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Savinska L.,NASU Institute of Molecular Biology and Genetics | Savinska L.,State Key Laboratory of Molecular and Cellular Biology | Skorokhod O.,NASU Institute of Molecular Biology and Genetics | Skorokhod O.,State Key Laboratory of Molecular and Cellular Biology | And 5 more authors.
Hybridoma | Year: 2012

Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa N-terminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies. © 2012 Copyright, Mary Ann Liebert, Inc.

Fedorenko Y.A.,Bogomolets Institute of Physiology of the NAS of Ukraine | Fedorenko Y.A.,State Key Laboratory of Molecular and Cellular Biology
Neurophysiology | Year: 2016

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a key role in intracellular calcium signaling. Up to the present time, the question on the existence of only one level of the unitary conductance of such receptors or a few levels of such conductance remained open. In experiments on the isolated nuclei of Purkinje cerebellar neurons of rats, we examined changes in the conductance of channels of these receptors localized on the internal membrane of the nuclear envelope, which were related to voltage variations. In all cases, these channels demonstrated only one level of the unitary conductance; no sublevels were found within a –100 mV to 100 mV range. Suppression of activity of IP3Rs at negative potentials is determined by a decrease in the probability of the open state of the channel. Thus, a hypothesis on the existence of a few levels of the IP3R channel conductance in the examined object has not been confirmed; the release of Ca2+ through channels of these receptors demonstrates a quantum nature. © 2016 Springer Science+Business Media New York

Prevarskaya N.,French Institute of Health and Medical Research | Prevarskaya N.,Lille University of Science and Technology | Ouadid-Ahidouch H.,Laboratory of Cellular and Molecular Physiology | Skryma R.,French Institute of Health and Medical Research | And 2 more authors.
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2014

Cancer involves defects in the mechanisms underlying cell proliferation, death and migration. Calcium ions are central to these phenomena, serving as major signalling agents with spatial localization, magnitude and temporal characteristics of calcium signals ultimately determining cell's fate. Cellular Ca2+ signalling is determined by the concerted action of a molecular Ca2+handling toolkit which includes: active energy-dependent Ca2+ transporters, Ca2+-permeable ion channels, Ca2+-binding and storage proteins, Ca2+dependent effectors. In cancer, because of mutations, aberrant expression, regulation and/or subcellular targeting of Ca2+-handling/transport protein(s) normal relationships among extracellular, cytosolic, endoplasmic reticulum and mitochondrial Ca2+ concentrations or spatio-temporal patterns of Ca2+ signalling become distorted. This causes deregulation of Ca2+dependent effectors that control signalling pathways determining cell's behaviour in a way to promote pathophysiological cancer hallmarks such as enhanced proliferation, survival and invasion. Despite the progress in our understanding of Ca2+ homeostasis remodelling in cancer cells as well as in identification of the key Ca2+-transport molecules promoting certain malignant phenotypes, there is still a lot of work to be done to transform fundamental findings and concepts into new Ca2+ transport-targeting tools for cancer diagnosis and treatment. © 2014 The Author(s) Published by the Royal Society.

Fedorenko O.A.,Bogomoletz Institute of Physiology | Fedorenko O.A.,State Key Laboratory of Molecular and Cellular Biology | Marchenko S.M.,Bogomoletz Institute of Physiology | Marchenko S.M.,State Key Laboratory of Molecular and Cellular Biology
Hippocampus | Year: 2014

Rise in Ca2+ concentration in the nucleus affects gene transcription and has been implicated in neuroprotection, transcription-dependent neuronal plasticity, and pain modulation, but the mechanism of regulation of nuclear Ca2+ remains poorly understood. The nuclear envelope is a part of the endoplasmic reticulum and may be one of the sources of nuclear Ca2+. Here, we studied ion channels in the nuclear membrane of hippocampal neurons using the patch-clamp technique. We have found that the nuclear membrane of CA1 pyramidal and dentate gyrus granule (DG), but not CA3 pyramidal neurons, was enriched in functional inositol 1,4,5-trisphosphate receptors/Ca2+-release channels (IP3Rs) localized mainly in the inner nuclear membrane. A single nuclear ryanodine receptor (RyR) has been detected only in DG granule neurons. Nuclei of the hippocampal neurons also expressed a variety of spontaneously active cation and anion channels specific for each type of neuron. In particular, large-conductance ion channels selective for monovalent cations (LCC) were coexpressed with IP3Rs. These data suggest that: (1) the nuclear membranes of hippocampal neurons contain distinct sets of ion channels, which are specific for each type of neuron; (2) IP3Rs, but not RyRs are targeted to the inner nuclear membrane of CA1 pyramidal and DG granule, but they were not found in the nuclear membranes of CA3 pyramidal neurons; (3) the nuclear envelope of these neurons is specialized to release Ca2+ into the nucleoplasm which may amplify Ca2+ signals entering the nucleus from the cytoplasm or generate Ca2+ transients on its own; (4) LCC channels are an integral part the of Ca2+-releasing machinery providing a route for counterflow of K{cyrillic}+ and thereby facilitating Ca2+ movement in and out of the Ca2+ store. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Stepanyuk A.R.,Bogomoletz Institute of Physiology | Stepanyuk A.R.,State Key Laboratory of Molecular and Cellular Biology | Belan P.V.,Bogomoletz Institute of Physiology | Belan P.V.,State Key Laboratory of Molecular and Cellular Biology | And 2 more authors.
PLoS ONE | Year: 2014

When dispersed and cultured in a multielectrode dish (MED), suprachiasmatic nucleus (SCN) neurons express fast oscillations of firing rate (FOFR; fast relative to the circadian cycle), with burst duration ,∼10 min, and interburst interval varying from 20 to 60 min in different cells but remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1 mm recording area of a MED exhibited correlated FOFR. Neither the mechanism of FOFR nor the mechanism of their synchronization among neurons is known. Based on recent data implicating vasoactive intestinal polypeptide (VIP) as a key intercellular synchronizing agent, we built a model in which VIP acts as both a feedback regulator to generate FOFR in individual neurons, and a diffusible synchronizing agent to produce coherent electrical output of a neuronal network. In our model, VIP binding to its (VPAC2) receptors acts through Gs G-proteins to activate adenylyl cyclase (AC), increase intracellular cAMP, and open cyclic-nucleotide-gated (CNG) cation channels, thus depolarizing the cell and generating neuronal firing to release VIP. In parallel, slowly developing homologous desensitization and internalization of VPAC2receptors terminates elevation of cAMP and thereby provides an interpulse silent interval. Through mathematical modeling, we show that this VIP/VPAC2/AC/cAMP/CNG-channel mechanism is sufficient for generating reliable FOFR in single neurons. When our model for FOFR is combined with a published model of synchronization of circadian rhythms based on VIP/VPAC2and Per gene regulation synchronization of circadian rhythms is significantly accelerated. These results suggest that (a) auto/paracrine regulation by VIP/VPAC2and intracellular AC/cAMP/ CNG-channels are sufficient to provide robust FOFR and synchrony among neurons in a heterogeneous network, and (b) this system may also participate in synchronization of circadian rhythms. © 2014 Stepanyuk et al.

Fedorenko O.A.,Bogomoletz Institute of Physiology | Fedorenko O.A.,State Key Laboratory of Molecular and Cellular Biology | Popugaeva E.,Saint Petersburg State Polytechnic University | Enomoto M.,University of Toronto | And 3 more authors.
European Journal of Pharmacology | Year: 2014

The inositol-1,4,5-trisphosphate receptors (InsP3Rs) are the major intracellular Ca2+-release channels in cells. Activity of InsP3Rs is essential for elementary and global Ca2+ events in the cell. There are three InsP3Rs isoforms that are present in mammalian cells. In this review we will focus primarily on InsP3R type 1. The InsP3R1 is a predominant isoform in neurons and it is the most extensively studied isoform. Combination of biophysical and structural methods revealed key mechanisms of InsP3R function and modulation. Cell biological and biochemical studies lead to identification of a large number of InsP3R-binding proteins. InsP3Rs are involved in the regulation of numerous physiological processes, including learning and memory, proliferation, differentiation, development and cell death. Malfunction of InsP3R1 play a role in a number of neurodegenerative disorders and other disease states. InsP3Rs represent a potentially valuable drug target for treatment of these disorders and for modulating activity of neurons and other cells. Future studies will provide better understanding of physiological functions of InsP3Rs in health and disease. © 2013 Elsevier B.V. All rights reserved.

Stepanyuk A.R.,Bogomoletz Institute of Physiology | Stepanyuk A.R.,State Key Laboratory of Molecular and Cellular Biology | Borisyuk A.L.,Bogomoletz Institute of Physiology | Borisyuk A.L.,State Key Laboratory of Molecular and Cellular Biology | And 2 more authors.
PLoS ONE | Year: 2011

A new method is described that accurately estimates kinetic constants, conductance and number of ion channels from macroscopic currents. The method uses both the time course and the strength of correlations between different time points of macroscopic currents and utilizes the property of semiseparability of covariance matrix for computationally efficient estimation of current likelihood and its gradient. The number of calculation steps scales linearly with the number of channel states as opposed to the cubic dependence in a previously described method. Together with the likelihood gradient evaluation, which is almost independent of the number of model parameters, the new approach allows evaluation of kinetic models with very complex topologies. We demonstrate applicability of the method to analysis of synaptic currents by estimating accurately rate constants of a 7-state model used to simulate GABAergic macroscopic currents. © 2011 Stepanyuk et al.

Prevarskaya N.,French Institute of Health and Medical Research | Skryma R.,Lille University of Science and Technology | Shuba Y.,Ukrainian Academy of Sciences | Shuba Y.,State Key Laboratory of Molecular and Cellular Biology
Expert Opinion on Therapeutic Targets | Year: 2013

Introduction: Cancer is caused by defects in the mechanisms underlying cell proliferation, death and migration. Calcium ions are central to all of these phenomena, serving as major signalling agents with the spatial localisation, magnitude and temporal characteristics of calcium signals ultimately determining cell's fate. The transformation of a normal cell into a malignant derivative is associated with a major rearrangement of Ca2+ pumps, Na/Ca exchangers and Ca2+ channels, which leads to enhanced proliferation and invasion under compromised/impaired ability to die. Areas covered: This paper examines the changes in Ca2+ signalling and the mechanisms that underlie the passage from normal to pathological cell growth and death control. Understanding these changes and identifying the molecular players involved provide new perspectives for cancer treatment. Expert opinion: Despite compelling evidence that the disruption of Ca2+ homeostasis in cancer cells leads to the promotion of certain malignant phenotypes as well as the identification of key Ca2+-transporting molecules whose altered expression and/or function underlies pathological changes, the therapeutic utilisation of these findings for cancer treatment is still at its infancy. However, the rapid development of the field warrants the development of improved molecular Ca2+ transport-targeting tools for cancer diagnosis and treatment. © 2013 Informa UK, Ltd.

Storozhuk M.,Bogomoletz Institute of Physiology | Storozhuk M.,State Key Laboratory of Molecular and Cellular Biology | Kondratskaya E.,Bogomoletz Institute of Physiology | Kondratskaya E.,State Key Laboratory of Molecular and Cellular Biology | And 3 more authors.
Molecular Brain | Year: 2016

Rapid acidification occurring during synaptic vesicle release can activate acid-sensing ion channels (ASICs) both on pre- and postsynaptic neurons. In the latter case, a fraction of postsynaptic current would be mediated by cation-selective acid-sensing ion channels. Additionally, in both cases, activation of acid-sensing ion channels could modulate synaptic strength by affecting transmitter release and/or sensitivity of postsynaptic receptors. To address potential involvement of acid-sensing ion channels in mediation/modulation of synaptic transmission at hippocampal GABAergic synapses, we studied effects of three structurally different blockers of acid-sensing ion channels on evoked postsynaptic currents using the patch-clamp technique. We found that GABAergic postsynaptic currents, recorded below their reversal potential as inward currents, are suppressed by all the employed blockers of acid-sensing ion channels. These currents were suppressed by ~ 20 % in the presence of a novel blocker 5b (1 μM) and by ~30 % in the presence of either amiloride (25 μM) or diminazene (20 μM). In the same cells the suppression of postsynaptic currents, recorded above their reversal potential as outward currents was statistically insignificant. These results imply that the effects of blockers in our experiments are at least partially postsynaptic. On the other hand, in the case of mediation of a fraction of postsynaptic current by acid-sensing ion channels, an increase of outward currents would be expected under our experimental conditions. Our analysis of a bicuculline-resistant fraction of postsynaptic currents also suggests that effects of the blockers are predominantly modulatory. In this work we present evidence for the first time that acid-sensing ion channels play a functional role at hippocampal GABAergic synapses. The suppressing effect of the blockers of acid-sensing ion channels on GABAergic transmission is due, at least partially, to a postsynaptic but (predominantly) modulatory mechanism. We hypothesize that the modulatory effect is due to functional crosstalk between ASICs and GABAA-receptors recently reported in isolated neurons, however, verification of this hypothesis is necessary. © 2016 The Author(s).

PubMed | State Key Laboratory of Molecular and Cellular Biology
Type: | Journal: Advances in experimental medicine and biology | Year: 2012

Ca(2+) is a ubiquitous signaling ion that regulates a variety of neuronal functions by binding to and altering the state of effector proteins. Spatial relationships and temporal dynamics of Ca(2+) elevations determine many cellular responses of neurons to chemical and electrical stimulation. There is a wealth of information regarding the properties and distribution of Ca(2+) channels, pumps, exchangers, and buffers that participate in Ca(2+) regulation. At the same time, new imaging techniques permit characterization of evoked Ca(2+) signals with increasing spatial and temporal resolution. However, understanding the mechanistic link between functional properties of Ca(2+) handling proteins and the stimulus-evoked Ca(2+) signals they orchestrate requires consideration of the way Ca(2+) handling mechanisms operate together as a system in native cells. A wide array of biophysical modeling approaches is available for studying this problem and can be used in a variety of ways. Models can be useful to explain the behavior of complex systems, to evaluate the role of individual Ca(2+) handling mechanisms, to extract valuable parameters, and to generate predictions that can be validated experimentally. In this review, we discuss recent advances in understanding the underlying mechanisms of Ca(2+) signaling in neurons via mathematical modeling. We emphasize the value of developing realistic models based on experimentally validated descriptions of Ca(2+) transport and buffering that can be tested and refined through new experiments to develop increasingly accurate biophysical descriptions of Ca(2+) signaling in neurons.

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