Calcium Signalling

Question 1

What is glycogenolysis (in striatal muscle)

Answer 1

1. The process of breaking glycogen down into glucose
2. Process triggered by two types of stimulation, neural stimulation and hormonal stimulation
3. Neural stimulation of GCPR leads to Ca2+ signalling, which activates glycogen phosphorylase kinase (GPK), which phosphorylates glycogen phosphorylase (GP) leading to increased glycogen degradation
4. Hormonal stimulation of B-adrenergic receptors like epinephrine, increases PKA, lowering activity of glycogen synthase (GS), causing decreased glycogen synthesis.

Question 2

Explain how the Store-operated Ca2+ channel, Orai1, is activated

Answer 2

1. STIM1 is an integral protein in the ER membrane and it controls Orai1 activation
2. When ER lumen Ca2+ concentrations are high, STIM1 is bound by Ca2+, maintaining STIM1 in an inactive form bound to cytosolic proteins and microtubules
3. When low, Ca2+ do not bind STIM1, causing protein to oligomerise and lose attachments to proteins and microtubules
4. Results in STIM1 extending through the cytosol and binding to Orai1 (integral plasma membrane protein) via CAD domains on the STIM1 protein
5. Orai1 opens, allowing extracellular Ca2+ to diffuse into the cytosol

Question 3

What are the roles of SERCA pumps and calreticulin in storing Ca2+ in the ER

Answer 3

1. (Sarco endorplasmic reticulum calcium pumps) are located in the ER membrane and require ATP to pump calcium from the cytosol into the ER lumen
2. Calreticulin is a chaperone protein that intercalates with calcium allowing the maintenance of high Ca2+ conc
3. IP3R calcium gated channel is an ER membrane protein that allows Ca2+ back into the cytosol

Question 4

Explain the interlinkage of ER and mitochondrial calcium signalling that allow for calcium to be recycled around these organelles

Answer 4

1. IP3 binds IP3 gated Ca2+ channels causing calcium to move from ER to cytosol
2. IP3GC on the mitochondrial associated membranes (MAMs) are also opened by IP3 binding
3. The Ca2+ that pass through the MAMs are transported into the mitochondrial intermembrane space by VDAC channels (voltage dependent channels)
4. When intermembrane space Ca2+ concentrations are high, Ca2+ uniporter channels in the inner membrane open and allow Ca2+ into the matrix
5. The reverse from the matrix to the intermembrane space occurs through Ca2+/Na+ and Ca2+/H+ antiporters, and from intermembrane space to cytosol by VDAC channels
6. ATP-powered Ca2+ pumps in the ER membrane transport Ca2+ from the cytosol back into the ER
7. These processes ensure high Ca2+ ER conc, and low Ca2+ cytosolic levels

Question 5

What is the role of calmodulin and how does it interact with calcium

Answer 5

1. Calmodulin has 4 EF hand motifs which can each bind Ca2+
2. Induces conformational changes allowing calmodulin to bind to helices from other proteins
3. Ca2+/CaM binds to an inactive kinase, called CaMKII, becoming active-CaM bound
4. Active-CaM bound kinase autophosphorylates (without help of an enzyme) to form active-CaM trapped
5. It then loses Ca2+/CaM to form active-Ca2+ Independent
6. Then further autophosphorylated to form active-capped CaMKII
7. This active capped form cannot interact with Ca2+/CaM, and must be phosphatased to restart the cycle
8. CaMKII involved in long term potentiation
9. CaMKII diffuses into dendrite head where it is phosphorylated and activated explained in steps 3-7.
10. Phosphorylated CaMKII leads RAS-ERK (Gprotein-Kinase) signalling and the phosphorylation of stargazin, which binds AMPARs and anchors them to the presynpatic membrane (bound to PSD95) ready to influence synaptic transmission
11. Phosphorylated CaMKII also phosphorylates parts of the AMPARs which increases their channel conductance

Question 6

Give an explanation how Calmodulin is used in plants

Answer 6

1. Flavonoids in plants are secreted out of the cell where Rhizobium (nitrogen fixing bacteria) then release NOD factors
2. NOD factors lead to Ca2+ signalling in plant cells, binding to calmodulin, which then is involved in activating CaMK
3. CaMK is involved in two major pathways
4. Binding with phosphorylated cyclops transcription factor and DELLA proteins lead to arbuscular development (RAM1 genes) - for fungal symbiosis
5. Can bind to cyclops transcription factor on its own which leads to nodulation (NIN genes)

Question 7

How can we measure Ca2+ concentration in cells

Answer 7

1. GFP can be linked to M13 protein and calmodulin
2. M13 binds to Ca2+ activated calmodulin
3. Fusion of M13 and Ca2+/CaM leads to intense green fluorescence when Ca2+ conc is high
4. Weak fluorescence when Ca2+ conc is low
5. Degree of fluorescence can be quantified and linked to Ca2+ concentration