A Carboxy-terminal Inter-Helix Linker As the Site of Phosphatidylinositol 4,5-Bisphosphate Action on Kv7 (M-type) K+ Channels

The regulation of M-type (KCNQ [Kv7]) K+ channels by phosphatidylinositol 4,5-bisphosphate (PIP2) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP2 on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP2 sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP2 action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative PIP2-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kir channels, and to the seven-β-sheet barrel motif common to other PIP2-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel–PIP2 interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels

TRPC3 determines osmosensitive [Ca2+]i signaling in the collecting duct and contributes to urinary concentration

It is well-established that the kidney collecting duct (CD) plays a central role in regulation of systemic water homeostasis. Aquaporin 2 (AQP2)-dependent water reabsorption in the CD critically depends on the arginine vasopressin (AVP) antidiuretic input and the presence of a favorable osmotic gradient at the apical plasma membrane with tubular lumen being hypotonic compared to the cytosol. This osmotic difference creates a mechanical force leading to an increase in [Ca2+]i in CD cells. The significance of the osmosensitive [Ca2+]i signaling for renal water transport and urinary concentration remain unknown. To examine molecular mechanism and physiological relevance of osmosensitivity in the CD, we implemented simultaneous direct measurements of [Ca2+]i dynamics and the rate of cell swelling as a readout of the AQP2-dependent water reabsorption in freshly isolated split-opened CDs of wild type and genetically manipulated animals and combined this with immunofluorescent detection of AVP-induced AQP2 trafficking and assessment of systemic water balance. We identified the critical role of the Ca2+-permeable TRPC3 channel in osmosensitivity and water permeability in the CD. We further demonstrated that TRPC3 -/- mice exhibit impaired urinary concentration, larger urinary volume and a greater weight loss in response to water deprivation despite increased AVP levels and AQP2 abundance. TRPC3 deletion interfered with AQP2 translocation to the plasma membrane in response to water deprivation. In summary, we provide compelling multicomponent evidence in support of a critical contribution of TRPC3 in the CD for osmosensitivity and renal water handling.Fil: Tomilin, Viktor N.. University of Texas; Estados UnidosFil: Mamenko, Mykola. Augusta University; Estados UnidosFil: Zaika, Oleg. University of Texas; Estados UnidosFil: Ren, Guohui. University of Texas; Estados UnidosFil: Marrelli, Sean P.. University of Texas; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Pochynyuk, Oleh. University of Texas; Estados Unido

Temperate bacteriophages collected by outer membrane vesicles in Komagataeibacter intermedius

The acetic acid bacteria have mainly relevance for bacterial cellulose production and fermented bio-products manufacture. The purpose of this study was to identify temperate bacteriophages in a cellulose-producing bacterial strain Komagataeibacter intermedius IMBG180. Prophages from K. intermedius IMBG180 were induced with mitomycin C and nalidixic acid. Transmission electron microscopy analysis exhibited tailed bacteriophages belonging to Myoviridae. A PCR assay targeting the capsid gene of the myoviruses proved phylogenetic position of induced phages. Nalidixic acid was poor inducer of prophages, however, it induced the OMV-like particles release. Size of OMVs depended on an antibiotic applied for phage induction and varied in the range of 30–80 and 120–200 nm. Inside some of them, tails of phages have been visible. Under conditions, inducing prophages, OMVs acted as the collectors of formed phage particles, using outer membrane receptors for phage detection (in this case, outer membrane siderophore receptor), and fulfilled therefore ―a cleaning,‖ as well as defensive functions, preventing bacteriophage spread outside population. This is the first description of myoviruses affiliated to K. intermedius, as well as outer membrane vesicles interaction with phages within this host.This research was supported by grant 47/2014 from National Academy of Sciences, Ukraine.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1521-4028hb2016Biochemistr

Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

Update to the knowledge of Odonata of Tuva and southern Krasnoyarskiy Kray, Siberia, Russia

Sympetrum fonscolombii is for the first time reported for Tuva (Tyva Republic, Russia), as found in the UbsuNur Depression. New data are provided on Odonata of the Turan or TuranUyuk Depression of Tuva, including the first record of Somatochlora exuberata in Tuva beyond the Todzha Depression and Coenagrion armatum, C. ecornutum, Aeshna juncea, A. grandis, Somatochlora graeseri and Libellula quadrimaculata for the first time reported for the Turan Depression. New distributional data and comments on Ophiogomphus spinicornis Selys, 1878 are added. Somatochlora alpestris found at Lake Oyskoe is for the first time reported for the southern Krasnoyarskiy Kray

Odonatological expeditions to the Tyva Republic (Tuva) 2000-2002

The Tyva Republic, within the Russian Federation, has acquired its present name in 1991. It was formerly known as the Tuvinian Autonomous Region within the USSR. Its common names in the past centuries were Uryankhaiskii Krai or Soyotskii Krai. In modern Russian, it is known under the name Tuva (not Tyva), which will be used further in the text. This country, for example, embraces the sources of the great Siberian river Yenisei and its capital, the town of Kyzyl, represents the geographical centre of Asia. To date, this vast and clear cut territory was hitherto almost unexplored in odonatological respect. In particular, it was not visited by the outstanding Siberian odonatologist B. F. Belyshev. Scarce materials from the recent Tuvinian territory were mentioned by Valle (1942), who considered it to be Mongolian. In the only paper concerning the dragonflies of Tuva in particular (two localities in its very south) 14 species were reported (Zaika & Kosterin 1990). The "transitional form between Ophiogomphus serpentinus and O. reductus” considered in that work, however, turned out to be the Chinese-Mongolian species Ophiogomphus spinicornis (Kosterin, 1999). Finally, one of us published two works on the general lentic fauna of the Ubsu-Nur Hollow in general (Zaika, 1996) and Lake Tere-Khol' in particular (Zaika, 1999), where 26 and 9 species were listed, respectively. The latter work was based on larval identification. Because this is difficult, this information should be taken with caution. The presence of species such as Ischnura pumilio and Cordulia aenea was not confirmed by records of adults and may be erroneous

Analysis of the features of application of vibration diagnostic methods of induction motors of transportation infrastructure using mathematical modeling

In the work, studies were carried out on the use of vibration diagnostic methods for monitoring the state of induction motors with a squirrel-cage rotor, operated in electric drives of transport equipment. The most common and difficult-to-diagnose damage to an induction motor is turn-to-turn short circuits in the stator winding, which require timely determination and establishment of the degree of damage to prevent an emergency shutdown of the equipment. The main purpose of the study is to establish the most effective areas of application of vibration diagnostic methods in determining the technical condition of the stator of induction motors under load. The experiments were carried out using simulation modeling for cases of turn-to-turn short circuits in one and two phases simultaneously, as well as with the influence of a low-quality supply voltage system on vibration parameters. The results of the work are relevant for further improvement of systems for diagnostic control of drives of transport equipment to increase the efficiency and reliability of their work

Purinergic activation of Ca2+-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron.

Mechanical forces are known to induce increases of [Ca(2+)](i) in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca(2+)](i) in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca(2+) peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca(2+)](i). Furthermore, ATP-induced Ca(2+)-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca(2+)](i) similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca(2+)](i) response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca(2+) responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca(2+) appeared to contribute to the generation of ATP-induced Ca(2+) response in ASDN cells. Importantly, flow- and hypotonic-induced Ca(2+) elevations are markedly blunted in P2Y2 -/- mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca(2+)](i) elevation during purinergic stimulation. Consistent with this, ATP-induced Ca(2+) plateau are dramatically attenuated in TRV4 -/- mice. Inhibition of TRPC channels with 10 µM BTP2 also decreased ATP-induced Ca(2+) plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN