily. Finally, BeKm-1 provides a specific tool not only to determine the functional role that ERG channels play in target tissues or to develop the molecular pharmacology of this channel, but also to determine the subcellular localization of this channel in heart and brain.

    ACKNOWLEDGEMENTS

Enterokinase was kindly provided by Dr. A. Mikhaylova. cDNA encoding hERG1 was a generous gift from G. Robertson. cDNAs encoding KCNQ2, KCNQ3, and KCNQ4 were kindly provided by Dr. Thomas Jentsch. cDNA encoding rELK1 was generously provided by Dr. Birgit Engeland, and cDNA for the hEAG was kindly provided by Dr. Jacqueline Fischer-Lougheed. KCNQ1 was from Dr. Jacques Barhanin. We thank Dr. E. D. Nosyreva for assistance in the preparation of this manuscript.

    FOOTNOTES

* This work was supported in part by the Russian Foundation of Fundamental Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF276623.

§ To whom correspondence should be addressed. Tel.: 7-095-3364022; Fax: 7-095-3307301; E-mail: july@ibch.ru.

Dagger Dagger  Supported by the Danish Heart Foundation.

Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M005973200

    ABBREVIATIONS

The abbreviations used are: EAG, ether-a-go-go gene; ERG, EAG-related; ELK, EAG-like; KCNQ, KQT-like K+ channel; BK, IK, SK, high-, intermediate-, small-conductance Ca2+-activated K+ channels; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; ORF, open reading frame; bp, base pair(s); HPLC, high performance liquid chromatography; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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52. Goldstein, S. A. N., Pheasant, D. J., and Miller, C. (1994) Neuron 12, 1377-1388[Medline] [Order article via Infotrieve]
53. Olamendi-Portugal, T., Gomez-Lagunas, F., Gurrola, G. B., and Possani, L. D. (1998) Toxicon 36, 759-770[CrossRef][Medline] [Order article via Infotrieve]
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