Present and future distribution of the European pond turtle versus seven exotic freshwater turtles, with a focus on Eastern Europe.

Freshwater turtles are often used as terrarium pets, especially juveniles of exotic species. At the adult stage they are often released by their owners into the wild despite their high invasion potential. In Europe these thermophilic potentially invasive alien species occupy the habitats of the native European pond turtle Emys orbicularis (Linnaeus, 1758), with new records from the wild being made specifically in Eastern Europe (Latvia and Ukraine) during recent decades. Assessing the potential of alien freshwater turtles to establish in new territories is of great concern for preventing invasion risks while preserving native biodiversity in the present context of climate change. We explored this issue by identifying the present and future (by 2050) suitable habitats of the European pond turtle and several potentially invasive alien species of freshwater turtle already settled in Europe, using a geographic information system (GIS) modelling approach based on datasets from CliMond for climate, Near-global environmental information (NGEI) for freshwater ecosystems (EarthEnv) and Maxent modelling using open-access databases, data from the literature and original field data. Modelling was performed for seven species of alien freshwater turtles occurring from the extreme northern to southern borders of the European range of E. orbicularis: the pond slider Trachemys scripta (Thunberg and Schoepff, 1792), the river cooter Pseudemys concinna (Le Conte, 1830), the Florida red-bellied cooter Pseudemys nelsoni (Carr, 1938), the false map turtle Graptemys pseudogeographica (Gray, 1831), the Chinese softshell turtle Pelodiscus sinensis (Wiegmann, 1835), the Caspian turtle Mauremys caspica (Gmelin, 1774) and the Balkan terrapin Mauremys rivulata (Valenciennes, 1833). In Ukraine, the most Eastern limit of E. orbicularis distribution, were previously reported northern American originated T. scripta, M. rivulata, M. caspica, whereas in Latvia, Emys' most northern limit, were additionally reported P. concinna, P. nelsoni, G. pseudogeographica and Asia originated P. sinensis. The resulting Species Distribution Models (SDM) were of excellent performance (AUC > 0.8). Of these alien species, the most potentially successful in terms of range expansion throughout Europe were T. scripta (34.3% of potential range expansion), G. pseudogeographica (24.1%), and M. caspica (8.9%) and M. rivulata (4.3%) mainly in Eastern Europe, especially in the south of Ukraine (Odesa, Kherson, Zaporizhzhia regions, and Crimean Peninsula). Correlation between the built SDMs for the native E. orbicularis and the invasive alien T. scripta was reliably high, confirming the highly likely competition between these two species in places they cooccur. Moreover, a Multiple Regression Analysis revealed that by 2050, in most of Europe (from the western countries to Ukraine), the territory overlap between E. orbicularis and potentially invasive alien species of freshwater turtles will increase by 1.2 times, confirming higher competition in the future. Importantly, by 2050, Eastern Europe and Ukraine are predicted to be the areas with most suitable habitats for the European pond turtle yet with most limited overlap with the invasive alien species. We conclude that Eastern Europe and Ukraine are the most relevant priority conservation areas for the European pond turtle where it is now necessary to take protective measures to ensure safe habitat for this native species on the long-term.

Countrywide screening supports model-based predictions of the distribution of Batrachochytrium dendrobatidis in Ukraine.

The chytrid Batrachochytrium dendrobatidis (Bd) is a widespread fungus causing amphibian declines across the globe. Although data on Bd occurrence in Eastern Europe are scarce, a recent species distribution model (SDM) for Bd reported that western and north-western parts of Ukraine are highly suitable to the pathogen. We verified the SDM-predicted range of Bd in Ukraine by sampling amphibians across the country and screening for Bd using qPCR. A total of 446 amphibian samples (tissue and skin swabs) from 11 species were collected from 36 localities. We obtained qPCR-positive results for 33 samples including waterfrogs (Pelophylax esculentus complex) and fire- and yellow-bellied toads (Bombina spp.) from 8 localities. We found that Bd-positive localities had significantly higher predicted Bd habitat suitability than sites that were pathogen-free. Amplification and sequencing of the internal transcribed spacer (ITS) region of samples with the highest Bd load revealed matches with ITS haplotypes of the globally distributed BdGPL strain, and a single case of the BdASIA-2/BdBRAZIL haplotype. We found that Bd was non-randomly distributed across Ukraine, with infections present in the western and north-central forested peripheries of the country with a relatively cool, moist climate. On the other hand, our results suggest that Bd is absent or present in low abundance in the more continental central, southern and eastern regions of Ukraine, corroborating the model-predicted distribution of chytrid fungus. These areas could potentially serve as climatic refugia for Bd-susceptible amphibian hosts.

Interactions of the Kv1.1 Channel with Peptide Pore Blockers: A Fluorescent Analysis on Mammalian Cells.

The voltage-gated potassium channel Kv1.1, which is abundant in the CNS and peripheral nervous system, controls neuronal excitability and neuromuscular transmission and mediates a number of physiological functions in non-excitable cells. The development of some diseases is accompanied by changes in the expression level and/or activity of the channels in particular types of cells. To meet the requirements of studies related to the expression and localization of the Kv1.1 channels, we report on the subnanomolar affinity of hongotoxin 1 N-terminally labeled with Atto 488 fluorophore (A-HgTx) for the Kv1.1 channel and its applicability for fluorescent imaging of the channel in living cells. Taking into consideration the pharmacological potential of the Kv1.1 channel, a fluorescence-based analytical system was developed for the study of peptide ligands that block the ion conductivity of Kv1.1 and are potentially able to correct abnormal activity of the channel. The system is based on analysis of the competitive binding of the studied compounds and A-HgTx to the mKate2-tagged human Kv1.1 (S369T) channel, expressed in the plasma membrane of Neuro2a cells. The system was validated by measuring the affinities of the known Kv1.1-channel peptide blockers, such as agitoxin 2, kaliotoxin 1, hongotoxin 1, and margatoxin. Peptide pore blocker Ce1, from the venom of the scorpion Centruroides elegans, was shown to possess a nanomolar affinity for the Kv1.1 channel. It is reported that interactions of the Kv1.1 channel with the studied peptide blockers are not affected by the transition of the channel from the closed to open state. The conclusion is made that the structural rearrangements accompanying the channel transition into the open state do not change the conformation of the P-loop (including the selectivity filter) involved in the formation of the binding site of the peptide pore blockers.

Modeling the Distribution of the Chytrid Fungus Batrachochytrium dendrobatidis with Special Reference to Ukraine.

Amphibians are the most threatened group of vertebrates. While habitat loss poses the greatest threat to amphibians, a spreading fungal disease caused by Batrachochytrium dendrobatidis Longcore, Pessier & D.K. Nichols 1999 (Bd) is seriously affecting an increasing number of species. Although Bd is widely prevalent, there are identifiable heterogeneities in the pathogen's distribution that are linked to environmental parameters. Our objective was to identify conditions that affect the geographic distribution of this pathogen using species distribution models (SDMs) with a special focus on Eastern Europe. SDMs can help identify hotspots for future outbreaks of Bd but perhaps more importantly identify locations that may be environmental refuges ("coldspots") from infection. In general, climate is considered a major factor driving amphibian disease dynamics, but temperature in particular has received increased attention. Here, 42 environmental raster layers containing data on climate, soil, and human impact were used. The mean annual temperature range (or 'continentality') was found to have the strongest constraint on the geographic distribution of this pathogen. The modeling allowed to distinguish presumable locations that may be environmental refuges from infection and set up a framework to guide future search (sampling) of chytridiomycosis in Eastern Europe.

Top-down and bottom-up effects and relationships with local environmental factors in the water frog-helminth systems in Latvia.

Semi-aquatic European water frogs (Pelophylax spp.) harbour rich helminth infra-communities, whose effects on host population size in nature are poorly known. To study top-down and bottom-up effects, we conducted calling male water frog counts and parasitological investigations of helminths in waterbodies from different regions of Latvia, supplemented by descriptions of waterbody features and surrounding land use data. We performed a series of generalized linear model and zero-inflated negative binomial regressions to determine the best predictors for frog relative population size and helminth infra-communities. The highest-ranked (by Akaike information criterion correction, AICc) model explaining the water frog population size contained only waterbody variables, followed by the model containing only land use within 500 m, while the model containing helminth predictors had the lowest rank. Regarding helminth infection responses, the relative importance of the water frog population size varied from being non-significant (abundances of larval plagiorchiids and nematodes) to having a similar weight to waterbody features (abundances of larval diplostomids). In abundances of adult plagiorchiids and nematodes the best predictor was the host specimen size. Environmental factors had both direct effects from the habitat features (e.g., waterbody characteristics on frogs and diplostomids) and indirect effects through parasite-host interactions (impacts of anthropogenic habitats on frogs and helminths). Our study suggests the presence of synergy between top-down and bottom-up effects in the water frog-helminth system that creates a mutual dependence of frog and helminth population sizes and helps to balance helminth infections at a level that does not cause over-exploitation of the host resource.

AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels.

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated Kv1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-Kv1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-Kv1.1 with moderate dependence on pH in the 7.0-8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for Kv1.x (x = 1, 3, 6) channels and at micromolar concentrations for Kv1.2. AgTx2-GFP bound to Kv1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-Kv1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with Kv1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

Records of common species of amphibians and reptiles widespread in northern, central, western and southern Ukraine.

Background: The dataset includes records of amphibian and reptile species from northern, central, western and southern Ukraine made by Ukrainian herpetologist O. D. Nekrasova during her field trips in the period from 1996 to 2022. Chosen species were not included in the latest published edition of the Red Data Book of Ukraine (2009) and in the latest lists of such species prepared in 2019. The species mentioned in this dataset are characterised by wide range within the country, covering more than 70% of its territory according to spatial distribution modelling (GIS-modelling) made with the help of Maxent software. New information: The dataset highlights records of eight common species of herpetofauna of Ukraine collected by the first author for the last 26 years. Within the period from 1996 to 2022, O. D. Nekrasova collected and studied information and material on the herpetofauna of the northern, central, western and southern parts of Ukraine from a total of 3960 cadastral points (1707 - for three species of reptiles and 2253 - for five species of amphibians). These records, being now available for the international scientific community, will fill the gap in updated records of the mentioned species, being potentially useful for GIS-modelling, distribution modelling, clarification of conservation lists of national and local importance, further assessment of impact of the war on native biota etc.

Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells.

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels' structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the "mix and measure", format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K+-conducting pore, and analyzing their affinity.

Atto488-Agitoxin 2-A Fluorescent Ligand with Increased Selectivity for Kv1.3 Channel Binding Site.

Fluorescently labeled peptide blockers of ion channels are useful probes in studying the localization and functioning of the channels and in the performance of a search for new channel ligands with bioengineering screening systems. Here, we report on the properties of Atto488-agitoxin 2 (A-AgTx2), a derivative of the Kv1 channel blocker agitoxin 2 (AgTx2), which was N-terminally labeled with Atto 488 fluorophore. The interactions of A-AgTx2 with the outer binding sites of the potassium voltage-gated Kv1.x (x = 1, 3, 6) channels were studied using bioengineered hybrid KcsA-Kv1.x (x = 1, 3, 6) channels. In contrast to AgTx2, A-AgTx2 was shown to lose affinity for the Kv1.1 and Kv1.6 binding sites but to preserve it for the Kv1.3 site. Thus, Atto488 introduces two new functionalities to AgTx2: fluorescence and the selective targeting of the Kv1.3 channel, which is known for its pharmacological significance. In the case of A-AgTx2, fluorescent labeling served as an alternative to site-directed mutagenesis in modulating the pharmacological profile of the channel blocker. Although the affinity of A-AgTx2 for the Kv1.3 binding site was decreased as compared to the unlabeled AgTx2, its dissociation constant value was within a low nanomolar range (4.0 nM). The properties of A-AgTx2 allow one to use it for the search and study of Kv1.3 channel blockers as well as to consider it for the imaging of the Kv1.3 channel in cells and tissues.

GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers.

Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP-MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP-MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP-MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP-MgTx as a component of an analytical system based on the hybrid KcsA-Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP-MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.

An Extended dataset of occurrences of species listed in Resolution 6 of the Bern Convention from Ukraine.

Background: The dataset includes georeferenced occurrences of species listed in Annex I of Resolution 6 of the Bern Convention and, partly, in the Red Data Book of Ukraine. The dataset was compiled within the work of NGO "Ukrainian Nature Conservation Group" aimed to prepare a Shadow list of Emerald Network (European network Areas of Special Conservation Interest) in Ukraine - newly proposed territories aimed at conservation of particular species and habitats mentioned in Resolution 4 and 6 of the Bern Convention. The list was prepared in 2017-2020 for expanding the already existing Emerald Network of Ukraine. Based on actual registrations of flora and fauna collected and gathered by scientists and naturalists in a form of dataset, which is described in the following paper. New information: This dataset provides information about 29,938 occurrences of species from the territory of Ukraine listed in Annex I of Resolution 6 of the Bern Convention, as well as in the Red Data Book of Ukraine. This is the largest public dataset on occurrences of rare and endangered species from Ukraine till now. Data presented here laid the foundations for the proposal of 106 approved Emerald Network sites (2019), as well as for 148 Emerald Network sites that were nominated in 2020. New insights on the endemic species Centaureapseudoleucolepis Kleopow is provided, which was previously considered to be extinct, according to the IUCN Red List.

Bioengineered System for High Throughput Screening of Kv1 Ion Channel Blockers.

Screening drug candidates for their affinity and selectivity for a certain binding site is a crucial step in developing targeted therapy. Here, we created a screening assay for receptor binding that can be easily scaled up and automated for the high throughput screening of Kv channel blockers. It is based on the expression of the KcsA-Kv1 hybrid channel tagged with a fluorescent protein in the E. coli membrane. In order to make this channel accessible for the soluble compounds, E. coli were transformed into spheroplasts by disruption of the cellular peptidoglycan envelope. The assay was evaluated using a hybrid KcsA-Kv1.3 potassium channel tagged with a red fluorescent protein (TagRFP). The binding of Kv1.3 channel blockers was measured by flow cytometry either by using their fluorescent conjugates or by determining the ability of unconjugated compounds to displace fluorescently labeled blockers with a known affinity. A fraction of the occupied receptor was calculated with a dedicated pipeline available as a Jupyter notebook. Measured binding constants for agitoxin-2, charybdotoxin and kaliotoxin were in firm agreement with the earlier published data. By using a mid-range flow cytometer with manual sample handling, we measured and analyzed up to ten titration curves (eight data points each) in one day. Finally, we considered possibilities for multiplexing, scaling and automation of the assay.

Chimeras of KcsA and Kv1 as a bioengineering tool to study voltage-gated potassium channels and their ligands.

Chimeric potassium channels KcsA-Kv1, which are among the most intensively studied hybrid membrane proteins to date, were constructed by replacing a part of the pore domain of bacterial potassium channel KcsA (K channel of streptomyces A) with corresponding regions of the mammalian voltage-gated potassium channels belonging to the Kv1 subfamily. In this way, the pore blocker binding site of Kv1 channels was transferred to KcsA, opening up possibility to use the obtained hybrids as receptors of Kv1-channel pore blockers of different origin. In this review the recent progress in KcsA-Kv1 channel design and applications is discussed with a focus on the development of new assays for studying interactions of pore blockers with the channels. A summary of experimental data is presented demonstrating that hybrid channels reproduce the blocker-binding profiles of parental Kv1 channels. It is overviewed how the KcsA-Kv1 chimeras are used to get new insight into the structure of potassium channels, to determine molecular basis for high affinity and selectivity of binding of peptide blockers to Kv1 channels, as well as to identify new peptide ligands.

Comparative Femtosecond Spectroscopy of Primary Photoreactions of Exiguobacterium sibiricum Rhodopsin and Halobacterium salinarum Bacteriorhodopsin.

The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bRPM) and in recombinant form (bRrec). The primary intermediates of the ESR photocycle were similar to intermediates I, J, and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J, and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bRPM and 0.74 ps in bRrec. The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site.

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Helminth Species and Infracommunities in Frogs Pelophylax ridibundus and P. esculentus (Amphibia: Ranidae) in Northern Ukraine.

PURPOSE: The study aims to characterise and compare the helminth assemblages and helminth infracommunities in the marsh frog, Pelophylax ridibundus and the edible frog, P. esculentus collected in the northern part of Ukraine. METHODS: Occurrence and abundance of the helminths were analysed by calculating the prevalence, intensity, and mean abundance of infection; similarities between the infracommunities were estimated by the Bray-Curtis index and visualised using nMDS plots. Dissimilarities were estimated using the ANOSIM and SIMPER routines. RESULTS: In total, 27 helminth species were found in 143 frogs. Pelophylax ridibundus (n = 86) harboured 20 species of helminths, 24 species were found in P. esculentus (n = 57), and 17 species were shared by the two hosts. Oswaldocruzia bialata and larval Strigea sp. were absent in P. ridibundus, while they reached the prevalence of 30% and 10%, respectively, in P. esculentus. Cosmocerca ornata, Diplodiscus subclavatus, Opisthioglyphe ranae, and Codonocephalus urniger had significantly larger prevalence in P. ridibundus, whereas Haematoloechus asper was found to be more prevalent in P. esculentus. Acanthocephalus ranae, Icosiella neglecta, Haematoloechus variegatus, Pleurogenes claviger, Pleurogenoides medians, and Prosotocus confusus were equally common in both hosts. Helminth infracommunities in the two hosts had identical species richness (1-10 species, 4 on average); abundance was significantly higher in P. ridibundus. CONCLUSIONS: Helminth assemblages of the two hosts in northern Ukraine are rather similar; however, small but significant differences were found in their species composition, parameters of infection in some species, and structure of helminth infracommunities.

Scorpion toxins interact with nicotinic acetylcholine receptors.

Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage-gated ion channels, while most snake neurotoxins target ligand-gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α-bungarotoxin binding to both muscle-type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK-1 (α-KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ-KTx family) from Heterometrus laoticus, both being blockers of voltage-gated potassium channels. With an IC50 of 1.6 µm, OSK1 inhibits acetylcholine-induced current through mouse muscle-type nAChR heterologously expressed in Xenopus oocytes. Other well-characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity.

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.

Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels.

We have recently developed a simple and effective bioengineering approach to large-scale production of alpha-KTx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. This approach was successfully approved for different peptides containing three disulfide bonds. To extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction with tobacco etch virus (TEV) protease need to be found. For this, the interplay between efficiency of TEV hydrolysis and sensitivity of the target peptides to disulfide reducing agents was studied, and optimized protocols of TEV cleavage reaction were worked out. Maurotoxin and hetlaxin were produced in a folded form avoiding in vitro renaturation step with yields of 14 and 12 mg/liter of culture, respectively.

Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels.

Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.