The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Kalium 3.0 is a comprehensive depository of natural, artificial, and labeled polypeptides acting on potassium channels.

Here, we introduce the third release of Kalium database (http://kaliumdb.org/), a manually curated comprehensive depository that accumulates data on polypeptide ligands of potassium channels. The major goal of this amplitudinous update is to summarize findings for natural polypeptide ligands of K+ channels, as well as data for the artificial derivatives of these substances obtained over the decades of exploration. We manually analyzed more than 700 original manuscripts and systematized the information on mutagenesis, production of radio- and fluorescently labeled derivatives, and the molecular pharmacology of K+ channel ligands. As a result, data on more than 1200 substances were processed and added enriching the database content fivefold. We also included the electrophysiological data obtained on the understudied and neglected K+ channels including the heteromeric and concatenated channels. We associated target channels in Kalium with corresponding entries in the official database of the International Union of Basic and Clinical Pharmacology. Kalium was supplemented with an adaptive Statistics page, where users are able to obtain actual data output. Several other improvements were introduced, such as a color code to distinguish the range of ligand activity concentrations and advanced tools for filtration and sorting. Kalium is a fully open-access database, crosslinked to other databases of interest. It can be utilized as a convenient resource containing ample up-to-date information about polypeptide ligands of K+ channels.

A scorpion toxin affecting sodium channels shows double cis-trans isomerism.

Scorpion α-toxins (α-NaTx) inhibiting the inactivation of voltage-gated sodium channels (Nav ) are a well-studied family of small proteins. We previously showed that the structure of α-NaTx specificity module responsible for selective Nav binding is governed by an interplay between the nest and niche protein motifs. Here, we report the solution structure of the toxin Lqq4 from the venom of the scorpion Leiurus quinquestriatus. Unexpectedly, we find that this toxin presents an ensemble of long-lived structurally distinct states. We unequivocally assign these states to the alternative configurations (cis-trans isomers) of two peptide bonds: V56-P57 and C17-G18; neither of the cis isomers has been described in α-NaTx so far. We argue that the native conformational space of α-NaTx is wider than assumed previously.

Methionine-isoleucine dichotomy at a key position in scorpion toxins inhibiting voltage-gated potassium channels.

Previous studies have identified some key amino acid residues in scorpion toxins blocking potassium channels. In particular, the most numerous toxins belonging to the α-KTx family and affecting voltage-gated potassium channels (KV) present a conserved K-C-X-N motif in the C-terminal half of their sequence. Here, we show that the X position of this motif is almost always occupied by either methionine or isoleucine. We compare the activity of three pairs of peptides that differ just by this residue on a panel of KV1 channels and find that toxins bearing methionine affect preferentially KV1.1 and 1.6 isoforms. The refined K-C-M/I-N motif stands out as the principal structural element of α-KTx conferring high affinity and selectivity to KV channels.

Apamin structure and pharmacology revisited.

Apamin is often cited as one of the few substances selectively acting on small-conductance Ca2+-activated potassium channels (KCa2). However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels (KCa, KV, NaV, nAChR, ASIC, and others) and confirmed its unique selectivity to KCa2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity KCa2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a KCa2-selective pharmacological tool and as a template for drug design.

The Dynamics of Antimicrobial Resistance among Enterobacteriaceae Isolates in Russia: Results of the 2012-2018 INFORM and ATLAS International Program Studies.

Background: The increasing prevalence of multidrug-resistant Enterobacteriaceae limits the range of active antimicrobial agents, thus worsening clinical outcomes. The objective of this study was to identify the trends in antimicrobial resistance for Enterobacteriaceae in Russia using the databases for the International Network for Optimal Resistance Monitoring (INFORM) and Antimicrobial Testing Leadership and Surveillance (ATLAS) studies between 2012 and 2018. Methods: This subanalysis was performed for 3811 non-duplicate clinical isolates of Enterobacteriaceae to evaluate the in vitro activity of the main classes of antibiotics against relevant clinical isolates from hospitalized patients with complicated infections of different anatomical locations. Results: The lowest susceptibility was observed for colistin (0%), ampicillin (16.4%), and ampicillin/sulbactam (31.1%), whereas the best susceptibility was observed for all combinations containing avibactam (>96%). Among individual antimicrobials, doripenem (3.2%), tigecycline (1.6%), and meropenem (5.9%) exhibited the lowest resistance. Important trends included the decreasing resistance of Enterobacteriaceae to glycylcyclines and the increasing resistance to aminoglycosides and carbapenems. K. pneumoniae strains were most aggressive in terms of the percentage of strains having multidrug resistance (8.3−18.3%, depending on location) and the percentage of ESBL-positive strains (44.8−86.8%). Conclusions: The current patterns and trends of antimicrobial resistance in different bacterial species should be taken into consideration for timely updating of clinical guidelines and local treatment protocols to ensure effective antimicrobial therapy.

Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors.

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.

AMRmap: An Interactive Web Platform for Analysis of Antimicrobial Resistance Surveillance Data in Russia.

Surveillance of antimicrobial resistance (AMR) is crucial for identifying trends in resistance and developing strategies for prevention and treatment of infections. Globally, AMR surveillance systems differ in terms of organizational principles, comprehensiveness, accessibility, and usability of data presentation. Until recently, the data on AMR in Russia were scarcely available, especially to international community, despite the fact that the large prospective multicenter surveillance in Russia was conducted and data were accumulated for over 20 years. We describe the source of data, structure, and functionality of a new-generation web platform, called AMRmap (https://amrmap.net/), for analysis of AMR surveillance data in Russia. The developed platform currently comprises susceptibility data of >40,000 clinical isolates, and the data on abundance of key resistance determinants, including acquired carbapenemases in gram-negatives, are updated annually with information on >5,000 new isolates. The AMRmap allows smart data filtration by multiple parameters and provides interactive data analysis and visualization tools: MIC and S/I/R distribution plots, time-trends and regression plots, associated resistance plots, prevalence maps, statistical significance graphs, and tables.

Structure of MeuNaTxα-1 toxin from scorpion venom highlights the importance of the nest motif.

Old world scorpions produce an abundance of toxins called α-NaTx, which interfere with the fast inactivation of voltage-gated sodium channels. Their selectivity to channels of mammals or insects depends on a part of toxin named the specificity module. We report here the spatial structure of a major and broadly active toxin MeuNaTxα-1 from the venom of Mesobuthus eupeus. Notably, its specificity module is markedly different from other α-NaTx with known 3D structure. Close inspection shows that its conformation is a result of an interplay between protein motifs such as the nest and niche, which eventually shape α-NaTx structural diversity.

Scorpion toxin MeuNaTxα-1 sensitizes primary nociceptors by selective modulation of voltage-gated sodium channels.

Venoms are a rich source of highly specific toxins, which allow the identification of novel therapeutic targets. We have now applied high content screening (HCS) microscopy to identify toxins that modulate pain sensitization signaling in primary sensory neurons of rat and elucidated the underlying mechanism. A set of venoms and fractions thereof were analyzed for their ability to activate type II protein kinase A (PKA-II) and extracellular signal-regulated kinases (ERK1/2). We identified MeuNaTxα-1, a sodium channel-selective scorpion α-toxin from Mesobuthus eupeus, which affected both PKA-II and ERK1/2. Recombinant MeuNaTxα-1 showed identical activity to the native toxin on mammalian voltage-gated sodium channels expressed in Xenopus laevis oocytes and induced thermal hyperalgesia in adult mice. The effect of MeuNaTxα-1 on sensory neurons was dose-dependent and tetrodotoxin-sensitive. Application of inhibitors and toxin mutants with altered sodium channel selectivity demonstrated that signaling activation in sensory neurons depends on NaV 1.2 isoform. Accordingly, the toxin was more potent in neurons from newborn rats, where NaV 1.2 is expressed at a higher level. Our results demonstrate that HCS microscopy-based monitoring of intracellular signaling is a novel and powerful tool to identify and characterize venoms and their toxins affecting sensory neurons.

Tuning Scorpion Toxin Selectivity: Switching From KV1.1 to KV1.3.

Voltage-gated potassium channels (KVs) perform vital physiological functions and are targets in different disorders ranging from ataxia and arrhythmia to autoimmune diseases. An important issue is the search for and production of selective ligands of these channels. Peptide toxins found in scorpion venom named KTx excel in both potency and selectivity with respect to some potassium channel isoforms, which may present only minute differences in their structure. Despite several decades of research the molecular determinants of KTx selectivity are still poorly understood. Here we analyze MeKTx13-3 (Kalium ID: α-KTx 3.19) from the lesser Asian scorpion Mesobuthus eupeus, a high-affinity KV1.1 blocker (IC50 ~2 nM); it also affects KV1.2 (IC50 ~100 nM), 1.3 (~10 nM) and 1.6 (~60 nM). By constructing computer models of its complex with KV1.1-1.3 channels we identify specific contacts between the toxin and the three isoforms. We then perform mutagenesis to disturb the identified contacts with KV1.1 and 1.2 and produce recombinant MeKTx13-3_AAAR, which differs by four amino acid residues from the parent toxin. As predicted by the modeling, this derivative shows decreased activity on KV1.1 (IC50 ~550 nM) and 1.2 (~200 nM). It also has diminished activity on KV1.6 (~1500 nM) but preserves KV1.3 affinity as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. In effect, we convert a selective KV1.1 ligand into a new specific KV1.3 ligand. MeKTx13-3 and its derivatives are attractive tools to study the structure-function relationship in potassium channel blockers.

Scots pine as a promising indicator organism for biomonitoring of the polluted environment: A case study on chronically irradiated populations.

In this paper the main results of long-term (2003-2016) observations on Scots pine populations inhabiting sites affected by the Chernobyl accident are presented. Populations growing for many years under chronic radiation exposure are characterized by the enhanced mutation rates, increased genetic diversity, changes in the gene expression and in the level of genome-wide methylation, alterations in the temporal dynamics of cytogenetic abnormalities and genetic structure of populations. However, significant changes at the genetic level had no effects on enzymatic activity, morphological abnormalities, and reproductive ability of pine trees. The results presented increase our understanding of the long-term effects of chronic radiation exposure on plant populations in the wild nature and provide important information for the management and monitoring of radioactively contaminated territories.

Kalium 2.0, a comprehensive database of polypeptide ligands of potassium channels.

Potassium channels are the most diverse group of ion channels in humans. They take vital parts in numerous physiological processes and their malfunction gives rise to a range of pathologies. In addition to small molecules, there is a wide selection of several hundred polypeptide ligands binding to potassium channels, the majority of which have been isolated from animal venoms. Until recently, only scorpion toxins received focused attention being systematically assembled in the manually curated Kalium database, but there is a diversity of well-characterized potassium channel ligands originating from other sources. To address this issue, here we present the updated and improved Kalium 2.0 that covers virtually all known polypeptide ligands of potassium channels and reviews all available pharmacological data. In addition to an expansion, we have introduced several new features to the database including posttranslational modification annotation, indication of ligand mode of action, BLAST search, and possibility of data export.

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

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 (α-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of KV1.2 (IC50 ∼0.2 nM), while its activity against KV1.1, KV1.3, and KV1.6 is 10 000, 330 and 45 000 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 (α-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.

Chronic radiation exposure modifies temporal dynamics of cytogenetic but not reproductive indicators in Scots pine populations.

Over a period of 13 years (2003-2015), reproductive and cytogenetic effects are investigated in Scots pine populations growing in the Bryansk region of Russia radioactively contaminated as a result of the Chernobyl accident. In reference populations, the frequencies of cytogenetic abnormalities are shown to change with time in a cyclic manner. In chronically exposed populations, the cyclic patterns in temporal dynamics of cytogenetic abnormalities appear to be disturbed. In addition, a tendency to decrease in the frequencies of cytogenetic abnormalities with time as well as an increase in their variability with dose rate is revealed. In contrast, no significant impact of chronic radiation exposure on the time dynamics of reproductive indexes is detected. Finally, long-term observations on chronically exposed Scots pine populations revealed qualitative differences in the temporal dynamics of reproductive and cytogenetic indicators.

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.

Influence of long-term chronic exposure and weather conditions on Scots pine populations.

Over a period of 8 years (2007-2014), we were evaluating seed quality and morphological abnormalities in Scots pine trees affected as a result of the Chernobyl accident. The calculated dose rates for the trees at the study sites varied from background values at the reference sites to 40 mGy/year at the most contaminated site. We investigated whether radioactive contamination and/or weather factors could decrease the reproductive capacity or increase the frequency of morphological abnormalities of needles in pine trees. Scots pine seeds are characterized by high interannual variability of viability, which is largely determined by weather conditions. No consistent differences in reproductive capacity were detected between the impacted and reference populations. Brachyblasts with three needles were found only in the affected populations; however, their frequency was very low and only at the very border of significance at the p < 0.10 level.

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.

Lachesana tarabaevi, an expert in membrane-active toxins.

In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18-79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present 'idealized' amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61-79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule.