DOCSLIB.ORG

Short Duration Presynaptic Action Potentials Shape Calcium Dynamics and Transmitter Release at the Neuromuscular Junction in Healthy and Diseased States

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Short Duration Presynaptic Action Potentials Shape Calcium Dynamics and Transmitter Release at the Neuromuscular Junction in Healthy and Diseased States Title Page Short duration presynaptic action potentials shape calcium dynamics and transmitter release at the neuromuscular junction in healthy and diseased states by Scott Patrick Ginebaugh B.S., Wayne State University, 2016 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2021 Committee Page UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Scott Patrick Ginebaugh It was defended on January 27, 2021 and approved by James R. Faeder, Ph.D., Associate Professor, Department of Computational and Systems Biology, University of Pittsburgh Zachary Freyberg, M.D., Ph.D., Assistant Professor, Department of Psychiatry, University of Pittsburgh Ivet Bahar, Ph.D., Distinguished Professor & John K. Vries Chair, Department of Computational and Systems Biology, University of Pittsburgh Rozita Laghaei, Ph.D., Research Scientist, Pittsburgh Supercomputing Center, Carnegie Mellon University Dissertation Director: Stephen D. Meriney, Ph.D., Professor, Department of Neuroscience, University of Pittsburgh ii Copyright © by Scott Patrick Ginebaugh 2021 iii Short duration presynaptic action potentials shape calcium dynamics and transmitter release at the neuromuscular junction in healthy and diseased states Scott Patrick Ginebaugh, PhD University of Pittsburgh, 2021 The action potential (AP) waveform controls the opening of voltage-gated calcium channels (VGCCs) at presynaptic nerve terminals. The calcium ion flux through these VGCCs acts as a second messenger, triggering the release of neurotransmitter. The frog and mouse neuromuscular junctions (NMJs) have long been model synapses for the study of neurotransmission, but the presynaptic AP waveforms from these NMJs have never been recorded because the nerve terminals are too small impale with an electrode for electrophysiological recordings. Neurotransmission from nerve terminals occurs at highly organized structures called active zones (AZs), and the relationships between the AP, VGCCs, and neurotransmission at AZs are poorly understood. Understanding these relationships is important for the study of Lambert- Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ is decreased, leading to severe muscle weakness. This reduced neurotransmission is thought to be caused by an antibody-mediated removal of presynaptic VGCCs and disruption of AZ structure. Furthermore, 3,4-diaminopyridine (3,4-DAP; the FDA-approved treatment for LEMS) was thought to indirectly increase calcium flux into the AZs by broadening the presynaptic AP, but this mechanism has come under scrutiny. Here, we use voltage imaging to optically record AP waveforms from frog and mouse motoneuron terminals. We find that the AP waveforms from these terminals are very brief in duration. We hypothesize the brief duration of these APs helps prevent a depletion of docked synaptic vesicles in AZs by limiting calcium flux during APs, and iv is thus an important mechanism by which healthy NMJs maintain reliable neurotransmission during repeated stimulation. We also show that clinically relevant concentrations of 3,4-DAP increase calcium flux by broadening the AP. Next, we use our recorded AP waveforms to constrain computational models of mammalian AZs, and utilize these models to investigate the effects of LEMS on the AZ. We demonstrate that the disruption of AZ structure plays an essential role in LEMS pathology. Finally, we show that disrupting the calcium-sensing proteins in the AZ could result in LEMS pathology and hypothesize that anti-VGCC antibodies may not be solely responsible for LEMS in many LEMS patients. v Table of Contents Preface .......................................................................................................................................... xv 1.0 Introduction ............................................................................................................................. 1 1.1 Action potential propagation in neurons ...................................................................... 2 1.2 The active zone ................................................................................................................ 4 1.2.1 Voltage-gated calcium channels ..........................................................................4 1.2.2 Calcium-activated potassium channels ..............................................................6 1.2.3 Active zone proteins contributing to calcium sensing and vesicle release ......8 1.2.4 Coupling of active zone components ................................................................10 1.3 Properties of the neuromuscular junction ................................................................. 11 1.3.1 The frog neuromuscular junction .....................................................................12 1.3.2 The murine neuromuscular junction ...............................................................13 1.4 Lambert-Eaton Myasthenic Syndrome ...................................................................... 16 1.4.1 Clinical properties ..............................................................................................16 1.4.2 LEMS neuromuscular physiology ....................................................................18 1.5 Treatment of Lambert-Eaton Myasthenic Syndrome ............................................... 20 1.5.1 Symptomatic treatment with 3,4-Diaminopyridine ........................................20 1.5.2 Immunotherapies and cholinesterase inhibitors .............................................21 1.5.3 Potential for treatment with calcium channel agonists ..................................23 1.6 Voltage imaging ............................................................................................................ 24 1.7 Computational modeling of the active zone ............................................................... 26 1.7.1 The advantages of computational modeling ....................................................26 vi 1.7.2 Selecting a method for computational modeling .............................................27 2.0 The frog motor nerve terminal has very brief action potentials and three electrical regions predicted to differentially control transmitter release .......................................... 29 2.1 Introduction .................................................................................................................. 30 2.2 Materials and Methods ................................................................................................ 32 2.2.1 Cutaneous pectoris nerve-muscle preparations ..............................................32 2.2.2 Voltage imaging ..................................................................................................32 2.2.3 Image and data analysis.....................................................................................34 2.2.4 MCell simulations ..............................................................................................43 2.2.5 NEURON simulations ........................................................................................46 2.3 Results ............................................................................................................................ 47 2.3.1 Action potentials at the adult frog neuromuscular junction are brief in duration ........................................................................................................................47 2.3.2 Differences in action potential duration along the length of motor nerve terminal branches reveal three distinct electrical regions.......................................48 2.3.3 The propagation speed of the AP waveform does not change along the length of the terminal .............................................................................................................55 2.3.4 Motor nerve terminal width does not correlate with AP duration or propagation speed within the motor nerve terminal ...............................................57 2.4 Discussion ...................................................................................................................... 60 2.4.1 Very brief AP waveforms may aid in maintaining strength and reliability at the NMJ ........................................................................................................................60 vii 2.4.2 Changes in the width of the AP waveform can explain proximal-distal changes in neurotransmitter release .........................................................................62 2.4.3 Relationship between axon width, the duration of the AP waveform, and AP propagation speed .......................................................................................................66 3.0 A high affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs .......................... 68 3.1 Introduction .................................................................................................................. 69 3.2 Results ............................................................................................................................ 73 3.2.1 3,4-DAP effects on Kv3 potassium channels ....................................................73 3.2.2 3,4-DAP effects on the presynaptic AP waveform at the NMJ ......................75 3.2.3
Load more

Recommended publications
  • Massive Alterations of Sarcoplasmic Reticulum Free Calcium in Skeletal Muscle Fibers Lacking Calsequestrin Revealed by a Genetic
    Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe M. Canatoa,1, M. Scorzetoa,1, M. Giacomellob, F. Protasic,d, C. Reggiania,d, and G. J. M. Stienene,2 Departments of aHuman Anatomy and Physiology and bExperimental Veterinary Sciences, University of Padua, 35121 Padua, Italy; cCeSI, Department of Basic and Applied Medical Sciences, University G. d’Annunzio, I-66013 Chieti, Italy; dIIM-Interuniversity Institute of Myology and eLaboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, 1081BT, Amsterdam, The Netherlands Edited* by Clara Franzini-Armstrong, University of Pennsylvania Medical Center, Philadelphia, PA, and approved November 12, 2010 (received for review June 30, 2010) The cytosolic free Ca2+ transients elicited by muscle fiber excitation tion essential to maintain a low metabolic rate in quiescent ex- are well characterized, but little is known about the free [Ca2+] citable cells. In addition, CSQ has been shown to modulate RyR- dynamics within the sarcoplasmic reticulum (SR). A targetable mediated Ca2+ release from the SR (6). To understand the pivotal ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed role of CSQ in SR function, it is critical to determine free SR us to investigate SR Ca2+ dynamics and analyze the impact of cal- Ca2+ concentration, and this has been made possible by the ad- sequestrin (CSQ) on SR [Ca2+] in enzymatically dissociated flexor vent of a targetable ratiometric FRET-based indicator, such as digitorum brevis muscle fibers from WT and CSQ-KO mice lacking D1ER Cameleon (7). Seminal studies using this technique have isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)].
    [Show full text]
  • The Role of Altered Calcium-Handling in Mechanically-Induced
    THE ROLE OF ALTERED CALCIUM-HANDLING IN MECHANICALLY-INDUCED ARRHYTHMIAS DURING ACUTE REGIONAL ISCHEMIA by Peter A.R. Baumeister Submitted in partial fulfillment of requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia May 2016 Copyright by Peter A.R. Baumeister, 2016 TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ vi ABSTRACT .................................................................................................................. viii LIST OF ABBREVIATIONS USED ................................................................................. ix ACKNOWLEDGEMENTS ................................................................................................ xi CHAPTER 1: Introduction .................................................................................................. 1 1.1 Sudden Cardiac Death and Acute Regional Ischemia .............................................. 1 1.2 Conditions for Arrhythmogenesis ............................................................................ 3 1.2.I Abnormal Automaticity ........................................................................................ 3 1.2.II Triggered Activity - Afterdepolarizations........................................................... 5 1.2.III Reentrant Activity .............................................................................................. 9 1.2.IV Conclusion ......................................................................................................
    [Show full text]
  • Calcium and Heart Failure: How Did We Get Here and Where Are We Going?
    International Journal of Molecular Sciences Review Calcium and Heart Failure: How Did We Get Here and Where Are We Going? Natthaphat Siri-Angkul 1,2,3, Behzad Dadfar 4 , Riya Jaleel 5 , Jazna Naushad 6, Jaseela Parambathazhath 7, Angelia A. Doye 8, Lai-Hua Xie 1 and Judith K. Gwathmey 1,9,* 1 Department of Cell Biology and Molecular Medicine, Rutgers University-New Jersey Medical School, Newark, NJ 07103, USA 2 Cardiac Electrophysiology Research and Training Center, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 3 Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 4 Department of General Medicine, School of Medicine, Mazandaran University of Medical Sciences, Sari 1471655836, Iran 5 School of International Education, Zhengzhou University, Zhengzhou 450001, China 6 Weill Cornell Medicine Qatar, Doha P. O. Box 24144, Qatar 7 Process Dynamics Laboratories, Al Khor 679576, Qatar 8 Gwathmey Inc., Cambridge, MA 02138, USA 9 Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA * Correspondence: [email protected]; Tel.: +1-973-972-2411; Fax: +1-973-972-7489 Abstract: The occurrence and prevalence of heart failure remain high in the United States as well as globally. One person dies every 30 s from heart disease. Recognizing the importance of heart failure, clinicians and scientists have sought better therapeutic strategies and even cures for end-stage Citation: Siri-Angkul, N.; Dadfar, B.; heart failure. This exploration has resulted in many failed clinical trials testing novel classes of Jaleel, R.; Naushad, J.; pharmaceutical drugs and even gene therapy. As a result, along the way, there have been paradigm Parambathazhath, J.; Doye, A.A.; Xie, shifts toward and away from differing therapeutic approaches.
    [Show full text]
  • David Alfred Eisner
    CURRICULUM VITAE David Alfred Eisner BHF Professor of Cardiac Physiology University of Manchester Cardiac Physiology Unit 3.18 Core Technology Facility 46 Grafton St Manchester M13 9NT Contents Page Career details 2 Honours 2 Editorial Boards 2 Professional Appointments 3 Service within the University 4 Bibliometric Analysis 5 Research Synopsis 6 Research grants held 9 Invited seminars and lectures 12 Publications 15 David Eisner David Eisner Born: 3 January, 1955, Manchester, England 1973-1976 King's College Cambridge 1976 B.A. (class II.1) 1976-1979 Balliol College Oxford 1979 D.Phil. (Supervisor Prof D. Noble) 1979-1980 Postdoctoral Research Assistant- Physiological Laboratory, Cambridge (with Prof I.M. Glynn). 1981-1986 University Lecturer, University College London. 1982-1988 Visiting Assistant Professor, Department of Physiology, University of Maryland 1986-1990 Wellcome Senior Lecturer, University College London 1988- Research Associate Professor, Department of Physiology, University of Maryland 1989-1990 Reader in Physiology, University of London 1990-1999 Professor of Veterinary Biology, University of Liverpool 1992-1999 Head of Veterinary Preclinical Sciences Department 1999 - Professor of Cardiac Physiology, Department of Medicine, The University of Manchester 2000- British Heart Foundation Professor of Cardiac Physiology, The University of Manchester Honours 1975 Powell Prize and Senior Scholarship: King's College Cambridge 1985 Awarded Pfizer Prize for research in biology 1986 Wellcome Senior Lectureship 1988 Awarded Wellcome
    [Show full text]
  • X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin
    cells Article X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin Sarita Limbu 1, Benjamin L. Prosser 2, William J. Lederer 3, Christopher W. Ward 4 and Mohsin S. Jafri 1,3,* 1 School of Systems Biology and The Krasnow Institute for Advanced Study, George Mason University, Fairfax, VA 22030, USA; [email protected] 2 Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; [email protected] 3 Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD 20201, USA; [email protected] 4 Center for Biomedical Engineering and Technology and Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD 20201, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-703-993-8420 Abstract: The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination Citation: Limbu, S.; Prosser, B.L.; of modeling and experiment are further informed by the elimination of length-dependent changes Lederer, W.J.; Ward, C.W.; Jafri, M.S.
    [Show full text]
  • Probing Calmodulin in Living Neurones
    Probing Calmodulin In Living Neurones Jacobus Martinus Milikan A thesis submitted for the degree of Doctor of Philosophy in the University of London Department of Physiology University College London 2000 ProQuest Number: 10014385 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10014385 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT In this thesis I explored the role of the calcium binding protein calmodulin in neuronal signalling in detail. Nerve cells have a high calmodulin content and calmodulin is involved in neuronal processes such as the memory models Long Term Potentiation and Long Term Depression, and neurite outgrowth. Previous studies mainly relied on biochemical and pharmacological techniques and immunocytochemistry to study calmodulin function. I investigated the suitability of the potent calmodulin antagonist calmidazolium for studying the calmodulin-mediated processes in living pheochroma cytoma 12 (PCI2) cells. I used two fluorescently labelled calmodulins to study calmodulin function in hving neurones: FL-CaM, which does not change its fluorescence when it binds calcium, and TA-CaM, which increases its fluorescence upon calcium binding.
    [Show full text]
  • Adrenergic Stimulation in Cardiac Myocytes
    Cardiovascular Research (2014) 104, 347–354 doi:10.1093/cvr/cvu201 Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during b-adrenergic stimulation in cardiac myocytes Sarah J. Briston1, Katharine M. Dibb1, R. John Solaro2, David A. Eisner1, and Andrew W. Trafford1* 1Unit of Cardiac Physiology, Manchester Academic Health Science Centre, Core Technology Facility, 46 Grafton St, Manchester M13 9NT, UK; and 2Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA Received 24 January 2014; revised 24 July 2014; accepted 25 August 2014; online publish-ahead-of-print 2 September 2014 Time for primary review: 33 days Aims During activation of cardiac myocytes, less than 1% of cytosolic Ca is free; the rest is bound to buffers, largely SERCA, and troponin C. Signalling by phosphorylation, as occurs during b-adrenergic stimulation, changes the Ca-binding affinity of these proteins and may affect the systolic Ca transient. Our aim was to determine the effects of b-adrenergic stimulation on Ca buffering and to differentiate between the roles of SERCA and troponin. ..................................................................................................................................................................................... Methods Ca buffering was studied in cardiac myocytes from mice: wild-type (WT), phospholamban-knockout (PLN-KO), and mice and results expressing slow skeletal troponin I (ssTnI) that is not protein kinase A phosphorylatable. WT cells showed no change in Ca buffering in response to the b-adrenoceptor agonist isoproterenol (ISO). However, ISO decreased Ca buffering in PLN-KO myocytes, presumably unmasking the role of troponin. This effect was confirmed in WT cells in which SERCA activity was blocked with the application of thapsigargin.
    [Show full text]
  • Proquest Dissertations
    ÎTTTT u Ottawa L'Université canadienne Canada's university FACULTÉ DES ÉTUDES SUPÉRIEURES I=I FACULTY OF GRADUATE AND ETPOSTOCTORALES u Ottawa posdoctoral studies L'Université canadienne Canada's university Krystyna Anna Banas AUTEUR DE LA THESE / AUTHOR OF THESIS M.Cs. (Cellular and Molecular Medicine) GRADE /DEGREE Department of Cellular and Molecular Medicine FACULTE, ECOLE, DEPARTEMENT / FACULTY, SCHOOL, DEPARTMENT Katp Channel Kir6.2 Subunit Distribution Differs Between Muscles and Between Fiber Types in Skeletal Muscle TITRE DE LA THÈSE / TITLE OF THESIS Jean-Marc Renaud DIRECTEUR (DIRECTRICE) DE LA THESE / THESIS SUPERVISOR CO-DIRECTEUR (CO-DIRECTRICE) DE LA THESE / THESIS CO-SUPERVISOR JRàshmiVotharx William Staines Gary W. Slater Le Doyen de la Faculté des études supérieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral Studies KATp CHANNEL Kir6.2 SUBUNIT DISTRIBUTION DIFFERS BETWEEN MUSCLES AND BETWEEN FIBER TYPES IN SKELETAL MUSCLE By Krystyna Anna Banas Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment ofthe requirements for the degree of Master of Science Cellular and Molecular Medicine Faculty ofMedicine University of Ottawa Krystyna Anna Banas, Ottawa, Canada, 2009 Library and Archives Bibliothèque et ?F? Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-65505-4 Our file Notre référence ISBN:
    [Show full text]
  • Abnormal Calcium Handling in Muscular Dystrophy Kevin G Culligan and Kay Ohlendieck(1)
    Abnormal Calcium Handling in Muscular Dystrophy Kevin G Culligan and Kay Ohlendieck(1) Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dub- lin 2 and (1) Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland Abstract Duchenne muscular dystrophy, primarily caused by a deficiency in the membrane cy- toskeletal protein dystrophin, is the most frequent neuromuscular disorder in humans and is characterized by progressive muscle weakness and wasting. This review outlines patho- physiological mechanisms of abnormal calcium handling by the sarcolemma, sarcoplasmic reticulum and mitochondria that may contribute to muscle fibre destruction. Dystrophin- deficient skeletal muscle membranes succumb to exercise-induced membrane ruptures more frequently than those of normal fibres. These transient micro-ruptures allow for the insertion of calcium leak channels into the sarcolemma during the natural processes of cell membrane resealing. Ion leak channels give rise to localized calcium elevations, contribut- ing to a cycle of enhanced protease activity and leak channel activation. Within the sar- coplasmic reticulum, loss of the calsequestrin-like proteins may contribute to decreased lu- minal calcium buffering. This might indirectly amplify elevated free cytosolic calcium con- centrations. Mitochondria proximal to ryanodine receptor calcium release channel com- plexes, although thought to buffer channel leakiness, may give rise to an increase in pro- apoptotic signals, ultimately giving rise to muscle cell death. Here, we summarise the po- tential effects of abnormal calcium homeostasis on fibre degeneration in dystrophic skeletal muscle. Key words: calcium homeostasis, calcium leak channel, calsequestrin, Duchenne muscular dystrophy, dystrophin-glycoprotein complex. Basic Appl Myol 12 (4): 147-157, 2002 Duchenne muscular dystrophy (DMD) is the most quence [21, 82], giving rise to a 14kb transcript produc- common and severe form of a group of muscle wasting ing 79 exons.
    [Show full text]
  • Calsequestrin Depolymerizes When Calcium Is Depleted in the Sarcoplasmic Reticulum of Working Muscle
    Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle Carlo Mannoa, Lourdes C. Figueroaa, Dirk Gillespiea, Robert Fittsb, ChulHee Kangc, Clara Franzini-Armstrongd,1, and Eduardo Riosa,1 aSection of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago, IL 60612; bDepartment of Biology, Marquette University, Milwaukee, WI 53233; cDepartment of Chemistry, Washington State University, Pullman, WA 99164; and dDepartment of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104 Contributed by Clara Franzini-Armstrong, December 12, 2016 (sent for review October 7, 2016; reviewed by Sandor Gyorke and Vincenzo Sorrentino) Calsequestrin, the only known protein with cyclical storage and at the interfaces between calsequestrin monomers, requiring two supply of calcium as main role, is proposed to have other functions, monomers to achieve useful binding affinity (12). It follows that 2+ which remain unproven. Voluntary movement and the heart beat higher [Ca ] will promote polymerization, which, in turn, will require this calcium flow to be massive and fast. How does provide additional sites for calcium binding. If adequately tuned, this cooperative feature will provide optimal calcium release calsequestrin do it? To bind large amounts of calcium in vitro, 2+ ability at physiological [Ca ]SR (2). calsequestrin must polymerize and then depolymerize to release it. 2+ Does this rule apply inside the sarcoplasmic reticulum (SR) of a [Ca ]SR decreases moderately (13) during normal skeletal muscle function; depletion is greater in cardiac muscle, where it working cell? We answered using fluorescently tagged calsequestrin – expressed in muscles of mice. By FRAP and imaging we monitored reaches 50 90% (14).
    [Show full text]
  • Mechanisms of Calcium Handling During a Malignant Hyperthermia Event
    Mechanisms of calcium handling during a malignant hyperthermia event. Rocky Hyun Suk Choi M.S., Molecular Biosciences & Bioengineering B.S., Molecular Biology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine Abstract Malignant hyperthermia (MH) is a clinical syndrome of the skeletal muscle that presents as a hypermetabolic response to volatile gas anaesthetics such as halothane, isoflurane, and to the depolarizing muscle relaxant succinylcholine. Genetic predisposition mainly arises from mutations in the gene for skeletal muscle ryanodine receptor (RyR1); a 2+ calcium channel that is tightly regulated to release Ca for muscle contraction. MH susceptible RyR1 are more prone to open by RyR1 agonist, resulting in the uncontrolled release of Ca2+ from the sarcoplasmic reticulum (SR). The hypermetabolic state is due to the excessive consumption of adenosine triphosphate (ATP) as the muscle works to clear the high cytosolic Ca2+. Dantrolene has been used to great effect by clinicians in treating MH. However the mechanism of its action remains highly contested. This thesis examines Ca2+ handling in muscle fibers where RyR1 activity is changed due to mutations. RyR1 activity was also acutely changed by altering the cytoplasmic environment and by treating the muscle fiber with varying RyR1 modulators. The release of SR Ca2+ was monitored using fluorescent Ca2+ indicators in mechanically skinned fibers isolated from rat, toad and human muscle susceptible to MH. The results show that the inhibitory effect of dantrolene is dependent on cytosolic concentrations of 2+ 2+ Mg ([Mg ]cyto), an endogenous negative regulator of the RyR1.
    [Show full text]
  • Organellar Calcium Buffers
    Downloaded from http://cshperspectives.cshlp.org/ on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Organellar Calcium Buffers Daniel Prins and Marek Michalak Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Correspondence: [email protected] Ca2þ is an important intracellular messenger affecting many diverse processes. In eukaryotic cells, Ca2þ storage is achieved within specific intracellular organelles, especially the endo- plasmic/sarcoplasmic reticulum, in which Ca2þ is buffered by specific proteins known as Ca2þ buffers. Ca2þ buffers are a diverse group of proteins, varying in their affinities and capacities for Ca2þ, but they typically also carry out other functions within the cell. The wide range of organelles containing Ca2þ and the evidence supporting cross-talk between these organelles suggest the existence of a dynamic network of organellar Ca2þ signaling, mediated by a variety of organellar Ca2þ buffers. INTRACELLULAR Ca2þ DYNAMICS that they also serve other roles within the cell. These roles include catalyzing the correct fold- a2þ serves as an intracellular messenger in ing of other cellular proteins, regulating Ca2þ various cellular processes, including muscle C release and retention, and communicating in- contraction, gene expression, and fertilization formation about Ca2þ levels within organelles (Berridge et al. 2003). To use Ca2þ, the cell to other proteins. requires a readily mobilizable source of Ca2þ, the majority of which is found within the lumen of the endoplasmic reticulum (ER) and/or sar- ENDOPLASMIC RETICULUM coplasmic reticulum (SR), but is also located in the Golgi apparatus, peroxisomes, mitochon- The ER is a multifunctional organelle within the dria, and endolysosomal compartments.
    [Show full text]