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

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 Signal transduction

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

Physiological role of Ca ²⁺ I

• S. Ringer: in the presence of Ca ²⁺ frog heart maintained activity for hours

• Locke: removal of Ca ²⁺ inhibited neuromuscular transmission

• Kamada and Kimoshita (1943): introduction of Ca ²⁺

into muscle fibers cause contraction

• Otto Loewi: “Ca ²⁺ ist alles.”

• Ca ²⁺ - “second” second messenger

Physiological role of Ca ²⁺ 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 [Ca ²⁺ ] [Mg ²⁺ ]

Plasma, extracellular fluid 1-2mM 1mM

Intracellular cytoplasmic 50-100nM 0.5-1mM

Intracellular stores 30-300mM

Cytoplasmic Ca ²⁺ is kept low

• Ca ²⁺ -ATPases

− Plasma membrane

− ER (SERCA)

• Na ⁺ /Ca ²⁺ exchanger – plasma membrane

• Ionophores:

– lipid-soluble, membrane-permeable ion-carriers

– e.g. A23187 (524kDa), ionomycin (709kDa) isolated from

Streptomyces

Measuring intracellular Ca ²⁺

• Ca ²⁺ -sensitive photoproteins: Aequorin (Aequoria victoria) – Emits blue light when binds Ca ²⁺

– 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

Ca ²⁺ -channels in the ER

• Ryanodine receptor (RyR): 4x560kDa

– in excitable cells (skeletal and cardiac muscle) – Modulators: Ca ²⁺ , ATP, calmodulin, FKBP12

(immunophilin)

• IP ₃ receptor (IP ₃ R): 4x310kDa

Ca ²⁺ -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 Ca ²⁺ -signaling, Ca ²⁺ -channels

ER release channel

SERCA pump Ca²⁺ channel

(gated by ligands)

Soluble Ca²⁺-sensor proteins

NCX

Internal Ca²⁺ pool (~100 nM)

Nucleus Ca²⁺ channel

(gated by voltage)

Ca²⁺

Ca²⁺ channel (gated by the emptying of Ca²⁺ stores)

Ca²⁺

External Ca²⁺

pool (mM)

Ca²⁺

MNCX

Mitochondrion Uniporter

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

Endoplasmic reticulum

Ca²⁺

Ca²⁺

Ca²⁺

Store-operated Ca ²⁺ -entry (SOCE)

Store-operated Ca ²⁺ -entry (SOCE) = capacitative Ca ²⁺ entry (1986.)

• Intracellular stores depleted plasma membrane → Ca ²⁺ channels open:

− TRP (transient rec. potential) proteins,

− CRAC (Ca ²⁺ release-activated Ca ²⁺ current) channels e.g. Orai 1 (33kDa)

– STIM1 (77kDa): transmembrane protein in the ER, Ca ²⁺ -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 (Ca ²⁺ -influx factor) (1993.)

IP3

Hormone

Receptor Plasma membrane

Cytoplasm

IP3 opens Ca²⁺ channel

Lumen of smooth endoplasmatic reticulum

IP3R

DAG PKC

PIP₂

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

IP₃ IP₃

GTP

β 

 G protein

GTP

 PLC

Several pathways use the Ca ²⁺

signal

NFAT MEF2

CBP p300 P HDAC

Ca²⁺

RAS

IP3

Src

DAG Acethylcholine.

Glutamate, Serotonine,

ATP Ligand

gated channel Depolarization/

Voltage Voltage

gated channel DHPR

CRAC

Growth factors

RTK GPCR

Hormones, Neurotransmitters

Hormones, Neurotransmitters,

Growth factors, Osmolarity

Light, Odorants, Test molecules TRPC

TRPA TRPV

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

PLCβ PLC

β  G14/15

Gq/11

cAMP ATP

AC

GTP cGMP

GC

GC Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

IP3R IP3R

IP3R

PMR1 RyR

RyR RyR

Ca²⁺

Na⁺/H⁺ exchanger

PTP Mitochondrial

uniporter SERCA

Calm CamK-IV

Cain Ca²⁺

NAADP

GI/0

Gs,Golf, Gt

Ca²⁺

Ca²⁺

Ca²⁺

Ca²⁺

cADPR Sph

Ca²⁺

Ca²⁺

Antigen Antigen

CREB PKC

Ca²⁺

Na⁺

PIP2 PIP2 PIP2

DAG DAG DAG

PLC PLC

Ca ²⁺ -regulated target proteins I

Calmodulin-dependent:

• CaM kinases

• EF2 kinase

• Phosphorylase kinase

• MLCK

• Calcineurin→NFAT

• Plasma membrane Ca ²⁺ ATPases

• Adenylyl cyclase

• Cyclic nucleotide phosphodiesterase

• MAP-2

• Tau

• Fodrin

• Neuromodulin

• NOS

Ca ²⁺ -regulated target proteins I

Calmodulin-independent

• Calpain (Ca ²⁺ -activated Cys protease)

• Synaptotagmin – exocytosis

• DAG kinase – inactivation of DAG

• Ras Neuronal Ca ²⁺ sensors

• GEFs and GAPs

• Cytoskeletal proteins: a-actinin, gelsolin

Effector mechanisms of Ca ²⁺ - 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 Protein

phosphorylation CaM kinase I,II and IV Elongation factor-2 kinase

Phosphorylase kinase Myosin light chain kinase

Ca²⁺

Ca ²⁺ in phototransduction

Plasma membrane

Cytoplasm

Rhodopsin Rhodopsin*

Na⁺

cGMP

Ca²⁺

Ca²⁺

K⁺ 4 Na⁺

Ca²⁺

1 K⁺

cGMP-gated channel

Closure of channel Transducin Transducin*

PDE PDE*

Photon

Rhodopsin*

P

ATP RK

To Na⁺ pump

cGMP GTP

Guanylate cyclase

Na⁺, Ca²⁺, K⁺ excanger

5’GTP cGMP