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