Calcium Homeostasis 1

Question 1

What are the calcium stores called within cells

Answer 1

Normal cells - endoplasmic reticulum
Muscle cells - sarcoplasmic reticulum

Question 2

Where is the calcium concentration the highest

Answer 2

Inside the cell (20-300nM) compared to outside (2nM)

Question 3

What does a persistent high intracellular calcium suggest

Answer 3

Apoptosis

Question 4

How do calcium enter the cell

Answer 4

Down concentrated gradient into cell via voltage gated calcium channels

Question 5

How can the calcium levels increase inside the cell

Answer 5

Via release of calcium from the endoplasmic reticulum
OR
Down concentration gradient into cells via voltage-gated calcium channels

Question 6

How is calcium released from the endoplasmic reticulum

Answer 6

IP3 (produced by phospholipase C via classical G protien-coupled receptor. The IP3 binds to the IP3 receptor on the membrane of calcium stores which opens the IP3R as also a channel which therefore releases calcium

OR

Ryanodine receptor opening

Question 7

How is calcium decreased inside the cell (4 ways)

Answer 7

Calcium-sodium exchanger
Buffering calcium proteins
ATP-dependent pumps
Endoplasmic reticulum pumps - take calcium into ER to refill the calcium vesicles

Question 8

Where is calcium found in the blood

Answer 8

50% on protein-bound
Conc same as phosphorus

Question 9

Give the 3 main calcium pools

Answer 9

Inside ER
Blood - plasma proteins
Bone - most is mineral but 1% is free

Question 10

How does calcium get into ER or SR

Answer 10

Via high-affinity calcium uptake through calcium-ATPase transporters

Form of active transport

These pumps are called SERCA - sarco/endoplasmic reticulum Ca-ATPase

Question 11

What are the name of the calcium channels that pump calcium ions back out of cell when desired concentration is achieved

Answer 11

PMCA - transports calcium out of the cell via high-affinity calcium uptake through calcium-ATPase transporters

Question 12

How do sodium-calcium exchangers work

Answer 12

Get calcium out of the cell in exchange for sodium into the cell.
Work at high concentration - (low affinity) but
High capacity (large exchange so fast)

Works first to get bulk of calcium out

Question 13

How does the ATP-dependent calcium pump differ in its capacity and affinity

Answer 13

Works at low concentration so (Affinity is high)
Low capacity (small exchange so slow)

Basically fine tunes the calcium level

Question 14

How is calcium brought back into the calcium vesicles in the ER without increasing cytoplasmic calcium levels

Answer 14

STIM1 and Orai1 are proteins as part of a store-operated calcium entry -

IP3 opens IP3R to release calcium which empties the stores
The store emptying triggers Orai1 (Cell membrane) and STIM1 (ER membrane) to align and form a single channel, allowing calcium to flow into the endoplasmic reticulum

Question 15

Give 4 calcium binding sites

Answer 15

Membranes
Amino acids
Nucleotides
Binding proteins

Question 16

Give some features of calcium binding proteins

Answer 16

Rapid with high affinity
Some are free and some restricted
Free - parvalbumin, calbinding, calretinin, calmodulin
Restricted - calreticulin and calsequestrin

Question 17

What is the purpose of calcium binding proteins/sites

Answer 17

Keep enough extra “inactivated” calcium present to refill calcium vesicles

Question 18

What is the mitochondria’s role in calcium uptake

Answer 18

When calcium is of high conc then the mitochondria will be activated, so much so that if the calcium conc is too high then the mitochondria stops ATP synthesis and puts all efforts into bringing calcium back down to prevent sustained toxic periods of high calcium ion conc intracellularly

Question 19

How is IP3 activated to work on IP3R to release calcium from ER (2 ways)

Answer 19

Tyrosine kinase linked receptor e.g. epidermal growth factor
OR
G-protein coupled receptor e.g. glutamate receptor and olfactory receptors

Question 20

What is the purpose of different IP3 isoforms

Answer 20

Theres 4 types -
All have different affinity and sensitivity for calcium

Question 21

What can block IP3 formation

Answer 21

Lithium

Question 22

What can block IP3 binding

Answer 22

Heparin

Question 23

How does the IP3R open

Answer 23

IP3 and calcium bind to cytosolic side of receptor to open it

Question 24

Why is calcium binding to the IP3R important

Answer 24

Promotes channel closure via feedback inhibition - high cystolic calcium and calcium ATPase pump activity turns off the IP3R - causes calcium oscillations

Question 25

Why are these calcium oscillations important

Answer 25

Believed that specific oscillations may be able to turn gene expression on etc

Question 26

What is the structure of IP3

Answer 26

A tetramer - each with 6 membrane spanning domains
Central pore for calcium
Each tetramer has 4 IP3 binding sites which activates its opening

Question 27

How do IP3 channels actually open

Answer 27

Conformational change on IP3 binding, this hinges suppressor region away from gatekeeper region and allows channel to open.

Question 28

What role does STIM1/ORAI1 have in store-operated calcium entry in endothelial cells

WIDER READING - Abdullaev 2008

Answer 28

Knockdown of orai1, stim1 and stim2 showed that it inhibited EC proliferation and caused cell cycle arrest. Therefore store-operated calcium (Ca2+) entry is crucial for endothelial cell proliferation in mice.
Also shows that SOCE through STIM1-ORAI1 not TRPC1 as previously believed

Question 29

What role did STIM1 and ORAI1 have in T cells

WIDER READING - lioudyno et al 2008

Answer 29

STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or superantigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR)
Also showed area of higher calcium concentration at the interface

Question 30

What joins mitochondria and ER and how much is joined

WIDER READING - Rizzuto et al 1998

Answer 30

20% - mitochondria-associated membranes

Question 31

How does calcium enter the mitochondria once released by the IP3Rs

WIDER READING - De Pinto 1992

Answer 31

The Ca2+ released through IP3Rs is transferred to the mitochondrial intermembrane space by a class of mitochondrial porins known as voltage-dependent anion channels (VDACs), which form very abundant and large voltage-gated pores in the outer mitochondrial membrane at the ER-mitochondria contacts

Question 32

How does the tumour suppressor gene PTEN regulate apoptosis

WIDER READING - Bononi et al 2013

Answer 32

PTEN enhances ER-Mitochondria calcium transfer to cause apoptosis. It does this by dephosphorylating IP3Rs which causes increased calcium release to the mitochondria to kill the cell and prevent a tumour

Question 33

What was shown regarding calcium oscillations and gene expression

WIDER READING
- Doletsch et al 1998

Answer 33

We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-κB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-κB.