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 -