1. 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 /1/A

2. 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 /1/A
Tímea Berki and Ferenc Boldizsár
Signal transduction
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+ ist alles.”
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
[Ca2+]
[Mg2+]
Plasma, extracellular fluid
1-2mM
1mM
Intracellular cytoplasmic
50-100nM
0.5-1mM
Intracellular stores
30-300mM

5. 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

6. 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

7. 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

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/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

10. 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.)

11. 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

12. 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

13. 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

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. 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+

16. 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