Alexey I. Kuzmenkov

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

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. 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.

Diversity of Potassium Channel Ligands: Focus on Scorpion Toxins

Potassium (K+) channels are a widespread superfamily of integral membrane proteins that mediate selective transport of K+ ions through the cell membrane. They have been found in all living organisms from bacteria to higher multicellular animals, including humans. Not surprisingly, K+ channels bind ligands of different nature, such as metal ions, low molecular mass compounds, venom-derived peptides, and antibodies. Functionally these substances can be K+ channel pore blockers or modulators. Representatives of the first group occlude the channel pore, like a cork in a bottle, while the second group of ligands alters the operation of channels without physically blocking the ion current. A rich source of K+ channel ligands is venom of different animals: snakes, sea anemones, cone snails, bees, spiders, and scorpions. More than a half of the known K+ channel ligands of polypeptide nature are scorpion toxins (KTx), all of which are pore blockers. These compounds have become an indispensable molecular tool for the study of K+ channel structure and function. A recent special interest is the possibility of toxin application as drugs to treat diseases involving K+ channels or related to their dysfunction (channelopathies).

Kalium: a database of potassium channel toxins from scorpion venom

Kalium (http://kaliumdb.org/) is a manually curated database that accumulates data on potassium channel toxins purified from scorpion venom (KTx). This database is an open-access resource, and provides easy access to pages of other databases of interest, such as UniProt, PDB, NCBI Taxonomy Browser, and PubMed. General achievements of Kalium are a strict and easy regulation of KTx classification based on the unified nomenclature supported by researchers in the field, removal of peptides with partial sequence and entries supported by transcriptomic information only, classification of β-family toxins, and addition of a novel λ-family. Molecules presented in the database can be processed by the Clustal Omega server using a one-click option. Molecular masses of mature peptides are calculated and available activity data are compiled for all KTx. We believe that Kalium is not only of high interest to professional toxinologists, but also of general utility to the scientific community. Database URL: http://kaliumdb.org/

Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study

AbstractHuman voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K+-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch–clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the “mix and read” mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom. FigureThe scheme of a fluorescent analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K+-conducting pore.

C-Terminal residues in small potassium channel blockers OdK1 and OSK3 from scorpion venom fine-tune the selectivity

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity.

Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels

Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

KV1.2 channel-specific blocker from Mesobuthus eupeus scorpion venom: structural basis of selectivity

ABSTRACT Scorpion venom is an unmatched source of selective high‐affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage‐gated potassium channel KV1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11–1 (&agr;‐KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11–1 is a high‐affinity blocker of KV1.2 (IC50 ˜0.2nM), while its activity against KV1.1, KV1.3, and KV1.6 is 10000, 330 and 45000 fold lower, respectively, as measured using the voltage‐clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11–1 to its natural analog MeKTx11–3 (&agr;‐KTx 1.17) having 15 times lower activity and reduced selectivity to KV1.2. We produced MeKTx11–1 and MeKTx11–3 as well as their mutants MeKTx11–1(G9V) and MeKTx11–1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11–1 specificity were identified by molecular modeling of the toxin–channel complexes. Confirming our molecular modeling predictions, site‐directed transfer of these elements from the pore region of KV1.2 to KV1.3 resulted in the enhanced sensitivity of mutant KV1.3 channels to MeKTx11–1. We conclude that MeKTx11–1 may be used as a selective tool in neurobiology. HIGHLIGHTSMeKTx11–1 from Mesobuthus eupeus is a selective high‐affinity ligand of KV1.2.MeKTx11–3 is a natural analog of MeKTx11–1 that has lower activity and selectivity.Molecular modeling identified key structural elements underlying the selectivity.Mutant KV1.3 channels with residues from KV1.2 presented enhanced sensitivity.The P‐S6 loop is the most important channel region for selective toxin activity.

Labelled animal toxins as selective molecular markers of ion channels: Applications in neurobiology and beyond

Animal toxins are traditional and indispensible molecular tools that find application in different fields of biochemistry, neurobiology and pharmacology. These compounds possess several outstanding properties such as high affinity and selectivity with respect to particular molecular targets, most importantly ion channels and neuroreceptors, and stability. In addition to using toxins per se, a wide variety of labelled modifications have been obtained including radioactive and fluorescent derivatives. Here, we discuss the major types of labelled toxins, methods of their production and principal possibilities of application ranging from receptor localization and visualization to development of screening systems and diagnostic tools, and drug discovery.

Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

Artificial KcsA-Kv1.x (x = 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical “mix and read” systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.