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Work 1-10 of 15

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Article

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

by Juan A. Contreras-Vite; Silvia Cruz-Rangel; José J. De Jesus-Perez; Iván A. Arechiga Figueroa; Aldo A. Rodriguez-Menchaca; Patricia Perez-Cornejo; Harrison Hartzell Jr.; Jorge Arreola

2016

Subjects
  • Biology, Physiology
  • Biology, Cell
  • File Download
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Abstract:Close

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca 2+ ] i ), membrane depolarization, extracellular Cl − or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca 2+ . (b) The Cl − conductance is decreased after reducing extracellular Cl − concentration ([Cl − ] o ). (c) I Cl is regulated by physiological concentrations of [Cl − ] o . (d) In cells dialyzed with 0.2 μM [Ca 2+ ] i , Cl − has a bimodal effect: at [Cl − ] o < 30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl − ] o I Cl activation displays fast and slow kinetics. To explain the contribution of V m , Ca 2+ and Cl − to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and V m -dependent binding of two Ca 2+ ions coupled to a V m -dependent binding of an external Cl − ion, with V m -dependent transitions between states. Our model predicts that extracellular Cl − does not alter the apparent Ca 2+ affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl − acts by stabilizing the open configuration induced by Ca 2+ and by contributing to the V m dependence of activation.

Article

Functional and pharmacological properties of triheteromeric GluN1/2B/2D NMDA receptors

by Feng Yi; Subhrajit Bhattacharya; Charles M. Thompson; Stephen Traynelis; Kasper B. Hansen

2019

Subjects
  • Biology, Neuroscience
  • Biology, Physiology
  • Biology, Cell
  • File Download
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Abstract:Close

Key points: Triheteromeric NMDA receptors contain two GluN1 and two distinct GluN2 subunits and mediate excitatory neurotransmission in the CNS. Triheteromeric GluN1/2B/2D receptors have functional properties intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors. GluN1/2B/2D receptors are more sensitive to channel blockade by ketamine and memantine compared to GluN1/2B receptors in the presence of physiological Mg2+. GluN2B-selective antagonists produce robust inhibition of GluN1/2B/2D receptors, and the GluN2B-selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors. These insights into the properties of triheteromeric GluN1/2B/2D receptors are necessary to appreciate their physiological roles in neural circuit function and the actions of therapeutic agents targeting NMDA receptors. Abstract: Triheteromeric NMDA-type glutamate receptors that contain two GluN1 and two different GluN2 subunits contribute to excitatory neurotransmission in the adult CNS. In the present study, we report properties of the triheteromeric GluN1/2B/2D NMDA receptor subtype that is expressed in distinct neuronal populations throughout the CNS. We show that neither GluN2B, nor GluN2D dominate the functional properties of GluN1/2B/2D receptors because agonist potencies, open probability and the glutamate deactivation time course of GluN1/2B/2D receptors are intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors. Furthermore, channel blockade of GluN1/2B/2D by extracellular Mg2+ is intermediate compared to GluN1/2B and GluN1/2D, although GluN1/2B/2D is more sensitive to blockade by ketamine and memantine compared to GluN1/2B in the presence of physiological Mg2+. Subunit-selective allosteric modulators have distinct activity at GluN1/2B/2D receptors, including GluN2B-selective antagonists, ifenprodil, EVT-101 and CP-101-606, which inhibit with similar potencies but with different efficacies at GluN1/2B/2D (∼65% inhibition) compared to GluN1/2B (∼95% inhibition). Furthermore, the GluN2B-selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors. We show that these key features of allosteric modulation of recombinant GluN1/2B/2D receptors are also observed for NMDA receptors in hippocampal interneurons but not CA1 pyramidal cells, which is consistent with the expression of GluN1/2B/2D receptors in interneurons and GluN1/2A/2B receptors in pyramidal cells. Altogether, we uncover previously unknown functional and pharmacological properties of triheteromeric GluN1/2B/2D receptors that can facilitate advances in our understanding of their physiological roles in neural circuit function and therapeutic drug actions.

Article

Editorial: Non-Coding RNAs and Human Diseases

by Yujing Li; Ge Shan; Zhao-Qian Teng; Thomas Wingo

2020

Subjects
  • Biology, Genetics
  • Biology, Cell
  • Health Sciences, Immunology
  • Biology, Anatomy
  • File Download
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Abstract:Close

Non-coding RNA (ncRNA) are functional RNA molecules that are not translatable into proteins (Djebali et al., 2012; Lonsdale et al., 2013; Forrest et al., 2014). Initially, ncRNAs referred to tRNAs and rRNAs (Brown et al., 1992; St Laurent et al., 2015). Recent technical advances have led to the discovery and characterization of many new classes of ncRNAs (Hüttenhofer and Vogel, 2006). These new ncRNAs species include snRNAs, snoRNAs, miRNAs, siRNAs, piRNAs, exRNAs, long non-coding RNA (lncRNAs), scaRNAs, and circRNAs (He and Hannon, 2004; Gu et al., 2007; Esteller, 2011; Redzic et al., 2014; Wu and Yang, 2015). While not all of their functions are known, many of the ncRNA species appear to play essential roles regulating transcription and translation of genes and transcription of ncRNAs themselves. Thus, there is little surprise that ncRNAs are identified as playing important roles in normal physiologic processes, complex human traits, and human diseases (Diederichs et al., 2016; Li et al., 2018; Fernandes et al., 2019; Vijayan and Reddy, 2020). This special issue focused on the ncRNA, particularly circRNAs, lncRNAs, miRNAs, and their role in human disease. The aim of this issue is to provide a broad overview of current research on the diverse work being done to elucidate the role of ncRNAs in disease. A major theme that emerged was the potential role of miRNAs as prognostic markers or biomarkers of disease.

Article

Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition

by James D. Wade; Xiao-Kang Lun; Nevena Zivanovic; Eberhard Voit; Bernd Bodenmiller

2020

Subjects
  • Biology, Cell
  • Engineering, Biomedical
  • File Download
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Abstract:Close

Intracellular signaling pathways are at the core of cellular information processing. The states of these pathways and their inputs determine signaling dynamics and drive cell function. Within a cancerous tumor, many combinations of cell states and microenvironments can lead to dramatic variations in responses to treatment. Network rewiring has been thought to underlie these context-dependent differences in signaling; however, from a biochemical standpoint, rewiring of signaling networks should not be a prerequisite for heterogeneity in responses to stimuli. Here we address this conundrum by analyzing an in vitro model of the epithelial mesenchymal transition (EMT), a biological program implicated in increased tumor invasiveness, heterogeneity, and drug resistance. We used mass cytometry to measure EGF signaling dynamics in the ERK and AKT signaling pathways before and after induction of EMT in Py2T murine breast cancer cells. Analysis of the data with standard network inference methods suggested EMT-dependent network rewiring. In contrast, use of a modeling approach that adequately accounts for single-cell variation demonstrated that a single reaction-based pathway model with constant structure and near-constant parameters is sufficient to represent differences in EGF signaling across EMT. This result indicates that rewiring of the signaling network is not necessary for heterogeneous responses to a signal and that unifying reaction-based models should be employed for characterization of signaling in heterogeneous environments, such as cancer.

Article

The chloride-bicarbonate exchanger pendrin is increased in the kidney of the pregnant rat

by Crystal A. West; Jill W. Verlander; Susan Wall; Chris Baylis

2015

Subjects
  • Biology, Physiology
  • Biology, Cell
  • File Download
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Abstract:Close

New Findings: What is the central question of this study? Pregnancy requires a robust plasma volume expansion driven by renal sodium retention. In the late-pregnant kidney, the aldosterone-responsive epithelial Na+ channel is increased, whereas the sodium-chloride cotransporter is decreased. Pendrin has been shown to support sodium reabsorption in the distal nephron and compensate for loss of the sodium-chloride cotransporter. We investigated the expression and abundance of pendrin in the pregnant kidney. What is the main finding and its importance? Pendrin protein, apical localization and thiazide sensitivity are increased in pregnancy. This implicates a possible role for pendrin in supporting the renal sodium chloride reabsorption and plasma volume expansion of pregnancy. Pregnancy is characterized by cumulative plasma volume expansion as a result of renal sodium retention, driven by activation of aldosterone. We previously reported that the abundance and activity of the aldosterone-responsive epithelial Na+ channel is increased, whereas the sodium-chloride cotransporter (NCC) is decreased in the kidney of the late-pregnant rat. The chloride-bicarbonate exchanger pendrin is also aldosterone responsive and has been shown to support activity of the aldosterone-responsive epithelial Na+ channel and compensate for the loss of NCC. Additionally, pendrin coupled to the sodium-dependent chloride-bicarbonate exchanger (NDCBE) mediates thiazide-sensitive sodium reabsorption in the cortical collecting duct. In this study, we investigated pendrin and NDCBE transcript expression, pendrin protein abundance, pendrin cellular localization and thiazide sensitivity in virgin, mid-pregnant and late-pregnant rats to test the hypothesis that increased pendrin activity might occur in pregnancy. By RT-PCR, NDCBE and pendrin mRNA expression was unchanged from virgins, whereas pendrin protein abundance determined by Western blotting was increased in both mid- and late-pregnant rats. The apical localization of pendrin was also increased in late-pregnant rats compared with virgins by immunohistochemistry. Pregnant rats displayed an increased natriuretic response to hydrochlorothiazide compared with virgins. Given that NCC expression is decreased in late pregnancy, an increased thiazide sensitivity may be due to inhibition of upregulated pendrin-NDCBE-coupled sodium reabsorption. Thus, increased pendrin in pregnant rats may compensate for the decreased NCC and aid in the renal sodium chloride reabsorption of pregnancy.

Article

Modulation of kidney urea transporter UT-A3 activity by alpha2,6-sialylation

by Xiaoqian Qian; Jeff Sands; Xiang Song; Guangping Chen

2016

Subjects
  • Biology, Physiology
  • Biology, Cell
  • File Download
  • View Abstract

Abstract:Close

Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.

Article

Ploidy tug-of-war: Evolutionary and genetic environments influence the rate of ploidy drive in a human fungal pathogen

by Aleeza C. Gerstein; Heekyung Lim; Judith Berman; Meleah Hickman

2017

Subjects
  • Biology, Genetics
  • Health Sciences, Human Development
  • Biology, Cell
  • Biology, Microbiology
  • File Download
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Abstract:Close

Variation in baseline ploidy is seen throughout the tree of life, yet the factors that determine why one ploidy level is maintained over another remain poorly understood. Experimental evolution studies using asexual fungal microbes with manipulated ploidy levels intriguingly reveals a propensity to return to the historical baseline ploidy, a phenomenon that we term “ploidy drive.” We evolved haploid, diploid, and polyploid strains of the human fungal pathogen Candida albicans under three different nutrient limitation environments to test whether these conditions, hypothesized to select for low ploidy levels, could counteract ploidy drive. Strains generally maintained or acquired smaller genome sizes (measured as total nuclear DNA through flow cytometry) in minimal medium and under phosphorus depletion compared to in a complete medium, while mostly maintained or acquired increased genome sizes under nitrogen depletion. Improvements in fitness often ran counter to changes in genome size; in a number of scenarios lines that maintained their original genome size often increased in fitness more than lines that converged toward diploidy (the baseline ploidy of C. albicans). Combined, this work demonstrates a role for both the environment and genotype in determination of the rate of ploidy drive, and highlights questions that remain about the force(s) that cause genome size variation.

Article

Stimulatory Role of SPAK Signaling in the Regulation of Large Conductance Ca2+-Activated Potassium (BK) Channel Protein Expression in Kidney

by Ye Bi; Chunmei Li; Yiqian Zhang; Yunman Wang; Shan Chen; Qiang Yue; Robert Hoover Jr.; Xiaonan Wang; Eric Delpire; Douglas Eaton; Jieqiu Zhuang; Hui Cai

2020

Subjects
  • Biology, Physiology
  • Biology, Cell
  • Biology, Neuroscience
  • File Download
  • View Abstract

Abstract:Close

SPS1-related proline/alanine-rich kinase (SPAK) plays important roles in regulating the function of numerous ion channels and transporters. With-no-lysine (WNK) kinase phosphorylates SPAK kinase to active the SPAK signaling pathway. Our previous studies indicated that WNK kinases regulate the activity of the large-conductance Ca2+-activated K+ (BK) channel and its protein expression via the ERK1/2 signaling pathway. It remains largely unknown whether SPAK kinase directly modulates the BK protein expression in kidney. In this study, we investigated the effect of SPAK on renal BK protein expression in both HEK293 cells and mouse kidney. In HEK293 cells, siRNA-mediated knockdown of SPAK expression significantly reduced BK protein expression and increased ERK1/2 phosphorylation, whereas overexpression of SPAK significantly enhanced BK expression and decreased ERK1/2 phosphorylation in a dose-dependent manner. Knockdown of ERK1/2 prevented SPAK siRNA-mediated inhibition of BK expression. Similarly, pretreatment of HEK293 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 reversed the inhibitory effects of SPAK knockdown on BK expression. We also found that there is no BK channel activity in PCs of CCD in SPAK KO mice using the isolated split-open tubule single-cell patching. In addition, we found that BK protein abundance in the kidney of SPAK knockout mice was significantly decreased and ERK1/2 phosphorylation was significantly enhanced. A high-potassium diet significantly increased BK protein abundance and SPAK phosphorylation levels, while reducing ERK1/2 phosphorylation levels. These findings suggest that SPAK enhances BK protein expression by reducing ERK1/2 signaling-mediated lysosomal and proteasomal degradations of the BK channel.

Article

Environmental particulate (PM2.5) augments stiffness-induced alveolar epithelial cell mechanoactivation of transforming growth factor beta

by Marilyn M. Dysart; Boris R. Galvis; Armistead G. Russell; Thomas Barker

2014

Subjects
  • Biology, Cell
  • Psychology, Physiological
  • Engineering, Biomedical
  • Engineering, Environmental
  • File Download
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Abstract:Close

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.

Article

Bioelectrical understanding and engineering of cell biology

by Zoe Schofield; Gabriel N. Meloni; Peter Tran; Christian Zerfass; Giovanni Sena; Yoshikatsu Hayashi; Murray Grant; Sonia A. Contera; Shelley D. Minteer; Minsu Kim; Arthur Prindle; Paulo Rocha; Mustafa B. A. Djamgoz; Teuta Pilizota; Patrick R. Unwin; Munehiro Asally; Orkun S. Soyer

2020

Subjects
  • Biology, Cell
  • Biology, Physiology
  • Biophysics, Medical
  • Physics, Electricity and Magnetism
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Abstract:Close

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.
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