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Shuba Y.M.,Ukrainian Academy of Sciences | Shuba Y.M.,State Key Laboratory of Molecular and Cellular Physiology
Pflugers Archiv European Journal of Physiology | Year: 2014

Ca2+ entry is indispensable part of intracellular Ca 2+ signaling, which is vital for most of cellular functions. Low voltage-activated (LVA or T-type) calcium channels belong to the family of voltage-gated calcium channels (VGCCs) which provide Ca2+ entry in response to membrane depolarization. VGCCs are generally characterized by exceptional Ca2+ selectivity combined with high permeation rate, thought to be determined by the presence in their selectivity filter of a versatile Ca2+ binding site formed by four glutamate residues (EEEE motif). The subfamily of LVA channels includes three members, Cav3.1, Cav3.2 and Cav3.3. They all possess two aspartates instead of glutamates (i.e., EEDD motif) in their selectivity filter and are the least Ca2+-selective of all VGCCs. They also have the lowest conductance, weakly discriminate Ca 2+, Sr2+ and Ba2+ and demonstrate channel-specific sensitivity to divalent metal blockers, such as Ni 2+. The available data suggest that EEDD binding site of LVA channels is more rigid compared to EEEE one, and their selectivity permeation and block are determined by two supplementary low-affinity intrapore Ca2+ binding sites located above and below EEDD locus. In addition, LVA channels have extracellular metal binding site that allosterically regulates channel's gating, permeation and block depending on trace metals concentration. © 2014 Springer-Verlag.

Abeele F.V.,French Institute of Health and Medical Research | Kondratskyi A.,French Institute of Health and Medical Research | Dubois C.,French Institute of Health and Medical Research | Shapovalov G.,French Institute of Health and Medical Research | And 6 more authors.
Journal of Cell Science | Year: 2013

The mechanisms by which volatile general anaesthetics (VAs) produce a depression of central nervous system are beginning to be better understood, but little is known about a number of side effects. Here, we show that the cold receptor transient receptor potential melastatin 8 (TRPM8) undergoes a complex modulation by clinical concentrations of VAs in dorsal root ganglion neurons and HEK-293 cells heterologously expressing TRPM8. VAs produced a transient enhancement of TRPM8 through a depolarizing shift of its activation towards physiological membrane potentials, followed by a sustained TRPM8 inhibition. The stimulatory action of VAs engaged molecular determinants distinct from those used by the TRPM8 agonist. Transient TRPM8 activation by VAs could explain side effects such as inhibition of respiratory drive, shivering and the cooling sensation during the beginning of anaesthesia, whereas the second phase of VA action, that associated with sustained TRPM8 inhibition, might be responsible for hypothermia. Consistent with this, both hypothermia and the inhibition of respiratory drive induced by VAs are partially abolished in Trpm8-knockout animals. Thus, we propose TRPM8 as a new clinical target for diminishing common and serious complications of general anaesthesia. © 2013. Published by The Company of Biologists Ltd.

Philyppov I.B.,Ukrainian Academy of Sciences | Philyppov I.B.,State Key Laboratory of Molecular and Cellular Physiology | Paduraru O.N.,Ukrainian Academy of Sciences | Paduraru O.N.,State Key Laboratory of Molecular and Cellular Physiology | And 4 more authors.
Life Sciences | Year: 2012

Aims: TRPV1-expressing, capsaicin (CAP)-sensitive afferent fibers innervating bladder in addition to sensory function also exhibit efferent features consisting in TRPV1-dependent release of tachykinins (TAC) affecting detrusor smooth muscle (DSM) contractions. Our aim was to investigate the effects of two novel polypeptide inhibitors of TRPV1 from the venom of tropical sea anemone Heteractis crispa, APHC1 and APHC3, on the contractions of DSM from bladders of normal and diabetic rats. Main methods: Experiments were conducted on urothelium-devoid DSM strips from normal rats and rats 8 weeks after streptozotocin-induced diabetes by means of contraction force measurements. Key findings: Pre-exposure of DSM strips to APHC1 or APHC3 (200 nM) specifically inhibited CAP-induced, TRPV1-dependent contractions. Both peptides also transiently enhanced basal tone and spontaneous contractions of DSM strips followed by delayed suppression of electric field stimulation (EFS)-evoked nonadrenergic-noncholinergic (NANC) contractions. The decrease of the amplitude of EFS-evoked NANC contractions by APHC1 or APHC3 reached 38.5 ± 3.4% and 25.1 ± 1.6%, respectively, in normal DSM strips and 46.3 ± 3.3% and 43.9 ± 1.8%, respectively, in diabetic ones. APHC-peptide-induced transient enhancement of basal tone could be mimicked by serine protease inhibitor, 4-(2-aminoethyl)bezenesulfonyl fluoride (300 μM). Significance: Our results demonstrate that APHC1 and APHC3 may be considered as effective inhibitors of bladder contractility especially during diabetic cystopathy. Modality of action of APHC-polypeptides via the mechanisms involving decreased TRPV1-dependent release of TAC from bladder afferents and suppression of TAC degradation due to their activity as endogenous proteases inhibitors is proposed. © 2012 Elsevier Inc. All rights reserved.

Sharop B.R.,Ukrainian Academy of Sciences | Sharop B.R.,State Key Laboratory of Molecular and Cellular Physiology | Boldyriev O.I.,Ukrainian Academy of Sciences | Boldyriev O.I.,State Key Laboratory of Molecular and Cellular Physiology | And 6 more authors.
Epilepsy Research | Year: 2016

Absence seizures are the non-convulsive form of generalized epilepsy critically dependent on T-type calcium channels (Cav3) in thalamic neurons. In humans, absences accompany only childhood or adolescent epileptic syndromes-though in its polygenic rat models WAG/Rij and GAERS the opposite developmental pattern is observed. Hereby we address this issue by transcriptional and functional study of thalamic Cav3 in juvenile (i.e., free of seizures) rats of the absence-prone WAG/Rij strain and their coevals of the maternal Wistar strain. First, we measured the low voltage-activated (LVA) Ca2+ current in freshly isolated thalamocortical neurons from laterodorsal nucleus of thalamus. The difference between current densities in control (12.9±1.8pA/pF) and absence epilepsy (7.9±1.8pA/pF) groups reached ~39%. Second, we assessed the contribution of different T-channel isoforms into the reduction of Cav3-mediated current in WAG/Rij juveniles by means of RT PCR. The expression of all three LVA calcium channels was revealed with the prevalence of G and I isoforms. The expression level of G isoform (Cav3.1) was 35% smaller in WAG/Rij strain if compared to the control animals while that of H and I isoforms (Cav3.2 and Cav3.3, respectively) remained stable. The weakened expression of Cav3.1 in juveniles of WAG/Rij rats could represent a compensatory mechanism determining the pattern of the age dependency in the disease manifestation by this model of absence epilepsy. © 2015 Elsevier B.V.

Nosal O.V.,Ukrainian Academy of Sciences | Nosal O.V.,State Key Laboratory of Molecular and Cellular Physiology | Lyubanova O.P.,Ukrainian Academy of Sciences | Lyubanova O.P.,State Key Laboratory of Molecular and Cellular Physiology | And 3 more authors.
Cellular and Molecular Life Sciences | Year: 2013

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni2+-binding site with critical histidine-191 (H191) within the extracellular IS3-IS4 domain of the most Ni2+-sensitive Cav3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni2+ effect and its dependence on PDC remain uncertain. Here we compared Ni2+ action on the wild-type "Ni2+-insensitive" Cav3.1w/t channel and Cav3.1Q172H mutant having glutamine (Q) equivalent to H191 of Cav3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni2+ blockade of Ca2+, Sr 2+, or Ba2+ currents was assessed by electrophysiology. Inhibition of Cav3.1w/t by Ni2+ conformed to two sites binding. Ni2+ binding with high-affinity site (IC 50 = 0.03-3 μM depending on PDC) produced maximal inhibition of 20-30 % and was voltage-dependent, consistent with its location within the channel's pore. Most of the inhibition (70-80 %) was produced by Ni2+ binding with low-affinity site (IC50 = 240-700 μM). Q172H-mutation mainly affected low-affinity binding (IC50 = 120-160 μM). The IC50 of Ni2+ binding with both sites in the Cav3.1w/t and Cav3.1Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca2+, Sr2+, or Ba2+ with Ni2+. We conclude that differential Ni2+-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni2+, may be occupied by PDC, influencing in turn the channel's permeation. © 2012 Springer Basel.

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