Laboratory of ligand-receptor interactions

Department of molecular bases of neurosignalization

Head: Igor' Kasheverov, D.Sc

ligand-gated ion channels, nicotinic receptors, glycine receptor, peptide synthesis, radioligand assay, electrophysiology

Laboratory was organised in 2009 and is headed by Dr. Igor Kasheverov

The main scope of the Ligand-receptor interactions lab is structure-function relationships of natural and artificial peptide ligands of several Cys-loop receptors (nAChR, GlyR and GABAA). Construction of completely new active compounds based on known anticholinergic ligands is also in the field of our interest.

The lab conducts computer-aided design of new compounds based on diverse conotoxins of different structural classes and modelling of their complexes with nicotinic receptors (nAChR) and acetylcholine-binding proteins (AChBP). We have at our disposal eqipment for peptide synthesis, chromatography and mass-spectrometry, which is used for synthesys and characterization of active compounds. We also have electrophysiology and radioligand assay eqipment for ligans activities research.

Our lab contacts has lots of international collaborations in Germany, France, Finland, Greece, China and Vietnam. And we are open to new contacts!

Previously, more than forty diverse conotoxins analogs were synthetized and studied in the lab. Partly these studies could be represented by following reviews:

Tsetlin V., Utkin Y., Kasheverov I. (2009). Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptorsBiochem Pharmacol. 78(7), 720—731

Kasheverov I.E., Utkin Y.N., Tsetlin V.I. (2009). Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptorsCurr Pharm Des. 15(21), 2430—2452.

Igor' Kasheverov, D.Scdepart.
Yana Makarovar.
Maxim Zhmak, Ph.D.r.
Elena Kryukova, Ph.D.r.
Denis Kudryavtsev, Ph.D.r.
Dmitry KuzminPhD
Igor IvanovPhD
Dmitry LebedevPhD

Former members:

Alexej KhrushevPhD

Selected publications

  1. Kasheverov I.E., Chugunov A.O., Kudryavtsev D.S., Ivanov I.A., Zhmak M.N., Shelukhina I.V., Spirova E.N., Tabakmakher V.M., Zelepuga E.A., Efremov R.G., Tsetlin V.I. (2016). High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method. Sci Rep 6, 36848 [+]

    Despite some success for small molecules, elucidating structure-function relationships for biologically active peptides - the ligands for various targets in the organism - remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules' surface as projection maps, which enables the exposure of the structure-function dependencies. Here, we use PST to uncover the "activity pattern" in α-conotoxins - neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by "standard" structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.

  2. Lyukmanova E.N., Shulepko M.A., Shenkarev Z.O., Kasheverov I.E., Chugunov A.O., Kulbatskii D.S., Myshkin M.Y., Utkin Y.N., Efremov R.G., Tsetlin V.I., Arseniev A.S., Kirpichnikov M.P., Dolgikh D.A. (2016). Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors. Toxicon 119, 274–9 [+]

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.

  3. Lyukmanova E.N., Shulepko M.A., Kudryavtsev D., Bychkov M.L., Kulbatskii D.S., Kasheverov I.E., Astapova M.V., Feofanov A.V., Thomsen M.S., Mikkelsen J.D., Shenkarev Z.O., Tsetlin V.I., Dolgikh D.A., Kirpichnikov M.P. (2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

  4. Kudryavtsev D.S., Shelukhina I.V., Son L.V., Ojomoko L.O., Kryukova E.V., Lyukmanova E.N., Zhmak M.N., Dolgikh D.A., Ivanov I.A., Kasheverov I.E., Starkov V.G., Ramerstorfer J., Sieghart W., Tsetlin V.I., Utkin Y.N. (2015). Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic GABA Receptors. J. Biol. Chem. , [+]

    Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins (TFTs) from snake venoms, specifically stained the α1β3γ2 receptor; at 10 μM α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nM) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other TFTs: long α-neurotoxin Ls III and non-conventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and non-competitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accomodating under loop C of the receptors.

  5. Kasheverov I.E., Kudryavtsev D.S., Ivanov I.A., Zhmak M.N., Chugunov A.O., Tabakmakher V.M., Zelepuga E.A., Efremov R.G., Tsetlin V.I. (2015). Rational design of new ligands for nicotinic receptors on the basis of α-conotoxin PnIA. Dokl. Biochem. Biophys. 461, 106–9 [+]

    A variety of different subtypes of nicotinic acetylcholine receptors (nAChRs) and their involvement in a number of diseases and pathologies (Parkinson’s and Alzheimer’s diseases, schizophrenia, myasthenia, nicotine addiction) dictates the needs in potent and selective ligands for each subtype. These ligands can be used as a tool for detection and characterization of the distinct nAChR subtypes, as well as be the basis for drug design. Novel cholinergic ligands can emerge in the result of search among natural sources or design (with the use of modern computer modeling) on the basis of known molecules. The significance of the first way was confirmed in our hands by the detection of affinities of a set of marine alkaloids from sponges and ascidians towards some receptor subtypes. The most active of them — makaluvamines — showed micromolar affinity for muscle and neuronal α7 nAChRs. Application of the recently presented Protein Surface Topography method to known natural antagonist of some neuronal nAChRs — α-conotoxin PnIA — resulted in design of new potent analogs with nanomolar affinities for α7 nAChR. Radioactive derivatives of these analogs were successfully applied in radioligand tests for characterization of novel compounds and could be perspective as well for detection of α7 nAChR in the various preparations. Combining these two ways (search and design) was demonstrated in the synthesis of small peptide compounds on the basis of discovered by us in venom of Burmese Viper linear peptide azemiopsin — powerful blocker of muscle-type nAChRs. Some designed peptides have retained a certain affinity to receptor and showed high practical potential: in the absence of toxicity they contributed to the reduction of facial wrinkles (patent application for cosmetic use RU2013102410; PCT/RU2014/000032).

  6. Shelukhina I., Paddenberg R., Kummer W., Tsetlin V. (2014). Functional expression and axonal transport of α7 nAChRs by peptidergic nociceptors of rat dorsal root ganglion. Brain Struct Funct , [+]

    In recent pain studies on animal models, α7 nicotinic acetylcholine receptor (nAChR) agonists demonstrated analgesic, anti-hyperalgesic and anti-inflammatory effects, apparently acting through some peripheral receptors. Assuming possible involvement of α7 nAChRs on nociceptive sensory neurons, we investigated the morphological and neurochemical features of the α7 nAChR-expressing subpopulation of dorsal root ganglion (DRG) neurons and their ability to transport α7 nAChR axonally. In addition, α7 receptor activity and its putative role in pain signal neurotransmitter release were studied. Medium-sized α7 nAChR-expressing neurons prevailed, although the range covered all cell sizes. These cells accounted for one-fifth of total medium and large DRG neurons and <5 % of small ones. 83.2 % of α7 nAChR-expressing DRG neurons were peptidergic nociceptors (CGRP-immunopositive), one half of which had non-myelinated C-fibers and the other half had myelinated Aδ- and likely Aα/β-fibers, whereas 15.2 % were non-peptidergic C-fiber nociceptors binding isolectin B4. All non-peptidergic and a third of peptidergic α7 nAChR-bearing nociceptors expressed TRPV1, a capsaicin-sensitive noxious stimulus transducer. Nerve crush experiments demonstrated that CGRPergic DRG nociceptors axonally transported α7 nAChRs both to the spinal cord and periphery. α7 nAChRs in DRG neurons were functional as their specific agonist PNU282987 evoked calcium rise enhanced by α7-selective positive allosteric modulator PNU120596. However, α7 nAChRs do not modulate neurotransmitter CGRP and glutamate release from DRG neurons since nicotinic ligands affected neither their basal nor provoked levels, showing the necessity of further studies to elucidate the true role of α7 nAChRs in those neurons.

  7. Kudryavtsev D., Makarieva T., Utkina N., Santalova E., Kryukova E., Methfessel C., Tsetlin V., Stonik V., Kasheverov I. (2014). Marine natural products acting on the acetylcholine-binding protein and nicotinic receptors: from computer modeling to binding studies and electrophysiology. Mar Drugs 12 (4), 1859–75 [+]

    For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5-1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.

  8. Vulfius C.A., Kasheverov I.E., Starkov V.G., Osipov A.V., Andreeva T.V., Filkin S.Y., Gorbacheva E.V., Astashev M.E., Tsetlin V.I., Utkin Y.N. (2014). Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2. PLoS ONE 9 (12), e115428 [+]

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  9. Utkin Y.N., Weise C., Kasheverov I.E., Andreeva T.V., Kryukova E.V., Zhmak M.N., Starkov V.G., Hoang N.A., Bertrand D., Ramerstorfer J., Sieghart W., Thompson A.J., Lummis S.C., Tsetlin V.I. (2012). Azemiopsin from Azemiops feae viper venom, a novel polypeptide ligand of nicotinic acetylcholine receptor. J. Biol. Chem. 287 (32), 27079–86 [+]

    Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC(50) 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC(50) 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1εδ) than the fetal form (α1β1γδ), EC(50) being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABA(A) (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT(3) receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3-6, 8-11, and 13-14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

  10. Tsetlin V., Kuzmin D., Kasheverov I. (2011). Assembly of nicotinic and other Cys-loop receptors. J. Neurochem. , [+]

    J. Neurochem. (2011) 10.1111/j.1471-4159.2010.07060.x ABSTRACT: The Cys-loop receptor family consists of nicotinic acetylcholine receptors (nAChR), glycine receptor, GABA-A and some other receptors. They fulfill a plethora of functions, whereas their malfunctioning is associated with many diseases. All three domains - extracellular ligand-binding, membrane and cytoplasmic - of these ligand-gated ion channels play important roles in the receptor assembly, delivery to the membrane surface and functional activity. In this study, we discuss the role of these domains in the assembly of the Cys-loop receptors, most comprehensively for the nAChRs. Heterologous expression and mutations of large N-terminal fragments of various subunits demonstrated their leading role in the assembly, although getting an isolated well-structured pentameric ligand-binding domain is still a problem. The long intracellular loop between transmembrane fragments M3 and M4 participates in modulating the receptor function and in clusterization of the receptor complexes because of interactions with the intracellular proteins. The transmembrane fragments play different functional roles: M2 fragments outline the channel, M4 fragments, the most remote from the channel, modulate the channel function and contact the lipid environment. The interactions of aromatic residues in the M1 and M3 fragments with those of M4 are important for the correct assembly of glycine receptor α1 subunit and for the formation of functional pentaoligomer. The role of the three receptor domains is discussed in the light of electron microscopy structure of the Torpedo nAChR, X-ray structures of agonist and antagonist complexes with the acetylcholine-binding proteins and the X-ray structures of the prokaryotic Cys-loop receptors.

  11. Kasheverov I.E., Zhmak M.N., Khruschov A.Y., Tsetlin V.I. (2011). Design of new α-conotoxins: from computer modeling to synthesis of potent cholinergic compounds. Mar Drugs 9 (10), 1698–714 [+]

    A series of 14 new analogs of α-conotoxin PnIA Conus pennaceus was synthesized and tested for binding to the human α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine-binding proteins (AChBP) Lymnaea stagnalis and Aplysia californica. Based on computer modeling and the X-ray structure of the A. californica AChBP complex with the PnIA[A10L, D14K] analog, single and multiple amino acid substitutions were introduced in α-conotoxin PnIA aimed at compounds of higher affinity and selectivity. Three analogs, PnIA[L5H], PnIA[A10L, D14K] and PnIA[L5R, A10L, D14R], have high affinities for AChBPs or α7 nAChR, as found in competition with radioiodinated α-bungarotoxin. That is why we prepared radioiodinated derivatives of these α-conotoxins, demonstrated their specific binding and found that among the tested synthetic analogs, most had almost 10-fold higher affinity in competition with radioactive α-conotoxins as compared to competition with radioactive α-bungarotoxin. Thus, radioiodinated α-conotoxins are a more sensitive tool for checking the activity of novel α-conotoxins and other compounds quickly dissociating from the receptor complexes.

  12. Haeger S., Kuzmin D., DetroDassen S., Lang N., Kilb M., Tsetlin V., Betz H., Laube B., Schmalzing G. (2010). An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors. Nat. Struct. Mol. Biol. 17 (1), 90–8 [+]

    Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

  13. Kasheverov I.E., Zhmak M.N., Fish A., Rucktooa P., Khruschov A.Y., Osipov A.V., Ziganshin R.H., D'hoedt D., Bertrand D., Sixma T.K., Smit A.B., Tsetlin V.I. (2009). Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor. J. Neurochem. 111 (4), 934–44 [+]

    alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha7 nAChR-targeting alpha-conotoxin ImI, blocked alpha7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [(125)I]-alpha-bungarotoxin from human alpha7 nAChRs in the cell line GH(4)C(1) (IC(50) 17 and 23 microM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC(50) 2.0-9.0 microM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K(d) and IC(50) 2.5-8.2 microM). On Torpedo nAChR, alpha-conotoxin [(125)I]-ImII(W10Y) revealed specific binding (K(d) 1.5-6.1 microM) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC(50) 2.7, 2.2 and 3.1 microM, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [(125)I]-ImII(W10Y) only at higher concentrations (IC(50)> or = 90 microM), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.

  14. Kasheverov I.E., Zhmak M.N., Vulfius C.A., Gorbacheva E.V., Mordvintsev D.Y., Utkin Y.N., van Elk R., Smit A.B., Tsetlin V.I. (2006). Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors. FEBS J. 273 (19), 4470–81 [+]

    Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.

  15. Kasheverov I.E., Chiara D.C., Zhmak M.N., Maslennikov I.V., Pashkov V.S., Arseniev A.S., Utkin Y.N., Cohen J.B., Tsetlin V.I. (2006). alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor. FEBS J. 273 (7), 1373–88 [+]

    alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined.

  16. Celie P.H., Kasheverov I.E., Mordvintsev D.Y., Hogg R.C., van Nierop P., van Elk R., van Rossum-Fikkert S.E., Zhmak M.N., Bertrand D., Tsetlin V.I., Sixma T.K., Smit A.B. (2005). Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582–8 [+]

    For the first time the X-ray structure of acetylcholine-binding protein in complex with conotoxin was established. The data obtained allowed to reveal the main structural features of conotoxin interaction with acetylcholine receptors.


Igor' Kasheverov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 33, office. 236
  • Phone: +7(495)330-73-74
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Development of a new technique based on calcium imaging and functional characterization of mutant α7/α9 nAChRs with the use of this technique. (2017-12-15)

On the basis of the calcium imaging method, we developed a new technique that allows to effectively express functionally active "problematic" subtypes of nicotinic receptors (nAChRs) in cell lines. It involves co-expressing with the appropriate receptor subtype a chaperone and a fluorescent calcium sensor Case12. This technique allowed us to obtain 6 mutant forms of α7 nAChR with selected single substitutions of amino acid residues from α9 nAChR subtype. All the mutants together with the wild-type receptors were analyzed for affinity to acetylcholine and epibatidine using the developed technique. This helped to identify two key mutations - L119D and F187S which are responsible for selectivity of these nAChR subtypes to above-mentioned ligands. Computer simulations showed a significant change in the arrangement of ligands’ molecules in binding sites of these two mutant forms of the receptor, explaining the data obtained.


  1. Shelukhina I., Spirova E., Kudryavtsev D., Ojomoko L., Werner M., Methfessel C., Hollmann M., Tsetlin V. (2017). Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants. PLoS ONE 12 (8), e0181936 [+]

    Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117-119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.


SLURP-1 (81 amino-acid residues, 5 disulfides), identical in the amino-acid sequence to the endogenous human protein, has been synthesized and shown to differ from all known recombinant (2017-12-15)

SLURP-1 (81 amino acid residues, 5 disulfides) has been synthesized with the amino acid sequence identical to that of the endogenous human toxin-like protein. 1H-NMR revealed the same structure as in the recombinant rSLURP-1 bearing additional N-terminal Met0. These proteins  have some differences in molecular dynamics, but differ greatly in their activity towards distinct subtypes of nicotinic receptors. Our work in general stresses the necessity of maximal approach to the structure of naturally-occurring proteins to solve the mechanisms of their endogenous activities and choosing appropriate medical applications.  


  1. Durek , Shelukhina V., Tae , Thongyoo , Spirova N., Kudryavtsev S., Kasheverov E., Faure , Corringer , Craik J., Adams J., Tsetlin I. (2036). Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors. Sci. Rep. , [+]

    SLURP-1 (81 amino acid residues, 5 disulfides) has been synthesized with the amino acid sequence identical to that  of the endogenous human toxin-like protein.   1H-NMR revealed the same structure as in the recombinant rSLURP-1 bearing additional N-terminal Met0. These proteins  have some differences in molecular dynamics, but differ greatly in their activity towards distinct subtypes of nicotinic receptors. Our work in general stresses the necessity of maximal approach to the structure of naturally-occurring proteins  to solve the mechanisms of their endogenous activities and choosing appropriate medical applications.