An ERG channel inhibitor from the scorpion Buthus eupeus

Y.V. Korolkova; S.A. Kozlov; A.V. Lipkin; K.A. Pluzhnikov; J.K. Hadley; A.K. Filippov; D.A. Brown; Kamilla Angelo Pedersen; D. Strøbaek; Thomas Jespersen; Søren-Peter Olesen; B. Jensen Skanning; E.V. Grishin

doi:10.1074/jbc.M005973200

An ERG channel inhibitor from the scorpion Buthus eupeus

Y.V. Korolkova, S.A. Kozlov, A.V. Lipkin, K.A. Pluzhnikov, J.K. Hadley, A.K. Filippov, D.A. Brown, Kamilla Angelo Pedersen, D. Strøbaek, Thomas Jespersen, Søren-Peter Olesen, B. Jensen Skanning, E.V. Grishin

83 Citations (Scopus)

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 IC(50) 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.

Original language	English
Journal	Journal of Biological Chemistry
Volume	276
Issue number	13
Pages (from-to)	9868-76
Number of pages	9
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M005973200
Publication status	Published - 30 Mar 2001

Keywords

Amino Acid Sequence
Animals
Base Sequence
Cation Transport Proteins
Cell Line
Chromatography, High Pressure Liquid
Cloning, Molecular
DNA, Complementary
DNA-Binding Proteins
Dose-Response Relationship, Drug
Electrophysiology
Escherichia coli
Ether-A-Go-Go Potassium Channels
Humans
Inhibitory Concentration 50
KCNQ Potassium Channels
KCNQ1 Potassium Channel
Kinetics
Mass Spectrometry
Mice
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Open Reading Frames
Polymerase Chain Reaction
Potassium Channel Blockers
Potassium Channels
Potassium Channels, Voltage-Gated
Protein Sorting Signals
Protein Structure, Tertiary
RNA, Messenger
Rats
Recombinant Fusion Proteins
Scorpion Venoms
Scorpions
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Substrate Specificity
Time Factors
Trans-Activators
Tumor Cells, Cultured

Access to Document

10.1074/jbc.M005973200

Cite this

@article{45bdeb7074c711dbbee902004c4f4f50,

title = "An ERG channel inhibitor from the scorpion Buthus eupeus",

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 IC(50) 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.",

keywords = "Amino Acid Sequence, Animals, Base Sequence, Cation Transport Proteins, Cell Line, Chromatography, High Pressure Liquid, Cloning, Molecular, DNA, Complementary, DNA-Binding Proteins, Dose-Response Relationship, Drug, Electrophysiology, Escherichia coli, Ether-A-Go-Go Potassium Channels, Humans, Inhibitory Concentration 50, KCNQ Potassium Channels, KCNQ1 Potassium Channel, Kinetics, Mass Spectrometry, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Open Reading Frames, Polymerase Chain Reaction, Potassium Channel Blockers, Potassium Channels, Potassium Channels, Voltage-Gated, Protein Sorting Signals, Protein Structure, Tertiary, RNA, Messenger, Rats, Recombinant Fusion Proteins, Scorpion Venoms, Scorpions, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Substrate Specificity, Time Factors, Trans-Activators, Tumor Cells, Cultured",

author = "Y.V. Korolkova and S.A. Kozlov and A.V. Lipkin and K.A. Pluzhnikov and J.K. Hadley and A.K. Filippov and D.A. Brown and Pedersen, {Kamilla Angelo} and D. Str{\o}baek and Thomas Jespersen and S{\o}ren-Peter Olesen and {Jensen Skanning}, B. and E.V. Grishin",

year = "2001",

month = mar,

day = "30",

doi = "10.1074/jbc.M005973200",

language = "English",

volume = "276",

pages = "9868--76",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

number = "13",

}

TY - JOUR

T1 - An ERG channel inhibitor from the scorpion Buthus eupeus

AU - Korolkova, Y.V.

AU - Kozlov, S.A.

AU - Lipkin, A.V.

AU - Pluzhnikov, K.A.

AU - Hadley, J.K.

AU - Filippov, A.K.

AU - Brown, D.A.

AU - Pedersen, Kamilla Angelo

AU - Strøbaek, D.

AU - Jespersen, Thomas

AU - Olesen, Søren-Peter

AU - Jensen Skanning, B.

AU - Grishin, E.V.

PY - 2001/3/30

Y1 - 2001/3/30

N2 - 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 IC(50) 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.

AB - 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 IC(50) 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.

KW - Amino Acid Sequence

KW - Animals

KW - Base Sequence

KW - Cation Transport Proteins

KW - Cell Line

KW - Chromatography, High Pressure Liquid

KW - Cloning, Molecular

KW - DNA, Complementary

KW - DNA-Binding Proteins

KW - Dose-Response Relationship, Drug

KW - Electrophysiology

KW - Escherichia coli

KW - Ether-A-Go-Go Potassium Channels

KW - Humans

KW - Inhibitory Concentration 50

KW - KCNQ Potassium Channels

KW - KCNQ1 Potassium Channel

KW - Kinetics

KW - Mass Spectrometry

KW - Mice

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Mutation

KW - Open Reading Frames

KW - Polymerase Chain Reaction

KW - Potassium Channel Blockers

KW - Potassium Channels

KW - Potassium Channels, Voltage-Gated

KW - Protein Sorting Signals

KW - Protein Structure, Tertiary

KW - RNA, Messenger

KW - Rats

KW - Recombinant Fusion Proteins

KW - Scorpion Venoms

KW - Scorpions

KW - Sequence Analysis, DNA

KW - Sequence Homology, Amino Acid

KW - Substrate Specificity

KW - Time Factors

KW - Trans-Activators

KW - Tumor Cells, Cultured

U2 - 10.1074/jbc.M005973200

DO - 10.1074/jbc.M005973200

M3 - Journal article

C2 - 11136720

SN - 0021-9258

VL - 276

SP - 9868

EP - 9876

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 13

ER -

An ERG channel inhibitor from the scorpion Buthus eupeus

Abstract

Keywords

Access to Document

Fingerprint

Cite this