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The calcium signal

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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The calcium signal

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signaltransduction The calcium signal

  3. Physiological role of Ca2+I • S. Ringer: in the presence of Ca2+ frog heart maintained activity for hours • Locke: removal of Ca2+ inhibited neuromuscular transmission • Kamada and Kimoshita (1943): introduction of Ca2+ into muscle fibers cause contraction • Otto Loewi: “Ca2+ istalles.” • Ca2+ - “second” second messenger

  4. Physiological role of Ca2+II • 3 forms in the body: • Free • Bound • Trapped (hydroxiapathite in calcified tissues e.g. bones, teeth) • Hypercalcemia: reduced neuromuscular transmission, myocardial dysfunction, lethargy • Hypocalcemia:excitabilty of membranes ↑, tetany, seizures, death

  5. Cytoplasmic Ca2+ is kept low • Ca2+-ATPases • Plasmamembrane • ER (SERCA) • Na+/Ca2+ exchanger • plasma membrane • Ionophores: • lipid-soluble, membrane-permeable ion-carriers • e.g. A23187 (524kDa), ionomycin (709kDa) isolated fromStreptomyces

  6. Measuring intracellular Ca2+ • Ca2+-sensitive photoproteins: Aequorin(Aequoriavictoria) • Emits blue light when binds Ca2+ • First microinjected into target cell (eg. giant squid axon) • Fluorescent indicators:Quin-2, Fura-2 (UV); Fluo-3 (visible light) • Can be used for cell suspensions – the signal represents the summation of individual unsynchronized contributions • Sigle cell measurement – fluorescent/confocal microscope • Genetically engineered indicators • Aequorin-transfected cells • Calmodulin-Myosin light chain Kinase-GFP

  7. Ca2+-channels in the ER • Ryanodinereceptor (RyR): 4x560kDa • in excitable cells (skeletal and cardiac muscle) • Modulators: Ca2+, ATP, calmodulin, FKBP12(immunophilin) • IP3receptor (IP3R): 4x310kDa

  8. Ca2+-influx through plasma membrane channels • Voltage-operated channels (VOCCs) • Nerve and muscle cells • open upon depolarization • L, N, P/Q, R and T types • Receptor-operated channels (eg. Glutamate NMDA receptor) • TRPM2 channels • Activated by ADP-ribose • Oxidative stress

  9. Intra/extracellularcompartments of Ca2+-signaling, Ca2+-channels Ca2+ channel (gatedbythe emptying of Ca2+ stores) Ca2+ channel (gatedbyvoltage) External Ca2+ pool (mM) Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ channel (gated by ligands) NCX Internal Ca2+ pool (~100 nM) Soluble Ca2+-sensor proteins SERCA pump MNCX Ca2+ Ca2+ Ca2+ Nucleus Ca2+ Uniporter ER release channel Ca2+ Endoplasmic reticulum Mitochondrion

  10. Store-operated Ca2+-entry (SOCE) • Store-operatedCa2+-entry (SOCE)=capacitative Ca2+ entry (1986.) • Intracellular stores depleted plasma membrane →Ca2+ channels open: • TRP(transient rec. potential) proteins, • CRAC (Ca2+ release-activated Ca2+ current) channelse.g. Orai 1 (33kDa) • STIM1 (77kDa): transmembraneprotein in the ER, Ca2+-sensor • 3 potential mechanisms of STIM1 action: • Direct interaction between ER and plasma membrane • Movement of STIM1 from the ER to the plasma membrane • Soluble mediator : CIF (Ca2+-influx factor) (1993.)

  11. IP3 Hormone Plasma membrane Receptor PIP2 DAG PKC Cytoplasm PLC    G protein GTP IP3 GTP β Ca2+ IP3 IP3 opens Ca2+ channel IP3R Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Lumen of smooth endoplasmatic reticulum

  12. Acethylcholine. Glutamate, Serotonine, ATP Hormones, Neurotransmitters, Growth factors, Osmolarity Several pathways use the Ca2+ signal Depolarization/ Voltage Growth factors Hormones, Neurotransmitters Light, Odorants, Test molecules Antigen Antigen CRAC DHPR Voltage gated channel Ligand gated channel BCR TCR RTK GPCR TRPC TRPA TRPV PMCA NCX CNG GPCR Ca2+ Ca2+ Ca2+ G14/15 Gq/11 RAS Src GI/0 DAG Gs,Golf, Gt Na+ PIP2 PIP2 PIP2 PIP2 PLC PLC PLCβ PLC Ca2+ AC GC Ca2+ Ca2+ DAG DAG DAG GTP cAMP ATP cGMP Ca2+ Ca2+ Ca2+ GC ADP-Ribose, ArachidonicAcid, Sphingosine IP3  β IP3R Ca2+ RyR IP3R PKC NAADP Ca2+ Sph Ca2+ PMR1 Ca2+ Ca2+ RyR Ca2+ Ca2+ Ca2+ cADPR Ca2+ Ca2+ Ca2+ Ca2+ P CBP Ca2+ HDAC CREB CamK-IV RyR Calm NFAT MEF2 p300 Ca2+ SERCA Cain Geneexpression Mitochondrial uniporter Na+/H+ exchanger Ca2+ Hypertrophy Ca2+ IP3R PTP

  13. Ca2+-regulated target proteins I • Calmodulin-dependent: • CaMkinases • EF2 kinase • Phosphorylasekinase • MLCK • Calcineurin→NFAT • Plasma membrane Ca2+ATPases • Adenylylcyclase • Cyclic nucleotide phosphodiesterase • MAP-2 • Tau • Fodrin • Neuromodulin • NOS

  14. Ca2+-regulated target proteins I • Calmodulin-independent • Calpain (Ca2+-activated Cys protease) • Synaptotagmin – exocytosis • DAG kinase – inactivation of DAG • Ras Neuronal Ca2+ sensors • GEFs and GAPs • Cytoskeletal proteins: a-actinin, gelsolin

  15. Effectormechanisms of Ca2+-signaling Ca2+ Calmodulin Nitricoxideformation Cyclicnucleotide metabolism Protein dephosphorylation Protein phosphorylation Cytoskeleton Ca2+ transport CaMkinase I,II and IV Elongation factor-2 kinase Phosphorylasekinase Myosinlightchainkinase MAP-2 Tau Fodrin Neuromodulin Adenylyl cyclase Cyclic nuvleotide Phosphodiesterase Plasma membrane Ca2+ ATPases Calcineurin

  16. Ca2+ inphototransduction Plasmamembrane Cytoplasm To Na+ pump 4 Na+ Na+, Ca2+, K+ excanger Photon ATP Rhodopsin Rhodopsin* RK Ca2+ 1 K+ Transducin Transducin* Closure of channel K+ PDE PDE* Ca2+ Na+ cGMP Guanylate cyclase Rhodopsin* P Ca2+ cGMP-gated channel cGMP GTP 5’GTP cGMP

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