Calcium

Question 1

What does calcium play a role in?

Answer 1

Call signalling and physiology regulating tasks: 
Metabolism
Hormonal regulation
Membrane linked functions
Contractile and motile systems
Intracellular signalling functions

Question 2

Is the more calcium inside our outside the cell at rest? Give approx values

Answer 2

More outside - 2-2.5mM

Inside - 100nM

Question 3

What state is calcium typically found extracellularly?

Answer 3

It can exist either in the dissolved ionised state as free Ca2+ ions or more commonly as Ca2+ bound to proteins or other molecules

Question 4

There is always interchange going on between bound and unbound Ca2+ ions
What is the overall concentration of free Ca2+?

Answer 4

~1mM

Question 5

Why do the concs of Ca2+ in small cytoplasmic volumes vary so much?

Answer 5

This is because the cell is very actively having to control its levels so that the Ca2+ signals controlling the activation of processes or signalling pathways are at the right concentration at the right time and in the right place.

Question 6

What benefit is there of having a high conc of Ca2+ outside the cell?

Answer 6

Important signal source bc they provide conc gradient

Question 7

Where else is calcium stored other than extracellularly?

Answer 7

Cells themselves also possess intracellular Ca2+ stores that can be activated to provide access to a large reservoir source of Ca2+. This can be released rapidly in the required amounts to synchronously drive a range of cellular processes.
E.g. in the ER

Question 8

Name some cellular processes driven by Ca2+

Answer 8

Muscle contraction
Neurotransmitter release
Gi secretions
Hormonal release

Question 9

What are the main Ca2+ stores in cells?

Answer 9

Main - sarco/endoplasmic reticulum - conc ~300uM-1mM - rapid release store
Mitochondria - to buffer [Ca2+]i levels - not so much as a reservoir - non rapid release store

Question 10

What’s the advantage in having a large conc gradient of Ca2+ between the cytosol and outside/in organelles?

Answer 10

The advantage of these large gradients is that relatively large changes in cytosolic ‘sink’ concentration can be achieved with relatively small movement of calcium from the two major sources: the extracellular stores and SER/SR stores. This means that relatively little Ca2+ has to be removed from the cytosol to get back to basal levels.

Question 11

How does prolonged elevation of Ca2+ lead to cell death?

Answer 11

Nonetheless, this activity is energy expensive and loss of Ca2+ homeostasis will increase derangement in cell activity. If this loss is very marked, then it will lead to cell death. This is because prolonged elevation of Ca2+ levels would mean pathways and processes regulated by Ca2+ were constantly active leading to loss of coherent cell function. Furthermore, reversal of calcium flux into the mitochondrion occurs and that precipitates apoptosis.

Question 12

Are cell membranes normally permeable or impermeable to Ca2+?

Answer 12

Very impermeable - can only enter through highly selective channels/transporters

Question 13

What are the 3 major routes of calcium into the cell?

Answer 13

Voltage Operated Calcium Channels (VOCCs)
Ligand Gated Ion Channels (LGICs)
Store Operated Channels (SOCs)

Question 14

How does Ca2+ enter through a VOCC?

Answer 14

Open in response to depolarisation - Ca2+ then flows in down conc gradient very rapidly
This family varies in the amount of Ca2+ they can conduct

Question 15

How does calcium enter via a LGIC? Give an example

Answer 15

Most LGICs are activated by excitatory neurotransmitters and when these bind the channel opens to allow Ca2+ to flow into the intracellular sink. An important example is the NMDA channel. The NMDA channel carries large Ca2+ currents that can be sufficient to cause the neurone to ‘burn out’ through accumulation of excess [Ca2+] i.

Question 16

What are SOCs?

Answer 16

These are distinctive Ca2+ ion channels with very low conductances that operate over seconds. That is they are comparatively very slow. These are expressed in both excitable and non-excitable cells. In this latter group, they appear to be important in accessing extracellular Ca2+ when the SER stores for Ca2+ are depleted.
When this intracellular store or ‘source’ is depleted and the Ca2+ ATPase in the plasma membrane has been very active, then the cell has the option of calling on the extracellular ‘store’ for back up.

Question 17

How is the SOC activated and what is the relevance of its location?

Answer 17

The SOC is also interesting because it needs to be activated by a special Ca2+ sensing protein in the SER to operate. This protein detects when [Ca2+]SER is low and interacts via close apposition to the SOC channel .
Thus, SOCs are important in smooth muscle where prolonged states of stable contraction are required (i.e. when a sphincter needs to remain closed).

Question 18

Name the 2 important carriers for Ca2+ which re establish the very low basal [Ca2+]i

Answer 18

Plasma membrane Ca2+ ATPase (PMCA)

Sodium-Calcium Exchanger (NCX)

Question 19

How much ATP is required to transfer 1 Ca2+ ion via PMCA?

Answer 19

1 ATP transfers 1 Ca2+ ion

Question 20

Describe the affinity for Ca2+ of PMCA

Answer 20

It has a high affinity for Ca2+, this means it will have begun to substantially carry Ca2+ when [Ca2+ ]i is about 1 µM or 10-6 M.
Its affinity for Ca2+ ions is further optimised when it binds with calmodulin, a cytoplasmic Ca2+ sensing protein (see below).

As its expression in cells is normally quite low, it is generally considered to have more of a regulation role in ‘fine tuning’ [Ca2+]i.

Question 21

Does NCX use ATP?

Answer 21

No - it uses the electrochemical energy gradient provided by large extracellular Na+ conc

Question 22

How much Na+is exchanged for Ca2+ via NCX?

Answer 22

3 sodium in for 1 calcium out

Leads to increased [Na+]i

Question 23

What restores the sodium concentrations?

Answer 23

The cell can normally use NCX without major changes in electrochemical gradients because the [Na+]i and [Na+]o are subsequently restored via plasma membrane Na+/K+-ATPase (sodium pump) activity.

Question 24

describe the affinity for Ca2+ of NCX

Answer 24

The NCX pump has a lower affinity for Ca2+, but is extensively expressed in many cell types. Whilst it has a relatively lower affinity for Ca2+, it has an overall higher capacity for pumping Ca2+ out of the cell when these levels are reached.

Question 25

When and where is the NCX especially active?

Answer 25

Compared to the Ca2+-ATPase, it is considered to act as the primary pump of Ca2+ when free cytosolic levels are > 10 µM. The NCX is especially active in excitable tissues, such as nerve and muscle, where continuous large movements of Ca2+ underpin normal physiological activity.
In heavily depolarised cells, if [Na+]i increases significantly then the NCX can act in reverse to pump Na+ out and carry Ca2+ inwards.

Question 26

Name 3 ways to regulate Ca2+ entry from the SER/SR into cytosol

Answer 26

• GPCRs (Gq type) in the Plasma Membrane
• IP3 receptors in the SER/SR membrane
• Ryanodine Receptors in the SER/SR membrane

Question 27

Which type of GPCR contributes to regulation of CA2+ effluent and how?

Answer 27

Gq
In brief, when a suitable ligand binds to the Gq receptor type, it triggers production of inositol trisphosphate (abbreviated to IP3), from membrane phospholipids. IP3 then diffuses through the cytoplasm and binds with the IP3 receptor on the external face of the SER/SR membrane.

Question 28

What is the IP3 receptor?

Answer 28

The IP3 receptor (IP3R), is a Ligand Gated Ion Channel that opens to allow Ca2+ efflux out of the SER/SR and into the cytosol when IP3 binds with it. Its activity remains proportional to the ligand binding with the Gq receptor driving the synthesis of IP3. When this falls, so too does the activity of the IP3R. This route is the main way in which GPCR activation signals an increase in [Ca2+]i.

Question 29

What is a ryanodine receptor?

Answer 29

This receptor is named after the alkaloid, ryanodine, which was found to block this receptor. RyRs are also Ligand Gated Ion Channels for which cytosolic free Ca2+ itself acts as the ligand. RyRs are expressed across many cell types and are heavily expressed in the SR of muscle cells.

Question 30

Describe the activation of RyRs in smooth and cardiac muscle

Answer 30

In smooth and cardiac muscle, the triggering calcium signal comes through activation of the VGCCs. These are located in the t-tubules and their equivalents in smooth muscle cells (known as caveolae).

Question 31

What is CICR (in smooth and cardiac muscle)?

Answer 31

With depolarisation of the t-tubule, the VGCCs open and allow an influx of Ca2+. This Ca2+ then binds with the RyR which results in a very large synchronous outward flux of SR Ca2+ into the sarcoplasm (i.e muscle cytoplasm). This is known as: Calcium-Induced Calcium Release (CICR)

Question 32

Describe the activation of RyRs in skeletal muscle

Answer 32

In skeletal muscle, the RyR can still be activated by increased [Ca2+]i, however, there is a structural modification whereby the T-tubule VGCCs are directly physically coupled to the RyR receptor. This channel coupling means that when the VGCCs open, the RyRs also open and release massive amounts of Ca2+ ions into the sarcoplasm. The increase in [Ca2+]i then enables contraction by the interaction of the contractile proteins. This process is described in more detail elsewhere in the course.

Question 33

What do RyRs work in conjunction with?

Answer 33

The activity of RyRs can be modulated by other signalling molecules to increase Ca2+ release further. In conjunction, with the IP3 receptor in smooth muscle the RyR route serves as a further route for Ca2+ release.

Question 34

What is the SERCA?

Answer 34

The SER/SR membrane contains a single efficient carrier for Ca2+ that contribute to re-establishing the very low cytosolic basal [Ca2+]I of 10-7 M. This is:

Question 35

Describe the function of the SERCA

Answer 35

The SERCA is a particularly efficient and along with the plasma membrane NCX enables a rapid re-establishment of the basal [Ca2+]i following an external stimulus of the cell ceasing. Again, this is energy requiring as it is transporting Ca2+ against a very steep gradient.

Question 36

Name 2 major protein groups that bind with Ca2+

Answer 36

Calcium buffers

Calcium sensors/trigger proteins

Question 37

Why is the rate of Ca2+ movement through cytosol slower than expected?

Answer 37

As Ca2+ moves through the cytosol, its rate of diffusion is slower than expected. This is because of the presence of buffer proteins that act to ‘smooth’ out and dampen down the very rapid entry of Ca2+ throughout the cell and its compartments.

Question 38

Name some buffer proteins

Answer 38

These buffer proteins include: parvalbumin, calbindin, calsequestrin and calreticulin - the last two proteins bind Ca2+ in the SR and SER, respectively.

Question 39

Describe the function of buffers

Answer 39

These different Ca2+ buffering proteins (as individual proteins) can bind with up to 50 Ca2+ ions. Depending on their concentration in cytosol, the buffer proteins can reduce the spread of Ca2+ current from a channel exit (effectively  1mM) down to about 10 -100 µM to over 0.1-0.5 µm. As [Ca2+]i drops, the buffer bound Ca2+ is gradually released to prolong its cytosolic availability.

Question 40

What are calcium sensors/triggers?

Answer 40

Sometimes it is not calcium which exerts this control over many target proteins directly. It is instead, mediated through a Ca2+ binding protein that then itself goes on to regulate the other proteins. These are known as the ‘calcium sensors’ or ‘trigger’ proteins. This group of proteins include calmodulin, STIM1 and annexin. Calmodulin (often abbreviated to CaM) can interact with and regulate many different intracellular proteins.

Question 41

Describe calmodulin as an example of a calcium sensor

Answer 41

Calmodulin (CaM) can bind up to four Ca2+ ions - induce a conformational change in CaM.
This then enables CaM to interact with this very wide range of proteins.
Many of the proteins that CaM binds to and regulates are unable to bind calcium themselves. Therefore, CaM acts as both a calcium sensor and a signal transducer.
The transduction signal is proportional to the changes in [Ca2+]i.
A good example of the transducer activity of CaM is its modulation of the PMCA Ca2+-ATPase. When bound with Ca2+, CaM can then bind with PMCA and then further increase its sensitivity to [Ca2+ ]i. This increases the sensitivity of PMCA by a factor of about 10. This then results in 10 times the activity of the ‘pump’ when it is bound with CaM.