DOCSLIB.ORG

Understanding the Molecular Mechanism of Trp Channel

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

UNDERSTANDING THE MOLECULAR MECHANISM OF TRP CHANNEL ACTIVATION/INHIBITION BY STRUCTURAL ANALYSIS by AMRITA SAMANTA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisor: Vera Moiseenkova-Bell Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY August 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of AMRITA SAMANTA Candidate for the degree of Physiology and Biophysics* Witold Surewicz (Committee Chair) Sudha Chakrapani Vera Moiseenkova-Bell Phoebe Stewart Derek Taylor Isabelle Deschenes May 29, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables vii List of Figures viii Acknowledgments xi List of Abbreviations xiii Abstract xv Chapter 1: Introduction 1.1 The Transient Receptor Potential (TRP) family of ion channels 2 1.2 TRPC subfamily 2 1.2.1 TRPC1 3 1.2.2 TRPC4 & TRPC5 5 1.2.3 TRPC2, TRPC3, TRPC6 & TRPC7 6 1.3 TRPV Subfamily 7 1.3.1 TRPV1 7 1.3.2 TRPV2 10 1.3.3 TRPV3 & TRPV4 11 i 1.3.4 TRPV5 & TRPV6 12 1.4 TRPM Subfamily 13 1.4.1 TRPM1 & TRPM3 14 1.4.2 TRPM4 & TRPM5 15 1.4.3 TRPM2, TRPM6 & TRPM7 16 1.4.4 TRPM8 17 1.5 TRPA Subfamily 18 1.6 TRPML Subfamily 20 1.6.1 TRPML1 21 1.6.2 TRPML2 & TRPML3 22 1.7 TRPP Subfamily 23 1.7.1 Polycystin family 25 1.7.2 TRPP2, TRPP3 & TRPP5 27 1.8 Purpose of this study 29 1.9 Cryo Electron Microscopy 30 Figures 32 ii Chapter 2: Understanding the molecular mechanism of the TRPV2 channel gating during ligand activation by cryo-EM 2.1 Introduction 37 2.2 Materials and Methods 2.2.1 Stable cell line generation and flux assay 38 2.2.2 Protein expression and purification 40 2.2.3 Cryo-EM data collection 40 2.2.4 Image processing 41 2.2.5 Model building 42 2.3 Results 2.3.1 Role of the pore turret domain in TRPV2 activation and large 43 organic molecules uptake 2.3.2 Architecture of the TRPV2 in apo- and CBD-activated states 44 2.3.3 Conformational differences between apo- and CBD-activated 47 TRPV2 2.4 Discussion 49 Figures 52 iii Chapter 3: Understanding the ligand induced conformational changes of mouse TRPA1 channel during activation and inhibition 3.1 Introduction 71 3.2 Materials and Methods 3.2.1 Expression and purification of TRPA1 74 3.2.2 Limited proteolysis of TRPA1 75 3.2.3 LC-MS analysis 75 3.3 Results 3.3.1 Limited proteolysis and in-solution mass spectrometry to 77 determine ligand induced conformational changes in TRPA1 3.3.2 Analysis of NMM (electrophilic agonist) induced 79 conformational change in TRPA1 3.3.3 Analysis of PF-4840154 (non-electrophilic agonist) induced 80 conformational change in TRPA1 3.3.4 Analysis of menthol (non-electrophilic modulator) induced 82 conformational change in TRPA1 3.3.5 Analysis of A-967079 (non-electrophilic antagonist) induced 83 conformational change in TRPA1 3.4 Discussion 84 iv Table 89 Figures 90 Chapter 4: Discussion and future directions 4.1 Summary 103 4.2 Impact of this study 4.2.1 Application for structural studies of other memorable proteins 105 4.2.2 Application on health and disease 106 4.3 Future directions 4.3.1 Permeation of large organic cations through TRP channels 107 4.3.2 Charecterization of TRPV2 specific blocker and 108 understanding the mechanism of TRPV2 inhibition 4.3.3 Resolving structure of full length TRPA1 reconstituted into 109 nanodisc by using cryo EM 4.3.4 Regions of TRPA1 molecule affected by other electrophilic 110 and non-electrophilic ligands 4.4 Concluding remarks 110 Figures 112 Appendix 117 v Reference 119 vi List of Tables Table 3.1. Modulators of TRPA1 used in this study 89 vii List of Figures A schematic representation of the TRP superfamily of ion Figure 1.1 32 channels High resolution structures for TRP channels initially Figure 1.2 33 resolved A schematic representation of the TRP channel pore Figure 1.3 34 showing the two regions of constriction (gates) A schematic representation of the TRPP1 and TRPP2 Figure 1.4 interacting with each other through their C-terminal region and 35 forming the receptor-channel complex Functional characterization of wild type and truncated Figure 2.1 TRPV2 indicates the pore turret region is essential for 52 passage of both Ca2+ and large cations Figure 2.2 Summary flowchart of TRPV2 data processing 53 Figure 2.3 Resolution data for TRPV2 refinement 54 Cryo-EM analysis of apo and CBD-activated TRPV2 Figure 2.4 55 channel Figure 2.5 Structural details of apo and CBD-activated TRPV2 56 Structural comparison of 4.6Å apo TRPV2 and previously Figure 2.6 58 published ~5Å apo TRPV2 channel Figure 2.7 Putative CBD binding site 59 viii Cryo-EM density map comparison of apo and CBD- Figure 2.8 activated TRPV2 channel indicates large conformational 60 change in the TMD but not in the ARD Comparison of apo and CBD-activated TRPV2 models and Figure 2.9 62 TMD Figure 2.10 Architecture of the apo and CBD-activated TRPV2 pores 64 Structural comparison of full length apo TRPV2, truncated Figure 2.11 65 apo TRPV2 and apo TRPV1 pores Figure 2.12 Comparison of TRPV pore architectures 67 Figure 2.13 Comparison of the pore region of the TRPV channels 68 Electron density corresponding to regions of interest in the Figure 2.14 69 TRPV2 structures validates model assignment Figure 3.1 TRPA1 purification and limited proteolysis of pure TRPA1 90 Construction of a representation of the full-length TRPA1 Figure 3.2 91 channel Figure 3.3 Effect of NMM on TRPA1 conformation 92 Nano- LC-MS/MS spectrum of the modified peptides after Figure 3.4 94 Asp-N digestion of NMM-activated TRPA1 Effect of non-electrophilic modulators on TRPA1 Figure 3.5 96 conformation Figure 3.6 Effect of A-967079 on TRPA1 conformation 97 Dimer representations of TRPA1 showing additional Figure 3.7 99 regions of miscleavage upon ligand interaction ix Cartoon representation of TRPA1 showing the regions Figure 3.8 101 involved in channel gating Cartoon representation of gating mechanism of TRPV2 in Figure 4.1 112 presence of CBD A tree representing the mammalian TRP superfamily of ion Figure 4.2 113 channels with most of the high resolution structures Schematic representation of the principle of limited Figure 4.3 114 proteolysis followed by mass spectrometry Inhibition of TRPV2 activity by PL and preliminary cryo-EM Figure 4.4 115 data of PL-inhibited TRPV2 Cryo-EM analysis of CBD-activated and PL-inhibited Figure 4.5 116 TRPV2 channel x ACKNOWLEDGEMENTS I am especially thankful to my PhD advisor, Dr. Vera Moiseenkova-Bell, for her professional and personal support and guidance during my training as a graduate student in her lab. I would like to thank the previous and present lab members of the Moiseenkova-Bell lab: Dr. Matthew Cohen, who taught me some of the molecular biology techniques and helped me to think critically about projects during the initial few years of my journey; Dr. Kevin Huynh, who taught me a lot of the molecular biology techniques that I used to complete this dissertation; Monica Kane, who was an undergraduate in the lab and brought a lot of fresh energy to the lab; Taylor Hughes, who is a great lab mate and colleague I enjoyed working and learning new techniques together with in the lab; Dr. Ievgen Ignatenko, a great lab mate who is always ready to help. I was also fortunate to have the opportunity to train an undergraduate student, Connor Dawedeit and a high school student, Fatema Uddin, both of whom brought a lot of young energy to the lab. I would like to thank my dissertation committee members (Dr. Witold Surewicz, Dr. Sudha Chakrapani, Dr. Phoebe Stewart, Dr. Derek Taylor and Dr. Isabelle Deschenes) for guiding me to think critically, forcing me to stay focused and supporting me in completion of my dissertation. Additionally, I would like to thank Heather Holdaway and Dr. Sudheer Molugu, who trained me in cryo-EM microscopy; Dr. Janna Kiselar who helped me with mass spectrometry; Dr. George Dubyak who trained me in cell culture and Ca2+ flux assay measurements; Dr. David Lodowski for his collaboration and support, Dr. Yuhang Liu and Dr. Seungil Han for their collaboration and allowing to use their electron microscope. Moreover, I would like xi to acknowledge Dr. Sudha Chakrapani who taught me the technique of electrophysiology and gave me space in her lab during the last few months of my graduation when my lab relocated to UPenn. In addition, I would also like to thank Chakrapani lab members for being great colleagues to work with and good friends. Thank you to my family for believing in me; Diptendu konar – I could not have completed this journey without you and your constant support and Adhrit for being such a patient and understanding child. Finally, I would like to thank all the professors, friends and colleagues whom I did not mention here but who were essential for my success in graduate school. xii List of Abbreviations ADPKD Autosomal Dominant Polycystic Kidney Disease AITC Allyl Isothiocyanate ARD Ankyrin Repeat Domain CBD Cannabidiol Cryo-EM Cryo Electron Microscopy DADS Diallyl Disulphide GBM Glioblastoma Multiforme ICRAC Calcium Release-Activated Calcium Current IP3 Inositol Triphosphate ISOC Store Operated Calcium Current LEL Late Endosome and Lysosome LC-MS Liquid Chromatography Mass Spectrometry NaV channels Voltage gated Sodium channels NHERF Na+/H+ exchanger regulatory factor NOMPC No Mechanoreceptor Potential C PKD Polycystic Kidney Disease PL Piperlongumine xiii REJ Receptor for Egg Jelly RTX/DkTx Resiniferatoxin/Double-knot Toxin S1-S6 Transmembrane helices 1 through
Recommended publications
  • The Role of Transient Receptor Potential Cation Channels in Ca2þ Signaling

    The Role of Transient Receptor Potential Cation Channels in Ca2þ Signaling

    Downloaded from http://cshperspectives.cshlp.org/ on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press The Role of Transient Receptor Potential Cation Channels in Ca2þ Signaling Maarten Gees, Barbara Colsoul, and Bernd Nilius KU Leuven, Department of Molecular Cell Biology, Laboratory Ion Channel Research, Campus Gasthuisberg, Herestraat 49, bus 802, Leuven, Belgium Correspondence: [email protected] The 28 mammalian members of the super-family of transient receptor potential (TRP) channels are cation channels, mostly permeable to both monovalent and divalent cations, and can be subdivided into six main subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and the TRPA (ankyrin) groups. TRP channels are widely expressed in a large number of different tissues and cell types, and their biological roles appear to be equally diverse. In general, considered as poly- modal cell sensors, they play a much more diverse role than anticipated. Functionally, TRP channels, when activated, cause cell depolarization, which may trigger a plethora of voltage-dependent ion channels. Upon stimulation, Ca2þ permeable TRP channels 2þ 2þ 2þ generate changes in the intracellular Ca concentration, [Ca ]i,byCa entry via the plasma membrane. However, more and more evidence is arising that TRP channels are also located in intracellular organelles and serve as intracellular Ca2þ release channels. This review focuses on three major tasks of TRP channels: (1) the function of TRP channels as Ca2þ entry channels; (2) the electrogenic actions of TRPs; and (3) TRPs as Ca2þ release channels in intracellular organelles. ransient receptor potential (TRP) channels choanoflagellates, yeast, and fungi are primary Tconstitute a large and functionally versatile chemo-, thermo-, or mechanosensors (Cai 2008; family of cation-conducting channel proteins, Wheeler and Brownlee 2008; Chang et al.
  • Transient Receptor Potential Channel Promiscuity Frustrates Constellation

    Transient Receptor Potential Channel Promiscuity Frustrates Constellation

    the sole sensor responsible for noxious cold responses in M+A+ LETTER neurons (1). However, TRPA1 is also activated by cooling and underlies at least part of the noxious cold responsiveness of Transient receptor potential channel AITC-sensitive neurons (3). Third, the authors used nicardipine 2+ promiscuity frustrates to selectively inhibit CaV1-type voltage-gated Ca channels (1). However, several dihydropyridines, including nicardipine, also constellation pharmacology act as TRPA1 agonists (4). These considerations led us to propose an alternative Sensory neurons from the trigeminal and dorsal root ganglia molecular interpretation of the difference between M+A− (DRG) have nerve endings in the skin and mucosa, where they and M+A+ neurons, which is in much better agreement with detect environmental stimuli and convey this information to published work. In accord with the authors, we conclude that the central nervous system. Several members of the transient M+A− neurons express TRPM8 but lack expression of receptor potential (TRP) superfamily of ion channels act as TRPA1. In contrast to the authors, we propose that M+A+ prime molecular sensors for thermal and chemical stimuli in neurons express TRPA1 as the prime cold and menthol these sensory neurons. However, it is incompletely understood sensor (2, 3). This interpretation is consistent with published how TRP channel expression and modulation affect the stimulus observations that menthol responses in M+A− but not in sensitivities of distinct neuronal subtypes. M+A+ neurons are inhibited by TRPM8 antagonists (5) In a recent article, Teichert et al. (1) described a “constellation and that TRPA1-mediated responses to cold in neurons are pharmacology approach” to identify and characterize subtypes characterized by a higher (colder) threshold (3).
  • Ankrd9 Is a Metabolically-Controlled Regulator of Impdh2 Abundance and Macro-Assembly

    Ankrd9 Is a Metabolically-Controlled Regulator of Impdh2 Abundance and Macro-Assembly

    ANKRD9 IS A METABOLICALLY-CONTROLLED REGULATOR OF IMPDH2 ABUNDANCE AND MACRO-ASSEMBLY by Dawn Hayward A dissertation submitted to The Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy Baltimore, Maryland April 2019 ABSTRACT Members of a large family of Ankyrin Repeat Domains proteins (ANKRD) regulate numerous cellular processes by binding and changing properties of specific protein targets. We show that interactions with a target protein and the functional outcomes can be markedly altered by cells’ metabolic state. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 by binding to vesicles. Upon nutrient limitation, ANKRD9 loses association with vesicles and assembles with IMPDH2 into rod-like structures, in which IMPDH2 is stable. Inhibition of IMPDH2 with Ribavirin favors ANKRD9 binding to rods. The IMPDH2/ANKRD9 assembly is reversed by guanosine, which restores association of ANKRD9 with vesicles. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicles-to-rods transition as well as binding and regulation of IMPDH2. ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Thus, the status of guanosine pools affects the mode of ANKRD9 action towards IMPDH2. Advisor: Dr. Svetlana Lutsenko, Department of Physiology, Johns Hopkins University School of Medicine Second reader:
  • The Intracellular Ca2+ Release Channel TRPML1 Regulates Lower Urinary Tract Smooth Muscle Contractility

    The Intracellular Ca2+ Release Channel TRPML1 Regulates Lower Urinary Tract Smooth Muscle Contractility

    The intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility Caoimhin S. Griffina, Michael G. Alvaradoa, Evan Yamasakia, Bernard T. Drummb,c, Vivek Krishnana, Sher Alia, Eleanor M. Naglea, Kenton M. Sandersb, and Scott Earleya,1 aDepartment of Pharmacology, Center for Molecular and Cellular Signaling in the Cardiovascular System, Reno School of Medicine, University of Nevada, Reno, NV 89557-0318; bDepartment of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV 89557-0318; and cDepartment of Life & Health Sciences, Dundalk Institute of Technology, Louth, Ireland A91 K584 Edited by Mark T. Nelson, University of Vermont, Burlington, VT, and approved October 13, 2020 (received for review August 12, 2020) TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-perme- including dense granulomembranous storage bodies in neurons, able, nonselective cation channel that is predominantly localized to elevated plasma gastrin, vacuolization in the gastric mucosa, and the membranes of late endosomes and lysosomes (LELs). Intracellular retinal degeneration (14). Interestingly, however, an anatomical release of Ca2+ through TRPML1 is thought to be pivotal for mainte- examination of these mice reveals dramatically distended bladders nance of intravesicular acidic pH as well as the maturation, fusion, and (14), leading us to question how TRPML1, an intracellular Ca2+-re- trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice lease channel important in LEL function, affects bladder physiology. −/− (Mcoln1 ) induces a hyperdistended/hypertrophic bladder phenotype. The lower urinary tract (LUT) is composed of the urinary Here, we investigated this phenomenon further by exploring an un- bladder and urethra—structures that serve the simple, reciprocal conventional role for TRPML1 channels in the regulation of Ca2+-signal- functions of storing and voiding urine (15).
  • The Intracellular Ca2+ Release Channel TRPML1 Regulates Lower Urinary Tract Smooth Muscle Contractility

    The Intracellular Ca2+ Release Channel TRPML1 Regulates Lower Urinary Tract Smooth Muscle Contractility

    The intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility Caoimhin S. Griffina, Michael G. Alvaradoa, Evan Yamasakia, Bernard T. Drummb,c, Vivek Krishnana, Sher Alia, Eleanor M. Naglea, Kenton M. Sandersb, and Scott Earleya,1 aDepartment of Pharmacology, Center for Molecular and Cellular Signaling in the Cardiovascular System, Reno School of Medicine, University of Nevada, Reno, NV 89557-0318; bDepartment of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV 89557-0318; and cDepartment of Life & Health Sciences, Dundalk Institute of Technology, Louth, Ireland A91 K584 Edited by Mark T. Nelson, University of Vermont, Burlington, VT, and approved October 13, 2020 (received for review August 12, 2020) TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-perme- including dense granulomembranous storage bodies in neurons, able, nonselective cation channel that is predominantly localized to elevated plasma gastrin, vacuolization in the gastric mucosa, and the membranes of late endosomes and lysosomes (LELs). Intracellular retinal degeneration (14). Interestingly, however, an anatomical release of Ca2+ through TRPML1 is thought to be pivotal for mainte- examination of these mice reveals dramatically distended bladders nance of intravesicular acidic pH as well as the maturation, fusion, and (14), leading us to question how TRPML1, an intracellular Ca2+- trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice release channel important in LEL function, affects bladder −/− (Mcoln1 ) induces a hyperdistended/hypertrophic bladder phenotype. physiology. Here, we investigated this phenomenon further by exploring an un- The lower urinary tract (LUT) is composed of the urinary conventional role for TRPML1 channels in the regulation of Ca2+-signal- bladder and urethra—structures that serve the simple, reciprocal ing activity and contractility in bladder and urethral smooth muscle cells functions of storing and voiding urine (15).
  • Chapter Four – TRPA1 Channels: Chemical and Temperature Sensitivity

    Chapter Four – TRPA1 Channels: Chemical and Temperature Sensitivity

    CHAPTER FOUR TRPA1 Channels: Chemical and Temperature Sensitivity Willem J. Laursen1,2, Sviatoslav N. Bagriantsev1,* and Elena O. Gracheva1,2,* 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA 2Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT, USA *Corresponding author: E-mail: [email protected], [email protected] Contents 1. Introduction 90 2. Activation and Regulation of TRPA1 by Chemical Compounds 91 2.1 Chemical activation of TRPA1 by covalent modification 91 2.2 Noncovalent activation of TRPA1 97 2.3 Receptor-operated activation of TRPA1 99 3. Temperature Sensitivity of TRPA1 101 3.1 TRPA1 in mammals 101 3.2 TRPA1 in insects and worms 103 3.3 TRPA1 in fish, birds, reptiles, and amphibians 103 3.4 TRPA1: Molecular mechanism of temperature sensitivity 104 Acknowledgments 107 References 107 Abstract Transient receptor potential ankyrin 1 (TRPA1) is a polymodal excitatory ion channel found in sensory neurons of different organisms, ranging from worms to humans. Since its discovery as an uncharacterized transmembrane protein in human fibroblasts, TRPA1 has become one of the most intensively studied ion channels. Its function has been linked to regulation of heat and cold perception, mechanosensitivity, hearing, inflam- mation, pain, circadian rhythms, chemoreception, and other processes. Some of these proposed functions remain controversial, while others have gathered considerable experimental support. A truly polymodal ion channel, TRPA1 is activated by various stimuli, including electrophilic chemicals, oxygen, temperature, and mechanical force, yet the molecular mechanism of TRPA1 gating remains obscure. In this review, we discuss recent advances in the understanding of TRPA1 physiology, pharmacology, and molecular function.
  • Repeat Proteins Challenge the Concept of Structural Domains

    Repeat Proteins Challenge the Concept of Structural Domains

    844 Biochemical Society Transactions (2015) Volume 43, part 5 Repeat proteins challenge the concept of structural domains Rocıo´ Espada*1, R. Gonzalo Parra*1, Manfred J. Sippl†, Thierry Mora‡, Aleksandra M. Walczak§ and Diego U. Ferreiro*2 *Protein Physiology Lab, Dep de Qu´ımica Biologica, ´ Facultad de Ciencias Exactas y Naturales, UBA-CONICET-IQUIBICEN, Buenos Aires, C1430EGA, Argentina †Center of Applied Molecular Engineering, Division of Bioinformatics, Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria ‡Laboratoire de physique statistique, CNRS, UPMC and Ecole normale superieure, ´ 24 rue Lhomond, 75005 Paris, France §Laboratoire de physique theorique, ´ CNRS, UPMC and Ecole normale superieure, ´ 24 rue Lhomond, 75005 Paris, France Abstract Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular structure that do not fold independently, but rather co-operate in stabilizing structural forms that comprise several repeat-units. For many natural repeat-proteins, it has been shown that weak energetic links between repeats lead to the breakdown of co-operativity and the appearance of folding sub-domains within an apparently regular repeat array. The quasi-1D architecture of repeat-proteins is crucial in detailing how the local energetic balances can modulate the folding dynamics of these proteins, which can be related to the physiological behaviour of these ubiquitous biological systems. Introduction and between repeats, challenging the concept of structural It was early on noted that many natural proteins typically domain. collapse stretches of amino acid chains into compact units, defining structural domains [1]. These domains typically correlate with biological activities and many modern proteins can be described as composed by novel ‘domain arrange- Definition of the repeat-units ments’ [2].
  • Interplay Between Gating and Block of Ligand-Gated Ion Channels

    Interplay Between Gating and Block of Ligand-Gated Ion Channels

    brain sciences Review Interplay between Gating and Block of Ligand-Gated Ion Channels Matthew B. Phillips 1,2, Aparna Nigam 1 and Jon W. Johnson 1,2,* 1 Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA; [email protected] (M.B.P.); [email protected] (A.N.) 2 Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA * Correspondence: [email protected]; Tel.: +1-(412)-624-4295 Received: 27 October 2020; Accepted: 26 November 2020; Published: 1 December 2020 Abstract: Drugs that inhibit ion channel function by binding in the channel and preventing current flow, known as channel blockers, can be used as powerful tools for analysis of channel properties. Channel blockers are used to probe both the sophisticated structure and basic biophysical properties of ion channels. Gating, the mechanism that controls the opening and closing of ion channels, can be profoundly influenced by channel blocking drugs. Channel block and gating are reciprocally connected; gating controls access of channel blockers to their binding sites, and channel-blocking drugs can have profound and diverse effects on the rates of gating transitions and on the stability of channel open and closed states. This review synthesizes knowledge of the inherent intertwining of block and gating of excitatory ligand-gated ion channels, with a focus on the utility of channel blockers as analytic probes of ionotropic glutamate receptor channel function. Keywords: ligand-gated ion channel; channel block; channel gating; nicotinic acetylcholine receptor; ionotropic glutamate receptor; AMPA receptor; kainate receptor; NMDA receptor 1. Introduction Neuronal information processing depends on the distribution and properties of the ion channels found in neuronal membranes.
  • Submicroscopic Distribution of Ruthenium Red in Human Glioblastoma Multiforme*

    Submicroscopic Distribution of Ruthenium Red in Human Glioblastoma Multiforme*

    I. Neurosurg. / Volume 32 / February, 1970 Submicroscopic Distribution of Ruthenium Red in Human Glioblastoma Multiforme* WILLIAM BONDAREFF, M.D., PH.D. Department of Biostructure,. Northwestern University Medical School, Chicago, Illinois LECTRON microscope studies have forme is well documented,24 and more recent suggested the presence of a poly- electron microscope studies further demon- anionic intercellular substance in strate this complexity.8,11,13,18,19 The ultra- mammalian central nervous tissue. 3-6,~ The structure of the intercellular matrix of glio- composition of this material, which appears blastoma multiforme has not been described to occupy the extracellular space of the previously, but light microscope studies sug- brain, is not known, but chemical studies in- gest an intercellular matrix with staining dicate the presence of an acid mucopolysac- properties similar to those of connective charide. 2~ Its intercellular distribution has tissue. 11 been demonstrated by visualization in the electron microscope of electron-opaque Materials and Methods metal complexes (for example, of phospho- Specimens of nine suspected gliomas were tungstic acid '-'~ and ruthenium redO, but the obtained during operation at the Neurosurgi- relation of these complexes to plasma mem- cal Clinic, Sahlgrenska Hospital, Gothen- branes is obscured in central nervous tissue burg, Sweden. After surgical exposure of the by the sparsity of intercellular space. The di- neoplasm, a specimen was taken by sharp mensions of the extracellular space in brain dissection from an area thought not to be are not known with certainty, and the stain- necrotic. Electrocautery was avoided and able material localized within the intercellu- special care was exercised in the use of lar spaces may be either a plasma mem- clamps and hemostats.
  • 1 UST College of Science Department of Biological Sciences

    1 UST College of Science Department of Biological Sciences

    UST College of Science Department of Biological Sciences 1 Pharmacogenomics of Myofascial Pain Syndrome An Undergraduate Thesis Submitted to the Department of Biological Sciences College of Science University of Santo Tomas In Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in Biology Jose Marie V. Lazaga Marc Llandro C. Fernandez May 2021 UST College of Science Department of Biological Sciences 2 PANEL APPROVAL SHEET This undergraduate research manuscript entitled: Pharmacogenomics of Myofascial Pain Syndrome prepared and submitted by Jose Marie V. Lazaga and Marc Llandro C. Fernandez, was checked and has complied with the revisions and suggestions requested by panel members after thorough evaluation. This final version of the manuscript is hereby approved and accepted for submission in partial fulfillment of the requirements for the degree of Bachelor of Science in Biology. Noted by: Asst. Prof. Marilyn G. Rimando, PhD Research adviser, Bio/MicroSem 602-603 Approved by: Bio/MicroSem 603 panel member Bio/MicroSem 603 panel member Date: Date: UST College of Science Department of Biological Sciences 3 DECLARATION OF ORIGINALITY We hereby affirm that this submission is our own work and that, to the best of our knowledge and belief, it contains no material previously published or written by another person nor material to which a substantial extent has been accepted for award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text. We also declare that the intellectual content of this undergraduate research is the product of our work, even though we may have received assistance from others on style, presentation, and language expression.
  • Molecular Biology of Neuronal Voltage-Gated Calcium Channels

    Molecular Biology of Neuronal Voltage-Gated Calcium Channels

    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 30, No 3, 123-130, September 1998 Molecular biology of neuronal voltage-gated calcium channels Hemin Chin and is capable of directing expression of calcium channel activity in heterologous expression systems. In the central Genetics Research Branch, Division of Basic and Clinical Neuroscience Research, nervous system (CNS), VGCCs are expressed by five National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, distinct a1 subunit genes (α1A, α1B, α1C, α1D and α1E), U.S.A. which exhibit further variations due to alternative splicing of the primary RNA transcripts. The α1C and, α1D su b u n i t Accepted 3 August 1998 genes encode dihydropyridine (DHP)-sensitive L-type channels, while the three other α1 subunit genes (α1A, α1B and α1E) give rise to DHP-insensitive P/Q-, N- and R-type channels, respectively. The α2 and δ s u b u n i t proteins are produced by proteolytic cleavage of a larger precursor produced by the single α2-δ gene (Table 1). Introduction Three alternatively spliced variants of the α2 subunit are expressed in a tissue-specific manner. Two variants Calcium ions are important intracellular messengers have been isolated from the brain and skeletal muscle mediating a number of neuronal functions including neuro- (Kim et al., 1992; Williams et al., 1992), and a distinct transmitter release, neurosecretion, neuronal excitation, third splice variant which is expressed in glial cells has survival of eurons, and regulation of gene expression. been recently identified (Puro et al., 1996). In addition to The entry of calcium across the plasmamembrane in the gene encoding the skeletal muscle β subunit, three response to membrane depolarization or activation of 1 other β subunit genes (β2, β3 and β4) have been isolated neurotransmitter receptors represents a major pathway thus far.
  • A Structural Guide to Proteins of the NF-Kb Signaling Module

    A Structural Guide to Proteins of the NF-Kb Signaling Module

    Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press A Structural Guide to Proteins of the NF-kB Signaling Module Tom Huxford1 and Gourisankar Ghosh2 1Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, California 92182-1030 2Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0375 Correspondence: [email protected] The prosurvival transcription factor NF-kB specifically binds promoter DNA to activate target gene expression. NF-kB is regulated through interactions with IkB inhibitor proteins. Active proteolysis of these IkB proteins is, in turn, under the control of the IkB kinase complex (IKK). Together, these three molecules form the NF-kB signaling module. Studies aimed at charac- terizing the molecular mechanisms of NF-kB, IkB, and IKK in terms of their three-dimen- sional structures have lead to a greater understanding of this vital transcription factor system. F-kB is a master transcription factor that from the perspective of their three-dimensional Nresponds to diverse cell stimuli by activat- structures. ing the expression of stress response genes. Multiple signals, including cytokines, growth factors, engagement of the T-cell receptor, and NF-kB bacterial and viral products, induce NF-kB Introduction to NF-kB transcriptional activity (Hayden and Ghosh 2008). A point of convergence for the myriad NF-kB was discovered in the laboratory of of NF-kB inducing signals is the IkB kinase David Baltimore as a nuclear activity with bind- complex (IKK). Active IKK in turn controls ing specificity toward a ten-base-pair DNA transcription factor NF-kB by regulating pro- sequence 50-GGGACTTTCC-30 present within teolysis of the IkB inhibitor protein (Fig.