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Tikhonov D.B.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry | Zhorov B.S.,McMaster University
Biochimica et Biophysica Acta - Biomembranes | Year: 2014

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. © 2013 Elsevier B.V.

Borodkina A.,Russian Academy of Sciences | Shatrova A.,Russian Academy of Sciences | Abushik P.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry | Nikolsky N.,Saint Petersburg State Polytechnic University | Burova E.,Russian Academy of Sciences
Aging | Year: 2014

Human endometrium-derived mesenchymal stem cells (hMESCs) enter the premature senescence under sublethal oxidative stress, however underlying mechanism remains unknown. Here, we showed that exogenous H2O2 induces a rapid phosphorylation and co-localization of ATM, H2A.X, 53BP1 leading to DNA damage response (DDR) activation. DDR was accompanied with nuclear translocation of p-p53 followed by up-regulation of p21Waf1 and the permanent hypophosphorylation of pRb. Additionally, the increased p38MAPK/MAPKAPK-2 activation persisted in H2O2- treated cells. We suggest that both p53/p21/pRb and p38MAPK/MAPKAPK-2 pathways are responsible for establishing an irreversible cell cycle arrest that is typical of senescence. The process of further stabilization of senescence required prolonged DDR signaling activation that was provided by the permanent ROS production which in turn was regulated by both p38MAPK and the increased functional mitochondria. To reverse senescence, the pharmacological inhibition of p38MAPK was performed. Cell treatment with SB203580 was sufficient to recover partially senescence phenotype, to block the ROS elevation, to decrease the mitochondrial function, and finally to rescue proliferation. Thus, suppression of the p38MAPK pathway resulted in a partial prevention of H2O2-induced senescence of hMESCs. The current study is the first to reveal the molecular mechanism of the premature senescence of hMESCs in response to oxidative stress. © Borodkina et al.

Belyaeva E.A.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry
Acta Biochimica Polonica | Year: 2010

To elucidate the molecular mechanisms of the protective action of stigmatellin (an inhibitor of complex III of mitochondrial electron transport chain, mtETC) against the heavy metal-induced cytotoxicity, we tested its effectiveness against mitochondrial membrane permeabilization produced by heavy metal ions Cd 2+, Hg 2+, Cu 2+ and Zn 2+, as well as by Ca 2+ (in the presence of P i) or Se (in form of Na 2SeO 3) using isolated rat liver mitochondria. It was shown that stigmatellin modulated mitochondrial swelling produced by these metals/metalloids in the isotonic sucrose medium in the presence of ascor-bate plus tetramethyl-p-phenylenediamine (complex IV substrates added for energization of the mitochondria). It was found that stigmatellin and other mtETC inhibitors enhanced the mitochondrial swelling induced by selenite. However, in the same medium, all the mtETC inhibitors tested as well as cyclosporin A and bongkrekic acid did not significantly affect Cu 2+-induced swelling. In contrast, the high-amplitude swelling produced by Cd 2+, Hg 2+, Zn 2+, or Ca 2+ plus P i was significantly depressed by these inhibitors. Significant differences in the action of these metals/metalloids on the redox status of pyridine nucleotides, transmembrane potential and mitochondrial respiration were also observed. In the light of these results as well as the data from the recent literature, our hypothesis on a possible involvement of the respiratory supercomplex, formed by complex I (P-site) and complex III (S-site) in the mitochondrial permeabilization mediated by the mitochondrial transition pore, is updated.

Shpakov A.O.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry
Tsitologiya | Year: 2012

The role of hormonal signaling systems in the etiology and pathogenesis of diabetes mellitus (DM) and the neurodegenerative diseases induced by them is currently poorly understood. It is generally accepted that the main causes of diabetes of types 1 and 2 and their CNS complications are disturbances in signaling systems regulated by insulin, leptin and glutamate. However, in recent years, there are many evidences in favor of participation of the brain signaling systems regulated by biogenic amines and cholinergic receptor agonists in the development of these diseases. The alterations in the expression and functional activity of signal proteins, the components of these systems, as well as the disturbances of the biosynthesis, transport and degradation of signal molecules for their regulation contribute significantly to the pathological processes in the brain in DM, and in some cases themselves are a trigger for the development of this disease. The main factors that cause dysfunctions of dopaminergic, serotonergic, adrenergic and cholinergic systems of the brain in DM are hyperglycemia and insulin resistance, and hypoglycemia that occurs as a result of inadequate insulin therapy. This review is devoted to the functional state of the brain signaling systems regulated by biogenic amines and cholinergic agonists in DM, as well as the relationship between the changes in these systems and the development of neurodegenerative processes in the diabetic brain.

Tikhonov D.B.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry | Zhorov B.S.,McMaster University
Molecular Pharmacology | Year: 2012

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. Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics.

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