Denis B Tikhonov

University of Debrecen, Debreczyn, Hajdú-Bihar, Hungary

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Publications (99)

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    Elina I. Nagaeva · Natalia N. Potapieva · Maxim V. Nikolaev · [...] · Denis B. Tikhonov
    [Show abstract] [Hide abstract] ABSTRACT: Acid-sensing ion channels (ASICs) are involved in numerous physiological and pathological processes in the central nervous system. Development of pharmacological tools capable to inhibit or potentiate these channels is important for our knowledge about roles of ASICs in the neuronal network and can be promising for treatment of some disorders. Recently we described four hydrophobic monoamines that potentiate and inhibit ASICs depending on subunit composition of the channel and peculiarities of the drug structure. In the present work we performed structure-activity relationship analysis using derivatives of adamantane, phenylcyclohexyl and 9-aminoacridine to reveal the main determinants of action of amine-containing compounds on recombinant ASIC1a and ASIC2a homomers expressed in CHO cells. We found that the most active compounds are monocations with protonatable aminogroup. In general, potentiators and inhibitors of ASIC1a we found, but only potentiators for ASIC2a. Flat aromatic structure of the headgroup determines inhibition of ASIC1a while “V-shape” structure of the hydrophobic moiety favors potentiation of ASIC2a. Moreover, for some series of monoamines there was a correlation between action on ASIC1a and ASIC2a, the weaker ASIC1a inhibition, the stronger ASIC2a potentiation. Decay of response was accelerated by ASIC1a inhibitors as well as by potentiators. All compounds potentiating ASIC2a slowed down desensitization. Our results suggest that hydrophobic amines cause complex action on ASICs.
    Full-text Article · Jun 2016 · European journal of pharmacology
  • B.S. Zhorov · D.B. Tikhonov
    [Show abstract] [Hide abstract] ABSTRACT: Voltage-gated sodium channels are targets for many toxins and medically important drugs. Despite decades of intensive studies in industry and academia, atomic mechanisms of action are still not completely understood. The major cause is a lack of high-resolution structures of eukaryotic channels and their complexes with ligands. In these circumstances a useful approach is homology modeling that employs as templates X-ray structures of potassium channels and prokaryotic sodium channels. On one hand, due to inherent limitations of this approach, results should be treated with caution. In particular, models should be tested against relevant experimental data. On the other hand, docking of drugs and toxins in homology models provides a unique possibility to integrate diverse experimental data provided by mutational analysis, electrophysiology, and studies of structure-activity relations. Here we describe how homology modeling advanced our understanding of mechanisms of several classes of ligands. These include tetrodotoxins and mu-conotoxins that block the outer pore, local anesthetics that block of the inner pore, batrachotoxin that binds in the inner pore but, paradoxically, activates the channel, pyrethroid insecticides that activate the channel by binding at lipid-exposed repeat interfaces, and scorpion alpha and beta-toxins, which bind between the pore and voltage-sensing domains and modify the channel gating. We emphasize importance of experimental data for elaborating the models.
    Chapter · Mar 2016
  • Vyacheslav S. Korkosh · Boris S. Zhorov · Denis B. Tikhonov
    [Show abstract] [Hide abstract] ABSTRACT: Mechanisms of sodium channel block by local anesthetics (LAs) are still a matter of intensive studies. In the absence of high-resolution structures of eukaryotic channels, atomic details of LA-channel interactions are analyzed using homology modeling. LAs are predicted to access the closed channel through a sidewalk (fenestration) between the channel repeats, bind in a horizontal orientation, and leave its aromatic moiety in the interface. Recent X-ray structure of a bacterial sodium channel NavMs with a cationic molecule Pl1, which is structurally similar to LAs, has confirmed this theoretical prediction and demonstrated a reduced selectivity filter occupancy by the permeant ions in the Pl1-bound channel. However, the nature of the antagonism between LAs and permeant ions are still unclear. Here we used the NavMs structure and Monte Carlo energy minimizations to model Pl1 binding. Our computations predict that Pl1 can displace permeant ion(s) from the selectivity filter by both steric and electrostatic mechanisms. We hypothesize that the electrostatic mechanism is more general, because it is applicable to many LAs and related drugs, which lack a moiety capable to enter the selectivity filter and sterically displace the permeant ion. The electrostatic mechanism is also consistent with the data that various cationic blockers of potassium channels bind in the inner pore without entering the selectivity filter.
    Article · Mar 2016 · European journal of pharmacology
  • V. S. Korkosh · B. S. Zhorov · D. B. Tikhonov
    [Show abstract] [Hide abstract] ABSTRACT: Comparative analysis of structure and function of macromolecules, such as proteins, is an integral part of modern evolutionary biology. The first and critical step in understanding evolution of homologous proteins is their amino acid sequence alignment. However, standard algorithms fail to provide unambiguous sequence alignment for proteins of poor homology. More reliable results can be provided by comparing experimental 3D structures obtained at atomic resolution with the aid of X-ray structural analysis. If such structures are lacking, homology modeling is used which considers indirect experimental data on functional roles of individual amino acid residues. An important problem is that sequence alignment, which reflects genetic modifications, not necessarily corresponds to functional homology, which depends on 3D structures critical for natural selection. Since the alignment techniques relying only on the analysis of primary structures carry no information on the functional properties of proteins, the inclusion of 3D structures into consideration is of utmost importance. Here we consider several ion channels as examples to demonstrate that alignment of their 3D structures can significantly improve sequence alignment obtained by traditional methods.
    Article · Jan 2016 · Journal of Evolutionary Biochemistry and Physiology
  • V. S. Korkosh · B. S. Zhorov · D. B. Tikhonov
    [Show abstract] [Hide abstract] ABSTRACT: The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.
    Article · Dec 2015 · European Biophysics Journal
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    [Show abstract] [Hide abstract] ABSTRACT: Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 μg/g in experiments on house fly Musca domestica larvae and with LD50 20 μg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca(2+) currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca(2+) current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca(2+) channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended β-hairpin loop and short β-hairpin loop which are capable of making "scissors-like mutual motions".
    Full-text Article · Nov 2015 · Scientific Reports
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    S. L. Malkin · K. Kh. Kim · D. B. Tikhonov · A. V. Zaitsev
    [Show abstract] [Hide abstract] 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.
    Full-text Article · Nov 2015 · Journal of Evolutionary Biochemistry and Physiology
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    Sergey L Malkin · Kira K Kim · Denis B Tikhonov · [...] · Aleksey V Zaitsev
    [Show abstract] [Hide abstract] 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.
    Full-text Article · Jun 2015 · Neuroscience
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    E I Nagaeva · N N Potapieva · D B Tikhonov
    [Show abstract] [Hide abstract] 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.
    Full-text Article · Jun 2015 · Acta Naturae
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    [Show abstract] [Hide abstract] ABSTRACT: Neuronal nicotinic acetylcholine receptors (nAChRs) are excitatory ion channels, which have wide subtype diversity due to the many possible subunit combinations, some heteromeric, others homomeric. Each subunit is composed of a large N-terminal segment containing the ACh binding site and four membrane-spanning segments named M1-M4. The pore lumen is lined mainly by the second of these membrane segments, M2. nAChRs play a critical role in many physiological and pathophysiological processes, thus, neuronal nAChRs have become targets for drug discovery and research (Romanelli et al., 2007; Unwin, 2013). The venom of the Egyptian digger wasp contains a toxin, philanthotoxin-433 (PhTX-433), that works as a strong non-competitive inhibitor of ionotropic glutamate receptors and nAChRs in their target prey (Strømgaard et al., 2000). PhTX-433 has a polyamine tail and aromatic head group. The main obstacle facing the use of the natural toxin (PhTX-433) as a candidate for drug development and understanding pharmacological characteristics of ionotropic receptors is the lack of subtype selectivity. The present study aims to investigate the activity and selectivity of twenty one synthetic analogues of PhTX-343 on rat neuronal α4β2 and α3β4 nAChRs expressed in Xenopus oocytes. We showed that the presence of positive charge in the polyamine tail of PhTX compounds is essential for nAChR subtype selectivity and their removal makes the molecule lose its selectivity. In addition, we identified the key regions and substitutions responsible for increasing PhTX-343 activity, cyclohexylalanine (IC 50 of 1 nM on α4β2 and 2 nM on α3β4), and selectivity, phenolic group (30 fold selectivity for α3β4 over α4β2). Analogues having cyclohexylalanine and a phenolic group in the head region showed IC 50 values in the low nano-molar and pico-molar (160-400 pM) range. These data suggest that PhTXs could serve as lead compounds for highly potent and selective inhibitors of N-nAChRs. (2000) Solid phase synthesis and biological evaluation of enantiomerically pure wasp toxin analogues PhTX-343 and PhTX-12. Chirality 12:93-102.
    Full-text Article · Jan 2015
  • [Show abstract] [Hide abstract] 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.
    Article · Dec 2014 · European Journal of Neuroscience
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    Tatiana B Tikhonova · Elina I Nagaeva · Oleg I Barygin · [...] · Denis B Tikhonov
    [Show abstract] [Hide abstract] 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.
    Full-text Article · Sep 2014 · Neuropharmacology
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    Viacheslav S Korkosh · Boris S Zhorov · Denis B Tikhonov
    [Show abstract] [Hide abstract] 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.
    Full-text Article · Sep 2014 · The Journal of General Physiology
  • Henrik Franzyk · John W. Grzeskowiak · Denis B. Tikhonov · [...] · Ian R. Mellor
    [Show abstract] [Hide abstract] 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.
    Article · Jul 2014 · ChemMedChem
  • Denis B Tikhonov · Iva Bruhova · Daniel P Garden · Boris S Zhorov
    [Show abstract] [Hide abstract] 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.
    Article · Apr 2014 · Pflügers Archiv - European Journal of Physiology
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    Full-text Article · Jan 2014 · Biophysical Journal
  • Vyacheslav S. Korkosh · Boris S. Zhorov · Denis B. Tikhonov
    Article · Jan 2014 · Biophysical Journal
  • Denis B. Tikhonov · Boris S Zhorov
    [Show abstract] [Hide abstract] 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.
    Article · Dec 2013 · Biochimica et Biophysica Acta
  • Boris S Zhorov · Denis B Tikhonov
    [Show abstract] [Hide abstract] 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.
    Article · Jan 2013 · Trends in Pharmacological Sciences
  • Denis B. Tikhonov · Iva Bruhova · Daniel P. Garden · Boris S. Zhorov
    Article · Jan 2013 · Biophysical Journal