Denis B Tikhonov

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (85)240.98 Total impact

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    S. L. Malkin · K. Kh. Kim · D. B. Tikhonov · A. V. Zaitsev
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    ABSTRACT: Quantum analysis of postsynaptic currents is important for fundamental and applied studies of synaptic transmission and plasticity. In the present work, we investigated the possibility of using the characteristics of spontaneous excitatory postsynaptic currents (EPSCs) for estimation of quantum parameters of excitatory synaptic transmission in different types of neurons from rat prefrontal cortex slices. By blocking spontaneous spiking activity in slices by tetrodotoxin, we showed that spontaneous and miniature EPSCs in the prefrontal cortex neurons did not differ in their properties. Therefore, both spontaneous and miniature responses can be used for estimation of quantum parameters of excitatory synaptic transmission in this preparation. We also revealed that excitatory spontaneous responses of pyramidal cells were two times lower by amplitude, had a twice lower coefficient of variation and exhibited much slower kinetics than responses of the fast-spiking and regular-spiking interneurons. Possible mechanisms of these differences are discussed.
    Journal of Evolutionary Biochemistry and Physiology 11/2015; 50(6):506-514. DOI:10.1134/S0022093014060052 · 0.37 Impact Factor
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    ABSTRACT: Properties of excitatory synaptic responses in fast-spiking interneurons (FSIs) and pyramidal neurons (PNs) are different; however, the mechanisms and determinants of this diversity have not been fully investigated. In the present study, voltage-clamp recording of miniature excitatory post-synaptic currents (mEPSCs) was performed of layer 2-3 FSIs and PNs in the medial prefrontal cortex of rats aged 19-22 days. The average mEPSCs in the FSIs exhibited amplitudes that were two times larger than those of the PNs and with much faster rise and decay. The mEPSC amplitude distributions in both cell types were asymmetric and in FSIs, the distributions were more skewed and had two-times larger coefficients of variation than in the PNs. In PNs but not in FSIs, the amplitude distributions were fitted well by different skewed unimodal functions that have been used previously for this purpose. In the FSIs, the distributions were well approximated only by a sum of two such functions, suggesting the presence of at least two subpopulations of events with different modal amplitudes. According to our estimates, two-thirds of the mEPSCs in FSIs belong to the high-amplitude subpopulation, and the modal amplitude in this subpopulation is approximately two times larger than that in the low-amplitude subpopulation. Using different statistical models, varying binning size, and data subsets, we confirmed the robustness and consistency of these findings. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
    Neuroscience 06/2015; 301. DOI:10.1016/j.neuroscience.2015.06.034 · 3.36 Impact Factor
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    E I Nagaeva · N N Potapieva · D B Tikhonov
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    ABSTRACT: Acid-sensing ion channels (ASICs) are widely distributed in both the central and peripheral nervous systems of vertebrates. The pharmacology of these receptors remains poorly investigated, while the search for new ASIC modulators is very important. Recently, we found that some monoamines, which are blockers of NMDA receptors, inhibit and/or potentiate acid-sensing ion channels, depending on the subunit composition of the channels. The effect of 9-aminoacridine, IEM-1921, IEM-2117, and memantine both on native receptors and on recombinant ASIC1a, ASIC2a, and ASIC3 homomers was studied. In the present study, we have investigated the effect of these four compounds on homomeric ASIC1b channels. Experiments were performed on recombinant receptors expressed in CHO cells using the whole-cell patch clamp technique. Only two compounds, 9-aminoacridine and memantine, inhibited ASIC1b channels. IEM-1921 and IEM-2117 were inactive even at a 1000 μM concentration. In most aspects, the effect of the compounds on ASIC1b was similar to their effect on ASIC1a. The distinguishing feature of homomeric ASIC1b channels is a steep activation-dependence, indicating cooperative activation by protons. In our experiments, the curve of the concentration dependence of ASIC1b inhibition by 9-aminoacridine also had a slope (Hill coefficient) of 3.8, unlike ASIC1a homomers, for which the Hill coefficient was close to 1. This finding indicates that the inhibitory effect of 9-aminoacridine is associated with changes in the activation properties of acid-sensing ion channels.
    Acta Naturae 06/2015; 7(2):95. · 1.00 Impact Factor
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    ABSTRACT: Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca2+-permeable and Ca2+-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca2+-impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC50 value for the inhibition of Ca2+-permeable AMPA receptors in giant striatal interneurons was 43 ± 7 μm. The inhibition of Ca2+-permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open-channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane-impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open-channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.
    European Journal of Neuroscience 12/2014; 41(7). DOI:10.1111/ejn.12817 · 3.18 Impact Factor
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    ABSTRACT: Acid-sensing ion channels (ASICs) are widely distributed in the peripheral and central nervous system. Although they are involved in many physiological functions, the actual processes that activate ASICs remain unclear. This is particularly true for brain ASICs, which produce only a transient response to a fast drop in pH and cannot mediate sustained current. Therefore, the search for ASIC inhibitors and, especially, potentiators/activators is important. We report that NMDA receptor channel blockers with a comparatively simple structure (9-aminoacridine, memantine, IEM-2117 and IEM-1921) potentiate and/or inhibit ASICs in submillimolar concentrations. The experiments were performed using the patch clamp technique on native ASICs from rat hippocampal interneurons and recombinant ASICs of different subunit compositions expressed in CHO cells. Native ASICs were potentiated by IEM-1921 and IEM-2117, and inhibited by memantine and 9-aminoacridine. Homomeric ASIC1a were inhibited by memantine, IEM-2117 and 9-aminoacridine while IEM-1921 was ineffective. In contrast, homomeric ASIC2a were potentiated by IEM-2117, memantine and IEM-1921, whereas 9-aminoacridine was inactive. The compounds caused a complex effect on ASIC3. 9-aminoacridine and IEM-1921 potentiated the steady-state response of ASIC3 and inhibited the peak component. IEM-2117 not only potentiated ASIC3-mediated currents caused by acidification but also evoked steady-state currents at neutral pH. Our results demonstrate that, depending on the subunit composition, ASICs can be activated or inhibited by simple compounds that possess only amino group and aromatic/hydrophobic moieties. This opens up the possibility to search for new ASIC modulators among a number of endogenous ligands.
    Neuropharmacology 09/2014; 89. DOI:10.1016/j.neuropharm.2014.08.018 · 5.11 Impact Factor
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    Viacheslav S Korkosh · Boris S Zhorov · Denis B Tikhonov
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    ABSTRACT: Voltage-gated sodium channels are targets for many drugs and toxins. However, the rational design of medically relevant channel modulators is hampered by the lack of x-ray structures of eukaryotic channels. Here, we used a homology model based on the x-ray structure of the NavAb prokaryotic sodium channel together with published experimental data to analyze interactions of the μ-conotoxins GIIIA, PIIIA, and KIIIA with the Nav1.4 eukaryotic channel. Using Monte Carlo energy minimizations and published experimentally defined pairwise contacts as distance constraints, we developed a model in which specific contacts between GIIIA and Nav1.4 were readily reproduced without deformation of the channel or toxin backbones. Computed energies of specific interactions between individual residues of GIIIA and the channel correlated with experimental estimates. The predicted complexes of PIIIA and KIIIA with Nav1.4 are consistent with a large body of experimental data. In particular, a model of Nav1.4 interactions with KIIIA and tetrodotoxin (TTX) indicated that TTX can pass between Nav1.4 and channel-bound KIIIA to reach its binding site at the selectivity filter. Our models also allowed us to explain experimental data that currently lack structural interpretations. For instance, consistent with the incomplete block observed with KIIIA and some GIIIA and PIIIA mutants, our computations predict an uninterrupted pathway for sodium ions between the extracellular space and the selectivity filter if at least one of the four outer carboxylates is not bound to the toxin. We found a good correlation between computational and experimental data on complete and incomplete channel block by native and mutant toxins. Thus, our study suggests similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels.
    The Journal of General Physiology 09/2014; 144(3):231-44. DOI:10.1085/jgp.201411226 · 4.79 Impact Factor
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    ABSTRACT: Philanthotoxin-433 (PhTX-433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype by using two-electrode voltage-clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX-343, with trans-cyclopropyl-PhTX-343 being the most potent (∼28-fold) and cis-cyclopropyl-PhTX-343 least potent (∼4-fold). Both cis- and trans-cyclopropyl-PhTX-444 had intermediate potency (both ∼12-fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX-444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.
    ChemMedChem 07/2014; 9(8). DOI:10.1002/cmdc.201402109 · 2.97 Impact Factor
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    Denis B Tikhonov · Iva Bruhova · Daniel P Garden · Boris S Zhorov
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    ABSTRACT: Voltage-gated sodium and calcium channels play key roles in the physiology of excitable cells. The alpha-1 subunit of these channels folds from a polypeptide chain of four homologous repeats. In each repeat, the cytoplasmic halves of the pore-lining helices contain exceptionally conserved asparagines. Such conservation implies important roles, which are unknown. Mutations of the asparagines affect activation and inactivation gating as well as the action of pore-targeting ligands, including local anesthetics and steroidal agonists batrachotoxin and veratridine. In the absence of the open-channel structures, underlying mechanisms are unclear. Here, we modeled the pore module of Cav1.2 and Nav1.4 channels and their mutants in the open and closed states using the X-ray structures of potassium and sodium channels as templates. The energy of each model was Monte Carlo-minimized. The asparagines do not face the pore in the modeled states. In the open-channel models, the asparagine residue in a given repeat forms an inter-repeat H-bond with a polar residue, which is typically nine positions downstream from the conserved asparagine in the preceding repeat. The H-bonds, which are strengthened by surrounding hydrophobic residues, would stabilize the open channel and shape the open-pore geometry. According to our calculation, the latter is much more sensitive to mutations of the asparagines than the closed-pore geometry. Rearrangement of inter-repeat contacts may explain effects of these mutations on the voltage dependence of activation and inactivation and action of pore-targeting ligands.
    Pflügers Archiv - European Journal of Physiology 04/2014; 467(2). DOI:10.1007/s00424-014-1508-0 · 4.10 Impact Factor
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    Biophysical Journal 01/2014; 106(2):338a. DOI:10.1016/j.bpj.2013.11.1935 · 3.97 Impact Factor
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    Vyacheslav S. Korkosh · Boris S. Zhorov · Denis B. Tikhonov
    Biophysical Journal 01/2014; 106(2):130a. DOI:10.1016/j.bpj.2013.11.764 · 3.97 Impact Factor
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    Denis B. Tikhonov · Boris S Zhorov
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    ABSTRACT: The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic "blocking particle". A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.
    Biochimica et Biophysica Acta 12/2013; 1838(3). DOI:10.1016/j.bbamem.2013.11.019 · 4.66 Impact Factor
  • Boris S Zhorov · Denis B Tikhonov
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    ABSTRACT: Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure-activity relationships, and effects of mutations in these ion channels.
    Trends in Pharmacological Sciences 01/2013; 34(3). DOI:10.1016/ · 11.54 Impact Factor
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    Denis B. Tikhonov · Iva Bruhova · Daniel P. Garden · Boris S. Zhorov
    Biophysical Journal 01/2013; 104(2):135-. DOI:10.1016/j.bpj.2012.11.773 · 3.97 Impact Factor
  • Denis B Tikhonov · Boris S Zhorov
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    ABSTRACT: The X-ray structure of the bacterial sodium channel NavAb provides a new template for the study of sodium and calcium channels. Unlike potassium channels, NavAb contains P2 helices in the outer-pore region. Because the sequence similarity between eukaryotic and prokaryotic sodium channels in this region is poor, the structural similarity is unclear. We analyzed it by using experimental data on tetrodotoxin block of sodium channels. Key tetrodotoxin-binding residues are outer carboxylates in repeats I, II, and IV, three positions downstream from the selectivity-filter residues. In a NavAb-based model of Nav1 channels derived from the sequence alignment without insertions/deletions, the outer carboxylates did not face the pore and therefore did not interact with tetrodotoxin. The hypothesis that the evolutionary appearance of Nav1 channels involved point deletions in an ancestral channel between the selectivity filter and the outer carboxylates allowed building of a NavAb-based model with tetrodotoxin-channel contacts similar to those proposed previously. This hypothesis also allowed us to reproduce in Nav1 the folding-stabilizing contacts between long-side chain residues in P1 and P2, which are seen in NavAb. The NavAb-based inner-pore model of Nav1 preserved major features of our previous KcsA-based models, including the access pathway for ligands through the repeat III/IV interface and their interactions with specific residues. Thus, structural properties of eukaryotic voltage-gated sodium channels that are suggested by functional data were reproduced in the NavAb-based models built by using the unaltered template structure but with adjusted sequence alignment. Sequences of eukaryotic calcium channels aligned with NavAb without insertions/deletions, which suggests that NavAb is a promising basis for the modeling of calcium channels.
    Molecular pharmacology 04/2012; 82(1):97-104. DOI:10.1124/mol.112.078212 · 4.13 Impact Factor
  • Maxim V Nikolaev · Lev G Magazanik · Denis B Tikhonov
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    ABSTRACT: The NMDA type of ionotropic glutamate receptors plays a unique role in synaptic functions because of high permeability for calcium and because of a voltage-dependent block by endogenous Mg(2+). Activity and voltage dependence of the NMDA receptor channel block by organic cations are strongly affected by competition with magnesium ions for the binding site in the channel pore. It complicates prediction of action of NMDA receptor channel blockers in vivo. In the present work we studied the NMDA receptor channel block in the presence of Mg(2+) by several organic blockers with different characteristics of voltage dependence and mechanism of action. The action of NMDA receptor channel antagonists was studied in native NMDA receptors of hippocampus CA1 pyramidal neurons isolated from rat brain slices. It was demonstrated that the IC(50) values of NMDA receptor channel blockers at -30 mV are increased 1.5-5 times compared with magnesium-free conditions. The voltage dependence of the channel block is decreased, abolished or even inversed in the presence of magnesium. Although simple competition between magnesium ion and organic channel blockers provides a general explanation of the observed effects, certain disagreements were revealed. Diversity in Mg(2+) effects on the NMDAR channel block by different organic cations reported herein likely reflects interaction of NMDAR channel blockers with additional binding site(s) and suggests that individual analysis in the presence of Mg(2+) is required for newly developed NMDAR channel blocking drugs.
    Neuropharmacology 04/2012; 62(5-6):2078-85. DOI:10.1016/j.neuropharm.2011.12.029 · 5.11 Impact Factor
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    Denis B. Tikhonov · Boris S. Zhorov
    Biophysical Journal 01/2012; 102(3):327-. DOI:10.1016/j.bpj.2011.11.1794 · 3.97 Impact Factor
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    Denis B. Tikhonov · Boris S. Zhorov
    Biophysical Journal 01/2012; 102(3):679-. DOI:10.1016/j.bpj.2011.11.3692 · 3.97 Impact Factor
  • Oleg I Barygin · Eugene V Grishin · Denis B Tikhonov
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    ABSTRACT: Binding of argiotoxin in the closed state of Ca(2+)-permeable AMPA receptor channels was studied using electrophysiological and molecular modeling approaches. Experimental study unambiguously revealed that argiotoxin is trapped in the closed AMPA receptor channels after agonist dissociation. Docking of the argiotoxin to the channel model based on recently published X-ray structure demonstrated that the drug can be effectively accommodated in the cavity of the closed channel only if the terminal moiety of the molecule penetrates in the narrow portion of the pore below the selectivity filter. Combining these results, we conclude that the selectivity filter of the AMPA receptor channels is not sterically occluded in the closed state.
    Biochemistry 08/2011; 50(38):8213-20. DOI:10.1021/bi200617v · 3.02 Impact Factor
  • T. B. Tikhonova · L. G. Magazanik · D. B. Tikhonov
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    ABSTRACT: The ionic composition of the medium can influence the actions of many pharmacological agents. We report here our studies of the effects of the extracellular sodium concentration on blockade of calcium-permeable ion channels of AMPA receptors by the cationic phenylcyclohexyl derivative IEM-1925. Experiments were performed using native receptors from striatal giant interneurons isolated from rat brain slices. Recordings were made by whole-cell patch voltage clamping. Replacement of some of the extracellular sodium with sucrose was found to increase the blocking action of IEM-1925. This effect depended on the membrane potential, increasing on hyperpolarization. Analysis of the kinetics of the interaction of IEM-1925 with channels showed that decreases in the extracellular sodium concentration had no effect on the stability of blocker–channel complexes, increases in blockade occurring because of increases in the association rate constant. These results lead to the conclusion that current-carrying sodium ions complete with the blocking IEM-1925 cation for the binding site in the channel. KEY WORDSsynaptic transmission–glutamate–AMPA–channel blockade–ionic composition of the extracellular medium
    Neuroscience and Behavioral Physiology 07/2011; 41(6):647-653. DOI:10.1007/s11055-011-9468-8
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    ABSTRACT: This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favour the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR-mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes – such as high-frequency synaptic activity or increase of the steady-state glutamate concentration – to be examined.
    The Journal of Physiology 04/2011; 589(Pt 7):1587-601. DOI:10.1113/jphysiol.2011.204362 · 5.04 Impact Factor

Publication Stats

1k Citations
240.98 Total Impact Points


  • 1998–2015
    • Russian Academy of Sciences
      • Institute of Evolutionary Physiology and Biochemistry
      Moskva, Moscow, Russia
  • 2012
    • University of Debrecen
      • Medical and Health Science Centre
      Debreczyn, Hajdú-Bihar, Hungary
  • 2004–2012
    • McMaster University
      • • Department of Biochemistry and Biomedical Sciences
      • • Department of Chemistry and Chemical Biology
      Hamilton, Ontario, Canada
  • 2006
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2002
    • University of Nottingham
      Nottigham, England, United Kingdom