An ERG channel inhibitor from the scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K+ current, BeKm-1, from t he venom of the Central Asian scorpion Buthus eupeus has been described pre viously (Fillipov A. K,, Kozlov, S, A, Pluzhnikov, K. A., Grishin, E. V., a nd 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 nucleotid es encoding the precursor of BeKm-1 was isolated using the rapid amplificat ion of cDNA ends polymerase chain reaction technique from mRNA obtained fro m scorpion telsons, Sequence analysis of the cDNA revealed that the precurs or contains a signal peptide of 21 amino acid residues. The mature toxin co nsists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these tox ins. The recombinant BeKm-1 was produced as a Protein A fusion product in t he periplasm of Escherichia coli. After cleavage and high performance liqui d chromatography purification, recombinant BeKm-1 displayed the same proper ties as the native toxin, Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 a nd 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 w as also studied. BeKm-1 inhibited hERG1 channels with an IC50 of 3.3 nM, bu t 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.