Term 2 Lecture 17: Calcium Signalling Flashcards
Process
cAMP can activate Na+/Ca²+ channels letting in ions
The Ca²+ then activates Ca²+ gated Cl- channels letting Cl- ions out
Ca²+ can also interact with antiporters e.g. Na+/ Ca²+ exchanger that exchanges a Ca²+ out for each Na+ in
Glycogenolysis in striatial muscle
Neural stimulation →Ca²+
→increase in → GPK*→increase in →GP
→ results in increased glycogen degeneration (to produce glucose)
Hormonal stimulation →cAMP
→increase in →PKA*
→stops production of GS
→results in decreased glucagon synthesis
*PKA phosphorylates GPK
Abbreviations:
PKA protein kinase A
GPK glycogen phosphorylase kinase
GP glycogen phosphorylase
GS glycogen synthase
Ca²+ signalling via neural stimulation and cAMP via epinephrine (hormonal) stimulation of beta adrenergic receptors increase glycogen degeneration in striatial muscle.
Glycogen phosphorylase kinase is regulated by Ca²+ and phosphorylation by cAMP dependent PKA
IP3 triggers Ca²+ release from the ER
PLC cleaves PIP2 into DAG and IP3
(Step 2 see previous lecture)
In turn IP3 activates IP3 gated Ca²+ channels in the ER membrane (step 3)
Ca²+ concentration in ER is much higher than in cytosol so Ca²+ is released into the cytosol (step 4)
(Ca²+ concentration in the cytosol can increase up to 100x from ~100nm to ~10 micro m)
STIM1 (stimulator protein 1) binds Ca²+ in the ER. Abundance of Ca²+ in the ER lumen maintains STIMli bound to cytosolic proteins and microtubules - away from the plasma membrane - bound in a monomeric singular form
When an IP3 gated ion channel opens causing sudden efflux of Ca²+ ions from ER into cytosol Ca²+ concentration decreases in the ER lumen. Binding to STIMl is reduced or absent and STIMl oligemrrises losing its attachment to cytosolic proteins and microtubules.
This enables binding via CAD domains to the store-operated channel (ORA-1) triggering its opening and the entrance of Ca²+ ions into the cytosol from the EC space. Causing more Ca²+ to flood into the cytosol.
Resulting in a 1000 fold increase in Ca²+ (when the 2 processes of Ca²+ entering are combined)
How does this work?
Ca²+ channels in plasma membrane allow flux of Ca²+ in/out.
ER stores Ca²+ at concentration higher than that of the cell and extracellular space via 2 processes:
1) SERCA pump (sarcoendoplasmic reticulum calcium ATPase) an ATP dependent pump that expends energy to get Ca²+ against the concentration gradient into the ER lumen
2) Ca²+ is stored via a chaperone protein called calreticulin which bind Ca²+ ions
Ca²+ signalling in mitochondria
1a) IP3 binds IP3 gated Ca²+ channels on ER
1b) IP3 gated channels in mitochondrial associated membranes (MAM) of the ER are opened by binding of Ca²+
2) Ca²+ is transported inter the intermembrane space via VDAC channels on outer membrane of mitochondria.
3) the high concentration of Ca²+ in the intermembrane space indices the opening of Ca²+ channels in the inner membrane resulting in the flow of Ca²+ into the mitochondrial membrane
4) Ca²+ is released from the mitochondrial matrix into the intermembrane space by Ca²+/N+ and Ca²+/H+ antiporters in the inner membrane then transferred into the cytosol through VDAC or other channels
In the outer membrane
5) finally pumping of Ca²+ ions from the cytosol by ATP powered SERCA pumps in the ER membrane restores the high ER and low cytosolic Ca²+ levels
Calcium oscillations
E.g. histamine signalling molecule (O.5micrometre) causes fast 100x increase in Ca²+ in cytosol as it enters from the ER followed by a rapid decrease back to baseline within one hour- negative feedback cooperation to regulate Ca²+ concentration in cell and it’s compartments
Cellular responses to hormone induced rise in cytosolic Ca²+ in various tissues
Tissue/Hormone for more Ca²+/ cellular response
Pancreas (acinar cells)/ acetylcholine/ secrete digestive enzymes e.g. amylase and trypsin
Parotid (salicary) gland/ acetylcholine/ secretion of amylase
Vascular or smooth muscle/ acetylcholine/ contraction
Liver/ vasopressin/ glycogen to glucose
Blood platelets/ thrombin/ aggregation, shape change and hormone secretion
Mast cells/ antigen/ histamine secretion
Fibroblasts/ peptide growth factors/
DNA synthesis and cell division
Nerve cells / many/ secretion of neurotransmitters
Calmodulin changed confirmation of Ca²+ binding
Calmodulin is a small protein important in binding Ca²+ it does this via 4 EF hand/helix -lool-helix motifs which can each bind one Ca²+ ion. This induces confirmational changes making Calmodulin capable of binding helices from specific proteins (peptides.) Helices come together to form a ring-like structure able to bind proteins when 4 Ca²+ are bound
Targets of Calmodulin: CAMKll
CAMKll is calcium Calmodulin protein kinase 2
It is inactive until in contact with calcium Calmodulin complex (Ca²+/CaM)
CAMKll is activated by binding 4 Ca²+/CaM it then undergoes protein autophosphorylation binding to Tyrosine at position 286 changing the confirmation allowing two more Ca²+/CaM to bind (6 in total
At this point each of the 6 subunits of the kinase is phosphorylated at T 286
The CAMKll is then active but held trapped in a Ca²+/CaM cage.
The CAMKll dissociates from the cage and remains phosphorylated it is now active.
Further autophosphorylation of T306 and T307 on each subunit occurs
This active capped CAMKll cannot be bound by Ca²+/CaM anymore and is fully active ready to fulfill its role.
Phosphatase regulates this by removing phosphate groups returning CAMKll to its inactive state.
CAMKll in long term potentiation in neurons
CAMKll participated in long-term potentiation (LTP) by anchoring receptors to the synapse and increasing their conductance e.g. important for memory
CAMKll activated by phosphorylation interactions with Ca²+/CaM enters the head of the neuron and
1) interferes/ modulates RAS (a g protein) signalling
2) phosphorylated CAMKll interacts with NMDA receptor opening Ca²+ channels allowing Ca²+ influx
Also it phosphorylates stargazin on AMPA receptors allowing PSD95 to bind to strargazin immobilising AMPA in the synapse region allowing influx.
Calmodulin and the formation of bacterial and arbuscular mycorrhizal symbioses
Calmodulin pathway is present in all eukaryotes
CCaMK in legumes is central to symbiotic relationships between legumes, bacteria and arbuscular mycorrhizae.
CCaMK is activated by Calmodulin that phosphorylates the transcription factor CYCLOPS to activate the nodule inception NIN or educed arbuscular mycorrhizae RAM1 genes.
See diagram middle of notebook 3
Using Calmodulin fusion protein to measure Ca²+ concentration
M13 peptide binds to Ca²+ activated Calmodulin. Fusion of these 2 proteins with GFP leads to relatively weak fluorescence in absence of Ca²+. However increase of Ca²+ concentration triggers binding of CaM to M13 and confirmational changes that increase fluorescence of GFP and this changed can be quantified
