1. Intracellular Ca2+ signalling
Vytášek 2009

2. Ca2+
is a universal intracellular signalling molecule (secondary messenger), which controls numerous developmental and cellular pathways
intracellular concentration of resting cell is nM, activated cells have Ca2+ concentration above 1M
concentration changes can activate extremely various signaling cascades not only in different types of cells but also in different areas of the same cell (e.g. Ca2+ elevation in nucleus of a neurone activates gene transcription and at the synapse causes neurotransmitter release)
Serum

3. Processes affected by changes in Ca2+
Learning and memory
Contraction and relaxation
Membrane excitability
Cell motility
Cytoplasmic and mitochondrial metabolism
Synthesis of proteins and lipids
Cell cycle
Apoptosis

4. Ca2+ binding cellular proteins
These proteins are conformationaly modulated by calcium binding
They exist on two forms :
- apo-form without calcium at intracellular Ca2+ concentration 50nM (resting state)
- calcium-form with bound Ca2+ after elevating of intracellular Ca2+ concentration up to 1000nM (activated state after stimulus)
The typical motif of polypeptide chain, by which Ca2+ is bound and which modulates the conformation of the appropriate protein, is EF-hand motif

5. EF-hand motif of calcium binding site
alfa-helix E forefinger loop around Ca - clenched middle finger alfahelx - F thumb

6. Proteins containing EF-hand domain
Calmodulin subfamily
calmodulin, troponin C, light chain of myosin
Calcineurin subfamily
calcineurin (phosphatase)
Other subfamilies
calpain (proteolytic enzyme), calbindin (storing cytosolic protein of Ca2+)

7. Calmodulin present in all cells of eukaryotes
a small, acidic protein approximately 148 amino acids long (17kDa)
highly conservated amino acid sequence
four EF-hand domains respectively four binding sites for calcium
binding of calcium modulates its conformation and then Ca2+-calmodulin is able or disable of the interaction with its target proteins and complex of Ca2+-calmodulin-target protein exhibits different functional properties than uncomplexed target protein

8. The conformational changes of calmodulin after binding of calcium ion

9. Ca2+ Blue circles

10. Proteins affected complexation with Ca2+-calmodulin
Protein kinases (CaMK)
Calcineurin (protein phosphatase 2B)
Adenylate cyclase
NO synthases
cAMP phoshodiesterase
Ion chanels
Receptors

11. Calcium/calmodulin-dependent protein kinases
CaMK enzyme family is activated by increase in intracellular Ca2+ and tranfers phosphate from ATP to serine or threonine residue on other proteins

12. Calcium/calmodulin-dependent protein kinases (CaMK) with narrow specifity
CaMLCK - myosin light chain kinase phoshorylates myosin light chain and this phoshorylation is sufficient for muscle contraction The enzyme exists on two forms - SkCaMLCK in skeletal muscle cells and SmCaMLCK is present in smooth muscle cells as well as in various other cells.
Both CaMLCKs are stimulated by binding Ca2+-calmodulin but this binding can be inhibited in the case of SmCaMLCK by its own phoshorylation (caused by various other kinases)

13. CaMK with narrow specifity
PhK - phosphorylase kinase
phosphorylates and activates glycogen phosphorylase (increases glycogen degradation)
eEF-2K (CaMKIII) - eukaryotic elongation factor 2 kinase
phosphorylates Thr-56 in eEF-2 (this factor promotes ribosomal translocation along mRNA during translation) and thus inhibits its activity. The activity of eEF-2K is increased by binding Ca2+-calmudulin and down-regulated by its own phosphorylation caused by various kinases

14. CaMK with broad specifity
CaMKI - Calcium/calmodulin-dependent protein kinase I
three isoforms are products of distinct genes. Several substrates for this enzyme were described but its physiological substrates and roles are still unknown
CaMKII- Calcium/calmodulin-dependent protein kinase II
Particularly important in the nervous system. It is localized in pre- and postsynaptic compartment. Four isoforms can phosphorylate various proteins in every part of a cell. It posses also autoregulatory properties. It is supposed that CaMKII plays inportant role on learning and memory

15. CaMK with broad specifity
CaMKIV - Calcium/calmodulin-dependent protein kinase IV
It contains nuclear localization sequence and therefore it is responsible for Ca2+-dependent phoshorylation of various nuclear transcription factors (but also phosphorylates cytosolic proteins). Autophosphorylation inhibits its activity
CaMKK - Calcium/calmodulin-dependent protein kinase kinase
dramatically increases the activity of CaMKI and CaMKIV after binding CaMKK to Ca2+-calmodulin and subsequent phosphorylation of CaMKI and CaMKIV

16. Calcineurin (protein phosphatase-2B)
Heterodimeric molecule with highly conserved domain structure
Iron-zinc active catalytic center
Dual protein phosphatase - dephosphorylates both phosphoserine/threonine and phosphotyrosine
Binding sites for calcium (four EF-hand domains in calcineurinB chain)
Preferentially dephosphorylates peptides with basic AA on N-terminal end and without acidic AA on C-terminus
Main substrate of calcineurin is NFAT (nuclear factor of activated T cells)

17. red sphere - calcium gray and blue spheres - iron and zinc
yellow ribon - CnA blue ribon - CnB

18. Calcineurin (protein phosphatase-2B)
The main function is the regulation of gene expression
Small activation is observed after increasing Ca2+
High activation after supplementation and complexation with Ca2+-calmodulin , increase in a fold of the Vmax . Afinity constant of this complex is more than 1010.
Its nhibitor is cyclosporin A (immunosupresory drug)
The gene activation pathway on T cells is increase Ca2+ - increased activity of Cn - dephosphorylation of NFAT in cytoplasma - transport of the complex NFAT-Cn to the nucleus - induction of cytokine genes (IL-2,3,4 , TNF-a, GM-CSF)

19. Adenylyl cyclase
enzyme synthetizing cAMP (the first identified secondary messenger )
nine isoforms are bound in plasma membrane and single isoform is found in cytoplasma
the activity of five membrane isoforms are influenced also by calcium
two isoforms 5 and 6 are inhibited by elevating intracellular Ca2+ concentration
two isoform 1 and 8 are activated by binding complex calcium-calmodulin
isoform 3 is inhibited by complex calcium-calmodulin-calmodulin kinase

20. The isoform-specific regulation of adenylyl cyclases by calcium

21. NO synthases (NOS) Generates nitric oxide from L-arginine
Three types of NOS are known nNOS, iNOS a eNOS
nNOS and eNOS are constitutively synthetized and they are known as calcium-dependent (its enzymatic activity is determined by calcium)
All three types can bind to calmodulin, but nNOS and eNOS are activated only by calmodulin with bound Ca2+.

22. Ca2+-calmodulin binding to nNOS

23. Modulation and effects of intracellular Ca2+

24. Increasing of the cytoplasmatic Ca2+ concentration
Transport of Ca2+ from extracellular space - transport can be mediated by calcium channel gated by agonist (e.g. hormone) or by voltage-gated channel
Liberating of Ca2+ from bound calcium in endoplasmatic/sarcoplasmatic reticulum - ER/SR are relatively abundant in calcium-binding proteins (class I and II) and Ca2+ is liberated on the cytoplasma after stimulus by IP3. (it is created from phosphatidylinositoldiphosphate by cleavage under catalysis of enzyme phospholipase C)

25. Signaling pathways of Ca2+
Cyclic ADP-ribose -RyR (rhyanodine)
Sarco/Endoplasmic Reticulum Ca2+-ATPase

26. Oscillation of intracellular Ca2+ concentration in the various
types of cells