Block A Lecture 1 - Calcium Signalling

Question 1

Are calcium ions found in high or low concentrations inside and outside the cell?

Answer 1

Calcium ions are found in high concentrations outside the cell and low concentrations inside the cell.

Remember: HOLI (high outside, low inside)
(Slide 4)

Question 2

How does the body maintain a low calcium concentration inside the cell?

Answer 2

The body actively pumps Ca2+ ions out of the cytosol into the extracellular space, the endoplasmic reticulum (ER) and sometimes into the mitochondria
(Slide 4)

Question 3

What occurs after the cell is stimulated to release calcium from intracellular stores and when calcium enters the cell through plasma membrane channels?

Answer 3

Signalling occurs
(Slide 4)

Question 4

What are 2 examples of how a cell can pump intracellular calcium out of the cell to maintain a low intracellular concentration?

Answer 4

The cell can either use ATP or it can couple the movement of the calcium ions to the “downhill” movement of sodium ions (Na+) via an anti-porter
(Slide 9)

Question 5

What is the difference between a symporter and an antiporter?

Answer 5

A symporter moves 2 or more molecules in the same direction whereas an antiporter moves 2 or more molecules in different directions
(Slide 10)

Question 6

What 2 places can cells store calcium?

Answer 6

Mainly in the endoplasmic reticulum (ER) but also in mitochondria
(Slide 12)

Question 7

What is the endoplasmic reticulum (ER)?

Answer 7

It’s an organelle in eukaryotic cells that forms an interconnected network of flattened, membrane-enclosed sacs or tube-like structures known as cisternae
(Slide 13)

Question 8

What are the membranes of the ER continuous with?

Answer 8

The outer nuclear membrane
(Slide 13)

Question 9

What ATPase pumps calcium into the endoplasmic reticulum from the cytosol?

Answer 9

Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA)
(Slide 14)

Question 10

What is an example of a storage protein which calcium binds?

Answer 10

Calsequestrin
(Slide 15)

Question 11

What do channels allow?

Answer 11

More rapid movement of ions across the membrane (~1000x faster than antiporters)
(Slide 19)

Question 12

What can some members of the G-protein coupled receptor family trigger in the context of calcium?

Answer 12

They can trigger elevations in calcium concentration
(Slide 23)

Question 13

Explain how a signalling molecule binding to a G-protein coupled receptor can result in intracellular calcium levels increasing and protein kinase C becoming activated.

Answer 13

1. The Signal molecule binding to the GPCR activates it, resulting in a change in conformation.
2. The activated G-protein α subunit directly activates phospholipase C
3. The activated phospholipase C hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) into Inositol 1,4,5-trisphosphate (IP3) and Diacylglycerol (DAG)
4. IP3 binds to receptors located on a calcium channel located on the endoplasmic reticulum (ER) which causes a conformational change, resulting in the opening of the channel and releases calcium ions into the cytosol.
5. DAG and the calcium released from the ER work together to activate protein kinase C (PKC) which goes on to phosphorylate various proteins.
   (Slide 24)

Question 14

What are the protein kinase C superfamily of proteins?

Answer 14

They are homologous proteins made up of domains
(Slide 29)

Question 15

How does DAG and Ca2+ ions activate members of the protein kinase C superfamily?

Answer 15

Calcium binds to the C2 domain, changing the shape of protein kinase C (PKC) and encouraging its binding to the plasma membrane by linking it to a negatively charged lipid known as phosphatidylserine.

At this point PKC is still inactive, but then DAG binds to the C1B domain, which causes a further conformational change, which activates the kinase.
(Slide 30)

Question 16

The activation of protein kinase C by DAG and calcium is localised and activated by 2 distinct events. What does this ensure?

Answer 16

That protein kinase C is not activated by accident.
(Slide 31)

Question 17

What do protein kinases usually do to signals?

Answer 17

They amplify them
(Slide 31)

Question 18

Where are beta cells found?

Answer 18

In the pancreas
(Slide 34)

Question 19

What is exocytosis?

Answer 19

A cellular process that moves large molecules and waste out of a cell’s cytoplasm and into the extracellular space involving intracellular vesicles containing said molecules / waste fusing with the plasma membrane
(Slide 34)

Question 20

Explain how glucose uptake by a beta cell can lead to the exocytosis of vesicles containing insulin.

Answer 20

1. Glucose entering the cell is broken down to generate ATP via glycolysis and respiration.
2. As the concentration of ATP rises, ATP-sensitive potassium channels close and the plasma membrane depolarises
3. Depolarisation of the plasma membrane leads to voltage-gated calcium channels opening, leading to a rapid influx of calcium ions entering the cell
4. The increased calcium concentration leads to vesicles containing insulin undergoing exocytosis and fusion with the plasma membrane
   (Slides 34 - 37)

Question 21

What are the 3 main types of calcium channels?

Answer 21

Voltage-operated channels
Second-messenger operated channels
Receptor-operated channels
(Slide 39)

Question 22

What are the 2 main things are calcium channel blockers used to treat?

Answer 22

High blood pressure
Irregular heartbeat
(also used for other syndromes)
(Slide 44)

Question 23

What are 2 effects which calcium channel blockers can have on the heart?

Answer 23

Lower the heart’s workload
Reduce the force of contraction of the heart
(Slide 45)

Question 24

How do calcium channels inhibitors lower the hearts workload?

Answer 24

They slow down heart beats, allowing the left ventricle to fill completely, lowering heart workload
(Slide 45)

Question 25

How do calcium channel blockers reduce blood pressure?

Answer 25

They reduce vasoconstriction by relaxing vascular smooth muscle, leading to vasodilation and therefore a lower blood pressure
(Slide 45)

Question 26

There are many different classes of calcium blockers. These can have different mechanisms of action to prevent calcium from using these channels. Briefly outline 2 of these.

Answer 26

They can plug the molecular pore of the channel to prevent calcium entry (e.g verapamil)
They can bind to the outside of the channel and modify the channel allosterically so calcium ends up lodging inside the channel (e.g amlodipine)
(Slide 48)

Question 27

What does FRET stand for?

Answer 27

Florescence Resonance Energy Transfer
(Slide 49)

Question 28

What is FRET?

Answer 28

It’s a technique used in biochemistry and cell biology to measure interactions between molecules or detect changes in molecular environments, such as fluctuations in calcium concentration
(Slide 49)

Question 29

How does FRET work?

Answer 29

When calcium binds to the calcium-sensing domain, the structure of the sensor changes, bringing the donor and acceptor fluorophores closer together. This proximity leads to FRET, where energy is transferred from the donor to the acceptor, and the acceptor emits fluorescence.
The intensity of fluorescence from the donor and acceptor changes in response to calcium binding, providing a quantitative measure of intracellular calcium levels.
(Slide 49)

Question 30

What 4 things does a genetically encoded calcium indicator consist of?

Answer 30

A donor fluorophore
An acceptor fluorophore
A calcium binding domain (e.g calmodulin)
A calcium sensitive peptide
(Slide 50)

Question 31

How does a genetically encoded calcium indicator work?

Answer 31

1. In the absence of calcium, the two fluorophores (donor and acceptor) are held apart by the calcium-binding domain and the calcium-sensitive peptide. In this state, the donor fluorophore emits light, but no FRET occurs, or FRET efficiency is very low.
2. When calcium binds to the indicator, it induces a conformational change in the calcium-binding domain (calmodulin) and the attached peptide (e.g., M13), bringing the donor and acceptor fluorophores closer together.
3. This close proximity (~1-10 nm) of the donor and acceptor, along with favorable spectral overlap and correct orientation, allows FRET to occur. The donor fluorophore (CFP) gets excited, and instead of emitting light itself, it transfers its energy to the acceptor fluorophore (YFP).
4. As a result, the acceptor fluorophore (YFP) emits fluorescence at its characteristic wavelength. By measuring this change in the emission ratio between the donor and acceptor (e.g., the ratio of YFP to CFP fluorescence), you can quantify the calcium concentration in real time.

(Slide 50)