Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1 AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY

Alexey I. Kuzmenkov; Alexander A. Vassilevski; Kseniya S. Kudryashova; Oksana V. Nekrasova; Steve Peigneur; Jan Tytgat; Alexey V. Feofanov; Mikhail P. Kirpichnikov; Eugene V. Grishin

doi:10.1074/jbc.M115.637611

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

AN INTEGRATED TRANSCRIPTOMIC AND PROTEOMIC STUDY*

From the ^‡Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia,
the ^§Biological Faculty, Lomonosov Moscow State University, Moscow 119992, Russia, and
the ^¶Laboratory of Toxicology and Pharmacology, University of Leuven, Leuven 3000, Belgium

↵1 To whom correspondence should be addressed. E-mail: avas{at}ibch.ru.

Background: Scorpion venoms are an ample source of toxins targeting potassium channels.

Results: A comprehensive search for new toxins was performed by combining transcriptomics and peptidomics with a fluorescent test system.

Conclusion: We identified five new high affinity potassium channel blockers in the venom of Mesobuthus eupeus.

Significance: The proposed integrated approach is of general utility for potassium channel pharmacology.

Abstract

The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus, and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here, we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands, and we compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis, we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β and inhibitor cystine knot types of fold. M. eupeus venom was then separated to individual components by multistage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore binding activity during venom separation. As a result, eight high affinity Kv1.1 channel blockers were identified, including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC₅₀ in the range of 1–780 nm) by the two-electrode voltage clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs.

Footnotes

↵2 Supported by grants G.0433.12 from F.W.O. Vlaanderen, Grant OT/12/081 from KU Leuven, and IUAP 7/10 from the Inter-University Attraction Poles Program, Belgian State, Belgian Science Policy.
↵* This work was supported in part by Russian Foundation for Basic Research Grants 14-04-32091 and 13-04-01857, Program for Fundamental Research of the Presidium of Russian Academy of Sciences No. 24, and by the Moscow State University Program of Development.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) KF612484–KF612542.
The protein sequences of KTx have been submitted to the UniProt Knowledgebase (UniProtKB) under the accession numbers C0HJQ4–C0HJQ8.