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

The calcium signal Tímea Berki and Ferenc Boldizsár Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signal transduction The calcium signal

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+ ist alles.” Ca2+ - “second” second messenger

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 [Ca2+] [Mg2+] Plasma, extracellular fluid 1-2mM 1mM Intracellular cytoplasmic 50-100nM 0.5-1mM Intracellular stores 30-300mM

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

Measuring intracellular Ca2+ Ca2+-sensitive photoproteins: Aequorin (Aequoria victoria) 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

Ca2+-channels in the ER Ryanodine receptor (RyR): 4x560kDa in excitable cells (skeletal and cardiac muscle) Modulators: Ca2+, ATP, calmodulin, FKBP12 (immunophilin) IP3 receptor (IP3R): 4x310kDa

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

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

Store-operated Ca2+-entry (SOCE) Store-operated Ca2+-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) channels e.g. Orai 1 (33kDa) STIM1 (77kDa): transmembrane protein 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.)

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

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

Ca2+-regulated target proteins I Calmodulin-dependent: CaM kinases EF2 kinase Phosphorylase kinase MLCK Calcineurin→NFAT Plasma membrane Ca2+ ATPases Adenylyl cyclase Cyclic nucleotide phosphodiesterase MAP-2 Tau Fodrin Neuromodulin NOS

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

Effector mechanisms of Ca2+-signaling Calmodulin Cyclic nucleotide metabolism Adenylyl cyclase Cyclic nuvleotide Phosphodiesterase Ca2+ transport Plasma membrane Ca2+ ATPases Protein dephosphorylation Calcineurin Cytoskeleton MAP-2 Tau Fodrin Neuromodulin Nitric oxide formation phosphorylation CaM kinase I,II and IV Elongation factor-2 kinase Phosphorylase kinase Myosin light chain kinase Ca2+

Ca2+ in phototransduction Plasma membrane Cytoplasm Rhodopsin Rhodopsin* Na+ cGMP Ca2+ K+ 4 Na+ 1 K+ cGMP-gated channel Closure of Transducin Transducin* PDE PDE* Photon P ATP RK To Na+ pump GTP Guanylate cyclase Na+, Ca2+, K+ excanger 5’GTP