Intracellular Ca2+ signalling Vytášek 2009
Ca2+ is a universal intracellular signalling molecule (secondary messenger), which controls numerous developmental and cellular pathways intracellular concentration of resting cell is 50-100nM, activated cells have Ca2+ concentration above 1M 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
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
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
EF-hand motif of calcium binding site 1-10. 2 alfa-helix E forefinger 11-21 loop around Ca - clenched middle finger 22-29 alfahelx - F thumb
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+)
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
The conformational changes of calmodulin after binding of calcium ion
Ca2+ Blue circles
Proteins affected complexation with Ca2+-calmodulin Protein kinases (CaMK) Calcineurin (protein phosphatase 2B) Adenylate cyclase NO synthases cAMP phoshodiesterase Ion chanels Receptors
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
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)
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
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
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
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)
red sphere - calcium gray and blue spheres - iron and zinc yellow ribon - CnA blue ribon - CnB
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 50-100-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)
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
The isoform-specific regulation of adenylyl cyclases by calcium
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+.
Ca2+-calmodulin binding to nNOS
Modulation and effects of intracellular Ca2+
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)
Signaling pathways of Ca2+ Cyclic ADP-ribose -RyR (rhyanodine) Sarco/Endoplasmic Reticulum Ca2+-ATPase
Oscillation of intracellular Ca2+ concentration in the various types of cells