Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
THE CALCIUM SIGNAL
Tímea Berki and Ferenc BoldizsárSignal transduction
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
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
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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 0.5-1mMIntracellular stores 30-300mM
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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
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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
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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
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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
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Intra/extracellular compartments of Ca2+-signaling, Ca2+-channels
ER releasechannel
SERCApump
Ca2+ channel(gated by ligands)
Soluble Ca2+-sensorproteins
NCX
Internal Ca2+ pool (~100 nM)
Nucleus
Ca2+ channel(gated by voltage)
Ca2+
Ca2+ channel(gated by theemptying ofCa2+ stores)
Ca2+
External Ca2+
pool (mM)Ca2+
MNCX
Mitochondrion
Uniporter
Ca2+Ca2+
Ca2+
Ca2+
Endoplasmicreticulum
Ca2+
Ca2+
Ca2+
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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.)
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IP3Hormone
Receptor Plasma membrane
Cytoplasm
IP3 opens Ca2+ channel
Lumen of smoothendoplasmatic reticulum
IP3R
DAG PKCPIP2
Ca2+
Ca2+Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
IP3
IP3
GTP
β
G proteinGTP
PLC
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Several pathways use the Ca2+ signal
NFAT MEF2CBPp300
PHDAC
Ca2+
RAS
IP3
Src
DAG
Acethylcholine.Glutamate,Serotonine,
ATPLigandgated
channel
Depolarization/Voltage
Voltagegated
channel
DHPRCRAC
Growthfactors
RTK GPCR
Hormones,Neurotransmitters
Hormones,Neurotransmitters,
Growth factors,Osmolarity
Light, Odorants,Test molecules
TRPCTRPATRPV
PMCA NCX CNG
Hypertrophy
Gene expression
BCR TCR GPCR
ADP-Ribose,Arachidonic Acid,
Sphingosine
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
PIP2
PLCβPLC
β G14/15Gq/11
cAMP ATP
AC
GTPcGMP
GC
GC
Ca2+
Ca2+
Ca2+Ca2+
Ca2+
IP3R
IP3R
IP3R
PMR1
RyR
RyR
RyR
Ca2+
Na+/H+
exchanger
PTP
Mitochondrialuniporter
SERCA
Calm CamK-IV
Cain
Ca2+
NAADP
GI/0Gs,Golf,
Gt
Ca2+
Ca2+
Ca2+
Ca2+
cADPR
Sph
Ca2+
Ca2+
AntigenAntigen
CREB
PKC
Ca2+
Na+PIP2 PIP2PIP2
DAG DAG DAG
PLC PLC
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Ca2+-regulated target proteins ICalmodulin-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
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Ca2+-regulated target proteins ICalmodulin-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
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Effector mechanisms of Ca2+-signaling
Calmodulin
Cyclic nucleotidemetabolism
Adenylyl cyclaseCyclic nuvleotide
Phosphodiesterase
Ca2+ transport
Plasma membraneCa2+ ATPases
Proteindephosphorylation
Calcineurin
Cytoskeleton
MAP-2Tau
FodrinNeuromodulin
Nitric oxide formationProteinphosphorylation
CaM kinase I,II and IVElongation factor-2 kinase
Phosphorylase kinaseMyosin light chain kinase
Ca2+
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Ca2+ in phototransductionPlasma membrane
Cytoplasm
Rhodopsin Rhodopsin*
Na+
cGMPCa2+
Ca2+
K+
4 Na+
Ca2+
1 K+
cGMP-gated channel
Closure ofchannel
Transducin Transducin*
PDE PDE*
Photon
Rhodopsin* PATP
RK
To Na+ pump
cGMP GTP
Guanylatecyclase
Na+, Ca2+,K+ excanger
5’GTP cGMP
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