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.
Highlights
Kϩ channels comprise a large, diverse group of integral membrane proteins, which are found in all cells
Ing of a large number of Kϩ channels has resulted in a classification into structural classes including the 2-transmembrane Kϩ channels, the 4-transmembrane Kϩ channels (2P domain channels), and the 6-transmembrane Kϩ channels
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
Summary
Native BeKm-1 toxin was purified from the scorpion venom as described previously [1]. Primer E2, 5Ј-CGAATTCTAAAAACAGTCGCAAAAACCATTCACGC-3Ј, 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). 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 ϫ 300 mm, Waters) using an acetonitrile gradient in 0.1% trifluoroacetic acid. Cells expressing KCNQ2/KCNQ3 channels were bathed in an extracellular Naϩ solution and the currents were activated by a 1-s step from a holding potential of Ϫ90 mV to Ϫ30 mV. Solutions Used in HEK-293 Cells—The composition of solutions used in experiments performed on HEK-293 cells consisted of extracellular Naϩ solution (mM): NaCl 140, KCl 4, CaCl2 2, MgCl2 1, and Hepes 10 (pH 7.4, titrated with NaOH), the extracellular Kϩ 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)
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