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.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, 117997, GSP-7, Moscow, Russia. july@ibch.ru
authors.forename: Y V
authors.initials: YV
authors.lastname: Korolkova
authors.forename: S A
authors.initials: SA
authors.lastname: Kozlov
authors.forename: A V
authors.initials: AV
authors.lastname: Lipkin
authors.forename: K A
authors.initials: KA
authors.lastname: Pluzhnikov
authors.forename: J K
authors.initials: JK
authors.lastname: Hadley
authors.forename: A K
authors.initials: AK
authors.lastname: Filippov
authors.forename: D A
authors.initials: DA
authors.lastname: Brown
authors.forename: K
authors.initials: K
authors.lastname: Angelo
authors.forename: D
authors.initials: D
authors.lastname: Strøbaek
authors.forename: T
authors.initials: T
authors.lastname: Jespersen
authors.forename: S P
authors.initials: SP
authors.lastname: Olesen
authors.forename: B S
authors.initials: BS
authors.lastname: Jensen
authors.forename: E V
authors.initials: EV
authors.lastname: Grishin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D027682:Cation Transport Proteins; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D020128:Inhibitory Concentration 50; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics*; D012605:Scorpions; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014407:Tumor Cells, Cultured
chemical_list: C425305:BeKm-1 toxin; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C092509:KCNH6 protein, human; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; C000606915:Kcnh2 protein, mouse; C496910:Kcnq1 protein, mouse; C496911:Kcnq1 protein, rat; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG